Unusual Excitation Wavelength Dependency of Quantum Yield in Water Soluble CdTe Quantum Dots

Unusual Excitation Wavelength Dependency of Quantum Yield in Water Soluble CdTe Quantum Dots | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Unusual Excitation Wavelength Dependency of Quantum Yield in Water Soluble CdTe Quantum Dots Chayan Nandi, Kush Kaushik, Jiban Mondal, Ritesh Bag, Shagun Sharma, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3999532/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Dec, 2024 Read the published version in Nanoscale → Version 1 posted You are reading this latest preprint version Abstract The Quantum yield (QY) of the semiconductor quantum dots (QDs) severely hampered by the inherent fluorescence intermittency. The QY of QDs usually increases with the increase in excitation wavelength. Here, we present an unusual behaviour where QY is found to decrease with increase in excitation wavelength in water soluble CdTe QDs (CQDs). Single particle measurement highlights the increase in permanent single dark particles at longer wavelength that comprehend the overall QY of the QDs in bulk solution. Fluorescence correlation spectroscopy further revealed an increase in number of dark particles at longer wavelength. The presence of H + ion in the water plays an important role in creating the permanently dark states in the CQDs. This observation was further supported by the cell internalization study of the QDs where much brighter images at shorter wavelength than longer wavelength were observed. A study of the excitation wavelength-dependent QY in QDs may reveal new insights into the applicability of QDs in different device fabrication cases. Physical sciences/Materials science/Nanoscale materials/Quantum dots Physical sciences/Nanoscience and technology/Nanoscale materials/Nanoparticles CdTe quantum dots quantum yield Dark fraction excitation dependency single particle fluorescence correlation spectroscopy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Semiconductor QDs are highly fluorescent nanostructures with unique optoelectronic properties. Due to their high QY 1 , high photostability 2 , broad absorption spectrum 3 with very narrow and tunable emission spectrum 4 , QDs have been employed in numerous applications such as light emitting diodes (LEDs), photonics, Solar cells, LASER, sensing and bioimaging agents 5 – 6 . Unfortunately, QY of the QDs is severely hampered by the inherent fluorescence intermittency (called blinking), which is the phenomenon of random switching between ON (bright) and OFF (dark) states of the emitter upon continuous excitation. Efforts have been made to understand the mechanism of such intermittency both at the ensemble and single particle level 7 – 12 . The unusual feature in this blinking phenomenon is the power law statistics of the ON and OFF time distributions 13 . Due to this fact, the time-averaged intensity of a single emitter is not reproducible in different experiments and implies a complex dynamic of the involved process and generally hampers the QY in almost all the QDs. A substantial effort has been made to manipulate the QY of QDs by modifying the surrounding environment, surface functionalization, change in pH, ligand length, electron-donating and withdrawing agents 15 – 18 . Recent reports suggest that excitation wavelength plays a very significant role in affecting the QY 7 , 8 , 15 – 19 . A general observation (Fig. 1 ) is that with the increase in excitation energy (shorter wavelength) the absorption of light by the QDs increases substantially, however, the emission intensity decreases monotonically in the reverse order from blue to the band edge emission. This is attributed to (a) exciting particles into continuum states, distinct from confined states 19 and (b) the charge carriers trapping in a dark state or a very stable trapped state that decreases the radiative recombination and thus decreases the QY of the material. Hoy et al. suggested that the QY in CdSe and CdSe/ZnS QDs of different sizes exhibited a proportional relationship with excitation energy 7 . Roy et al. performed excitation wavelength dependent single particle study on InP and CdSe’s core-shell and core alloy shell quantum dots. They observed that at longer wavelength, PLQY decreases with increment in ON truncation time and decrement in OFF truncation times. It was also shown that the core-alloy shell exhibited more ON fraction than the core-shell QD structures 16 . Often, these changes in QY, ON fraction, ON-OFF truncation times are attributed to high density of trap states at higher energy levels. De et al. performed the excitation-dependent ultra-fast dynamics in small-sized InP-based core/alloy-shell/shell QDs 19 . They showed that the excitons took a longer time to recombine for lower wavelength excitations in comparison to higher wavelength excitation and proposed that the high degree of trapping yielded lower QY values at low wavelength excitations. Surprisingly, the excitation wavelength-dependent emission was conflicted in describing the probable measurement error or artifacts due to the presence of QDs aggregates as well as excess surface ligands present in the solution that leads to scattering losses in QY measurement at shorter excitation wavelength 20 . The mechanism of photoluminescence intermittency and the relationship between ensemble quantum yields and the blinking behavior in the QDs were found to be deeply related to the surface of QDs 9 – 12 . Most of the above studies found the dependence of QY on single particle fluorescence ON-OFF time fraction variations that occurred due to the surface state modification. Interestingly, on the contrary, a few interesting reports showed that QY in QDs is hardly dependent on ON-OFF fractions. In the seminal paper by Ebenstein et al., complete dark particles were proposed using correlative light and atomic force microscopy for the first time. They revealed the presence of several particles which do not turn ON or emit light at all at any interval of time. The bright-to-dark fraction ratio in such QD samples was found to be directly proportional to ensemble QY 21 . Yao et al. discovered the existence of such particles in the solution phase with the help of fluorescence correlation spectroscopy and confocal fluorescence coincidence analysis. They proposed that the QY was majorly dependent upon the complete dark fractions across different QDs samples, whereas the individual QD brightness was almost constant. They observed insignificant differences in ON-OFF time fractions for individual QDs, thus suggesting the importance of complete dark fractions for the ensemble QY 22 . In another work, Durisic et al. were shown to form a completely dark state upon decreasing the pH, which is mainly responsible for the decrease in fluorescent ON-fraction as well as bright fraction, ultimately reducing the fluorescent QY 23 . On the other hand, Pons et al . showed the presence of completely dark particles that were mainly responsible for the QY change in the quantum dots 24 . Here, we present completely unusual behavior of the excitation wavelength dependent QY in marcaptosuccinic acid (MSA) surface functionalized CdTe QDs. We observed a decrease in the QY with the increase of the excitation wavelength and the QY near the band edge is much smaller in comparison to shorter wavelength excitation. Surprisingly, we didn’t observe any change in the ON-OFF time histogram, or mean-photon counts under different excitation wavelengths, rather, we demonstrated that some of the particles are in completely dark states, and don’t show any emission at the experimental time scale. We proposed that the more the excitation energy in the shorter wavelength lesser are dark particles, thus increasing the QY. We also confirmed the presence of a larger number of dark particles in solution phase at longer wavelength using fluorescence correlation spectroscopy (FCS), which is closely a single molecule solution-based technique, provides very insightful information on the exact number of "only emitted fluorescent particles" in the confocal volume. We validated our observation upon internalization of the QDs in HeLa cell. We observed that the cellular images are brighter at shorter wavelengths than the longer wavelength under the same experimental condition. Results and Discussion Water soluble mercaptosuccinic acid (MSA) capped QDs were synthesized following slightly modified protocol of Tan et al. 25 . The detailed synthesis protocol can be found in the supporting information. The formation of CQDs is confirmed by several characterization techniques. The UV-visible spectrum shows band edge absorption at 587 nm with a steady increase in absorption intensity towards the blue region and is well matched with the reported absorption spectrum ( Fig. S1 a ). The fluorescence emission spectrum showed maxima at 604 nm ( Fig. S1 b ) and closely matched the reported result. The CQDs solution can be seen as red-color under white light illumination and orange-red color under UV light ( Fig. S1 b inset ). XRD spectrum ( Fig. S1 c ) showed CQDs peak at 24.08, 39.7 and 46.97 degrees (2θ) representing (111), (220), and (311), thus confirming the Zinc blended structure of CQDs (JCPDS 75-2086). TEM micrograph ( Fig. S2) shows a uniform size distribution of CQDs with a mean particle size of ~ 3.8 ± 0.5 nm. AFM image ( Fig. S1 d ) shows uniform height profiles of individual CQDs ~ 3.5 nm. Excitation wavelength dependent ensemble photophysical properties We performed fluorescence QY, fluorescence lifetime, and single-particle level fluorescence studies at various excitation wavelengths to understand the effect of excitation wavelength dependency of the QY in CQDs at the ensemble and single-particle level. Fluorescence lifetime studies were performed, keeping the emission maxima fixed at 604 nm. Pulsed diode LEDs of wavelength 389 nm, 454 nm, and 574 nm were used to excite the CQDs sample. Figure 2 a represents the fluorescence decay curve of the CQDs. Interestingly, no significant difference in lifetime was observed when the excitation wavelength changed from 389 nm to 454 nm, and finally to 574 nm. A similar trend in fluorescence lifetime was also observed in CdSe QDs 8 . The emission decay profiles were fitted with tri-exponential model. Figure 2 b and Table 1 show the fitted lifetime components along with their fractions. We measured the QY at 389, 454, 488, and 532 nm wavelengths. We have performed the absolute QY measurements at above mentioned wavelengths using integrating sphere-based method. The detail of the measurement can be found in the supporting information ( Fig. S3 and Table S1 ). Absolute QY methods is best suited here for eliminating any unwanted error that may arise due to change in the wavelength. To our surprise, the QY was found to be a maximum of ~ 81% when excited at 389 nm, ~ 50% at 454 nm, ~ 32% at 488 nm and ~ 22% at 532 nm (Fig. 2 c and Table 1 ). We also have performed relative QY measurements following the standard methods and procedures for performing relative QY measurements 26 , as described in detail in supporting information ( Fig. S4 ). Interestingly, the QY was found to be a very closely similar to the measured absolute QY with maximum of 78% when excited at 389 nm, 57% at 454 nm, 33% at 488 nm and 22% at 532 nm (Fig. 2 c and Table 1 ). The comparable data can be seen in the Table 1 . We acknowledge that a comprehensive understanding of the surface properties, particularly the ligand density that varies with particle size, may mislead such observation of wavelength dependent QY. However, it is essential to note that our investigation is confined to a homogeneous particle size distribution within the acceptable range of a standard deviation of ~ 14% of around 175 particles. The observed excitation independent emission spectral signature along with narrow full width half maximum (FWHM) confirms the CQDs homogeneity in the sample ( Fig. S5 ). For our curiosity, the QY at each wavelength was measured in triplicate, and we got similar results with a 5% standard deviation in the measurement every time. The trend in QY values is quite unusual, as per the reported literature for other QDs 7 , 15 – 17 , 19 , 27 , 28 . However, it closely matched with the QY 36% when excited at 479 nm reported by Grabolle et al. 20 . In addition, as observed in our study, the decrease in QY was also observed by them, i.e., the QY decreased from 36–31% when the excitation wavelength changed from 479 nm to 502 nm. We calculated the radiative and non-radiative rates for each of the excitation wavelengths. Figure 2 d shows that the radiative decay rate is decreasing, while the non-radiative rate increases with the increase in the excitation wavelength. The calculated radiative and non-radiative decay rates at the ensemble level of the CQDs at different excitation wavelengths seem to be quite supportive to the observed change in the QY. However, the opposite trend of the change in the QY as per the vast literature 7 , 8 , 15 – 17 , 19 , 28 , 29 led us to carry out a detailed analysis of the observed phenomena. Table 1 Experimentally measured data of QY via relative and absolute method, Average lifetime, lifetime components, amplitudes, and derived values of k r and k nr . Lifetime values at 488 nm and 532 nm (red coloured) are extrapolated values. Steps of extrapolation are available in supplementary information. Lifetime components are written in ns, amplitude values are written in percentage (%), and radiative and non-radiative rates are reported in ns - 1 . Excitation wavelength (nm) Q.Y (%) 𝜏 avg (ns) 𝜏 1 (A) 𝜏 2 (A) 𝜏 3 (A) K r K nr Abs Rel [×10 − 2 ] ns − 1 389 81 78 23.75 4.34 (2.55) 24.84 (83.37) 51.46 (14.08) 3.28 0.93 454 50 57 23.99 4.76 (2.56) 24.54 (80.84) 60.41 (16.60) 2.38 1.80 488 32 33 24.16 -- -- -- 1.38 2.80 532 22 22 24.36 -- -- -- 0.90 3.20 574 -- -- 24.56 6.32 (3.84) 25.94 (84.63) 57.35 (11.52) -- -- Excitation wavelength dependent Single particle analysis Hence, next, we performed single particle level fluorescence spectroscopy using a very diluted solution of CQDs that were spin-coated over a glass coverslip and excited at two different wavelengths (488 nm and 532 nm). To better compare all the necessary single molecular parameters, the power density for both lasers was kept at 0.016 kW/cm 2 , and all the measurements were carried out on the same glass coverslip spin-coated samples. Since QDs blink at a single-particle level, it was technically not possible for us to visualize the same area under both excitations at this stage of time. Hence, we captured approximately seven videos of random areas with 5000 frames (250 s, 20 Hz), 20.48 µm x 20.48 µm area under both the excitation sources. After the analysis of single particle data, interesting observations were made. Figure 3 a (i and ii) showed representative real-time single particle intensity-time traces upon excitation at 488 nm and 532 nm, respectively. A substantial blinking phenomenon was observed in both cases. On contrary to the ensemble observation of QY, no significant difference in total photon counts (Fig. 3 b i and ii ) were observed. CQDs excited at 488 nm laser emitted 7554 ± 495 photons, while emitted 6475 ± 600 photons when excited at 532 nm laser. This little difference cannot explain the decrease in the QY observed and may exist due to lesser absorbance cross section of 532 in comparison to that of 488 nm. Experimental single particle localization precision was also measured with 488 nm and 561 nm excitations (Fig. 3 d and e ), and no difference was observed in localization precision value. The data was also similar to one of our previously reported localization precision for a slightly different-sized CQDs 6 . This can be explained as the number of photons are comparable and the localisation precision is heavily dependent on number of photons. We then plotted the probability density distribution curves of ON and OFF times (combined) under 488 nm and 532 nm excitations (Fig. 3 c i and ii ). Approximately 400 bright CQDs in 488 nm and ~ 250 bright CQDs in 532 nm excitation were analysed with time traces, photon counting statistics, and probability density histograms for ON-OFF dwell times. ON times followed truncated power law behaviour under both excitations with similar m on , t c values. The OFF-dwell time histogram follows inverse power law behaviour under both excitations with similar m off values. Surprisingly, we observed very close ON and OFF time distribution for both laser excitations. This observation is quite astonishing compared to what was observed earlier in reported QDs. This is also quite contradictory to the ensemble results, where the radiative rate was found to decrease with an increase in excitation wavelength. It is to be pointed out here that the change in QY in CQDs in earlier reports proposed several hypotheses. For example, GSH-capped CQDs showed an increase in QY with the concomitant increase in fluorescence lifetime when the average particle size increased during the course of reactions 30 . Thioglycolic acid (TGA) capped CQDs showed an increased ON (bright) state, while the OFF state remained constant with decreased excitation intensity. TGA-capped CQDs showed its improved QY by amine functionalization due to the increase in ON time by improving surface passivation 14 . The surrounding water molecules were found both to increase or decrease the QY depending on the surface passivation and defects state creation 31 . On the other hand, an optimum size of CQDs was proposed to have maximum QY, below and above which the QY was found to decrease substantially in considering the amount of surface states and its density 32 . The above observation on size 30 , 32 , surface passivation 14 , excitation intensity-dependent and concentration-dependent QY 33 was attributed to the trap states and the change in the ON-OFF time (bright fraction) at the single-particle level. On the contrary, our study found that ON and OFF states do not significantly change with the change in excitation wavelength. Instead, we proposed the coexistence of two optically distinct populations of CQDs: one type of particles that are permanently dark (non-emitting) particles and the other one is the intermittently luminescent particles (emissive) that showed inherent ON-OFF blinking statistics. A similar behaviour was observed in mercaptopropionic acid (MPA) capped CQDs when studied by fluorescence correlation spectroscopy (FCS) 34 . The apparent concentration of QDs using FCS technique was found to be far less than the actual concentration measured by the ensemble spectroscopic method and hence the observed reduced QY was proposed due to the presence of available dark particles in the solution but not the ON-OFF state time change 22 . On the other hand, correlative light, and atomic force microscopy in CdSe/ZnS QDs showed the absence of emission properties in optical microscope for some of the particles that were observed in atomic force microscopy 21 . Single particle number analysis using intensity projection Although we are not able to measure the number of single permanent dark CQDs directly onto the glass coverslip with the current experimental fluorescence microscopy setup, we speculated that they must have shown the same number of bright particles in a known area if there is no difference in dark/bright fraction under different laser excitations. Hence, we carried out an alternative approach to measure the number of particles in maximum intensity projection from the observed single-particle fluorescence videos of 5000 frames. Figure 4 a and b shows the maximum intensity projection of one video recorded for both excitation lasers at 488 nm and 532 nm. Surprisingly, we observed a quite significantly different number of bright CQDs under these excitations. We took 7–8 videos ( Fig. S6 ) for this measurement. We plotted the number of bright particles observed in each video as a box chart in Fig. 4 c, showing lesser number of bright particles in all videos of 532 nm excitations in comparison of the 488 excitation videos. The average number of bright particles found in 488 nm excitation was ~ 43, and 532 nm excitation showed ~ 33 bright particles. A different number of bright particles on the same glass coverslip, keeping the same power density for 488 nm and 532 nm excitations, advocates the presence of a greater number of dark particles of CQDs at 532 nm excitation in comparison to 488 nm excitation. Fluorescence antibunching, and 2nd order correlation analysis To ensure that the CQDs are emitting as a single emissive species, fluorescence antibunching studies were performed. We have performed total correlation FCS and fluorescence antibunching (Fig. 6 a and b) with 405, 488 and 532 nm CW laser excitations for a very diluted solution (< 0.6 average CQDs in confocal volume). There exists no correlation at the ~ ns time scale in all of the total correlation FCS traces confirming the presence of single emitter in solution. Figure 6 b also confirms the CQDs to be single emitter as all G 2 (0) values are ≤ 0.27, less than 0.5. Also, the two-level distribution of ON-OFF intensity time traces in Fig. 3 a (i and ii) supports the presence of single CQDs under observation. Mean total photons emitted by single CQDs and probability distribution of ON and OFF times at both excitation wavelengths show that the single CQDs from the bright fraction of CQDs emit a similar number of photons under different excitations. Whereas the bulk QY data suggests higher excitations showing lower QY, like the bright fraction of CQDs observed under single particle microscopy. This led us to believe that the major reason for excitation dependency for CQDs is the dark fraction of CQDs. Considering this, we have formulated the relation between QY Bulk and bright fraction as follows: $${QY}^{Bulk}\propto Bright fraction\propto no of bright particles$$ Therefore, we can use this relation between QY and the number of bright particles. $$at 488 nm excitation, 46.5 bright CQDs\gg 33.28\text{% }QY$$ $$at 532 nm excitation,32.8 bright CQDs\gg \frac{33.28}{46.5}\times 32.8=23.5\text{%}$$ The QY at 532 nm calculated with the help of dark fraction matches with the bulk QY observed at 532 nm. This observation of obtaining QY at 532 nm excitation only with the help of the number of bright particles at 532 nm, 488 nm excitations and with QY at 488 nm excitations reaffirms our speculation of dark fraction responsible for QY decrement at ensemble/bulk level for CQDs. Figure 5 represents a detailed understanding of the fact observed in our case on excitation-dependent measurements in CQDs, where the decrease in the QY directly correlates with the number of permanently dark states at higher wavelengths. Discussion on dark fraction studies It is to be emphasized that the QY of QDs may vary due to three main reasons at the single particle level: (a) A decrease in the ON time fraction, which has been commonly observed 7 , 8 , 15 – 19 , 27 – 29 , 35 ; (b) an increase in the dark fraction either partial or complete dark particles 21 , 22 , 24 ; and (c) a combination of both 23 , 36 above. In most cases, the change in QY is due to factors a and c, which can lead to a change in the radiative rate and non-radiative rate of fluorescent bright fraction. In our present study, the change in QY is mainly due to factor b alone with the formation of completely dark particles. In this case, the QY-lifetime relation \(\left(QY={k}_{r}\times \tau \right)\) can give a false sense of decreased radiative and increased non-radiative rates. Here, the bright fraction’s fluorescence lifetime remained constant, and their behaviour was also found to be same under various excitations at single particle level. This implies that the radiative and non-radiative rates of the bright fraction are constant across all settings in our case. Therefore, the change in QY is not due to a change in the radiative rate or non-radiative rate of bright fraction but rather to a change in the dark fraction (permanently dark particles) only. Also, since the complete CQDs solution contains both fractions, the overall rates (ensemble averaged) will vary from the bright and dark fractions’ radiative and non-radiative rates. This also signifies that the radiative and non-radiative rates determined in Fig. 2 d are actually apparent rates or (ensemble averaged). To address these issues, we have introduced the concept of apparent rates ( \({K}_{r}^{App} and {K}_{nr}^{App}\) ), and shown their relationship with the intrinsic rates of bright fraction ( \({k}_{r}^{B} and {k}_{nr}^{B}\) ) in the supplementary information. The relationships between the real and apparent rates are derived as follows: $${k}_{r}^{App}=\left(1-D\right)\times {k}_{r}^{B}$$ and $${k}_{nr}^{App}=\frac{D}{\tau }+\left(1-D\right)\times {k}_{nr}^{B}$$ where, $$D is the fluorescent dark fraction, \tau is the fluorescence lifetime$$ $${k}_{r}^{App}, {k}_{r}^{B} are the radiative rate for ensemble solution and Q{D}^{{\prime }}s bright fraction respectively$$ $${k}_{nr}^{App}, {k}_{nr}^{B} are the non-radiative rate for ensemble solution and Q{D}^{{\prime }}s bright fraction respectively$$ The above equation suggests that both apparent rates (radiative and non-radiative) are directly dependent upon the dark (or bright) fraction of the CQDs solution only. Also, when there is no dark fraction, the intrinsic radiative rates and non-radiative rates of bright fraction represent the apparent rates. Excitation wavelength dependent FCS studies and H + ion addition study All of the single particle analysis performed above was for the immobilized CQDs over glass coverslip. To understand that the formation of permanently dark particle is also occurring in solution phase, we calculated the number of fluorophores diffusing through the confocal volume in FCS measurement. FCS is a solution-based technique, which provides valuable information at closely single molecule level by averaging out a fewer number of particles (molecules) diffusing through the confocal volume. It provides quantitative information in the triplet state contribution, its lifetime and the number of fluorescent particles diffusing out through the confocal volume. Experimental details are given in the supplementary information.A drop of highly diluted CQDs sample was excited with 561 nm laser and recorded the FCS spectra. We then repeated the procedure with 488 nm and 405 nm excitations, respectively (Fig. 6 c). All these lasers were ensured to have the same laser power (2.6 µW). Fitting the FCS data with the triplet model revealed that the number of emissive species inside the confocal volume (~ 10 − 15 litre) increased from ~ 3 at 561 nm excitation to ~ 9 at 405 nm excitation (Fig. 6 d). Also, it must be considered that the higher wavelength excitation makes a larger confocal volume, as the size of spot is directly proportional to the wavelength of incident light \(\left[diffraction limit=\frac{Wavelength of light}{2 \times Numerical Aperture}\right]\) . Therefore, if there is no dark fraction, then the number of particles must be higher in higher excitations as more particles can now diffuse through confocal volume due to its increased size. Hence, the observation clearly supports the existence of higher dark particles of CQDs at longer wavelength in the solution, as was observed in ensemble QY and at single particle level data. next, we elucidated the reason for excitation wavelength dependent dark fraction formation. It has been reported by several studies that the H + ion plays an important role in QDs photophysical properties and can force the QD into permanently dark states 23 , 36 . We carried out a pH dependent emission studies for the CQDs at three different pH. We incubated the CQDs solution into pH-3, pH 7.4 and pH 11 and measured the fluorescence intensity initially for 10 minutes with a 5 second time interval at the fluorescence maxima (λ max ) and then measured the intensity for a longer period of about 3 hours. It is observed that at lower pH (~ 3), the fluorescence intensity dropped down (Fig. 7 d ) . Interestingly, no such behavior was observed either at pH 7.4 or pH 11. To our surprize, the fluorescence lifetime did not change iat any of the pH solution. Here we speculate that the H + ions are adsorbed over the CQDs surface forcing it to go in permanently dark state, and since different energy excitation may influence the CQDs-H + complex differently, the amount of dark fraction will also be varied. It is to be noted that, this complex is formed in the ground state and hence it is not influencing the fluorescence lifetime. Higher energy excitations can break this complex easily forcing darker fraction particle to go in the bright fraction causing more QY in the lower wavelength excitations in comparison to the higher wavelength excitations. Wavelength dependent Bioimaging of the QDs We finally ratified our hypothesis in a real bioimaging platform. We measured the brightness of the image of human cancer cells upon being treated with the CQDs. The detail of the cell imaging protocol and the experimental condition is presented in the supporting information. We have shown a confocal microscopy image of CQDs stained HEK cells excited at 488 nm and 561 nm laser ( Fig. S7) . It is clearly visible that the image obtained under 488 nm excitation is brighter and more intense than the intensity upon 561 nm excitation under the same excitation laser power and all other similar experimental condition. This result suggested that the number of particles at shorter wavelengths are more emissive in nature than at longer wavelengths. QDs are widely used in various modern technological applications, such as the development of quantum computers as single photon sources. However, if a QD has a large number of dark fractions, then the single photon source efficiency will be reduced, as more bright fraction is needed. Conclusion The bulk fluorescence measurements for CQDs suggested QY to decrease with an increase in excitation wavelength. This trend is unusual than the trend observed for different reported QDs. Single-particle level fluorescence studies showed fewer fluorescent CQDs on 532 nm excitation compared to 488 nm excitation. In contrast, the total photon emitted, the probability distribution of ON and OFF times of CQDs, and the experimental localization precision value remain similar at both excitations. This finding suggests that the CQDs which are emissive in both excitations exhibit the same behaviour, but the number of bright or emissive CQDs are different for these excitations. Also, the FCS analysis revealed fewer bright particles at longer wavelength in comparison to shorter wavelength in solution. This anomaly signifies that there is a fraction of CQDs which has turned completely dark at longer wavelength, causing the unusual excitation dependent QY decrement. These dark fraction formation in this report is a ground state phenomenon and may exist due to the presence of H + ions in the water. The different energy excitations alter the CQDs-H + complex differently and cause less particle to convert from permanently dark to bright at higher wavelength excitations. The dependence of QY on the excitation wavelength, due to the presence of a permanent dark fraction, may affect the selection of QDs for specific applications, as this can compromise the device functionality. Therefore, proper characterization of the fluorescence dark fraction is necessary, and the influence of other factors on the QY of different QDs must be thoroughly investigated. Declarations Conflict of Interest The authors declare no conflict of interest. Author contributions KK conceptualized and designed all the experiments. JM, and RKB performed all the bulk-level measurements with the help of KK. KK performed single particle measurements with the help of RKB and JM. KK performed FCS experiment. KK analysed the data with the help of JM, RKB, and SS. SS helped in several experiments. FA provided HEK cells for the imaging study. CKN guided the complete project. KK wrote the manuscript with the help of CKN. Acknowledgment CKN is thankful to Science and Engineering Research Board (SERB) core research grant (CRG) India for the project number CRG/2020/000268. CKN is thankful to the facilities of the AMRC center of IIT Mandi, India. KK and CKN acknowledge the Sophisticated Analytical and Technical Help Institutes (SATHI), IIT Delhi and Indian Science Technology and Engineering facilities Map (I-STEM) for absolute quantum yield, total correlation FCS and fluorescence antibunching measurements. KK, and SS, thank the Ministry of Education (MoE), India, for research scholarship. FA acknowledges the SERB for scholarship. KK also acknowledge fruitful discussions with Mr. Abdul, Ms. Richa, Dr. Aditya, Dr. Chethana, and Dr. Monika. 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Nano Lett 15(6):3953–3958 Mahler B, Spinicelli P, Buil S, Quelin X, Hermier J-P, Dubertret B (2008) Towards Non-Blinking Colloidal Quantum Dots. Nat Mater 7(8):659–664 Efros AL, Nesbitt DJ (2016) Origin and Control of Blinking in Quantum Dots. Nat Nanotechnol 11(8):661–671 Cichos F, Vonborczyskowski C, Orrit M (2007) Power-Law Intermittency of Single Emitters. Curr Opin Colloid Interface Sci 12(6):272–284 Mandal A, Tamai N (2011) Suppressed Blinking Behavior of Thioglycolic Acid Capped CdTe Quantum Dot by Amine Functionalization. Appl Phys Lett 99(26):263111 Roy D, Das A, De CK, Mandal S, Bangal PR, Mandal PK (2019) Why Does the Photoluminescence Efficiency Depend on Excitation Energy in Case of a Quantum Dot? A Case Study of CdSe-Based Core/Alloy Shell/Shell Quantum Dots Employing Ultrafast Pump–Probe Spectroscopy and Single Particle Spectroscopy. J Phys Chem C 123(11):6922–6933 Roy D, Ghosh S, De CK, Mukherjee S, Mandal S, Mandal PK (2022) Excitation-Energy-Dependent Photoluminescence Quantum Yield Is Inherent to Optically Robust Core/Alloy-Shell Quantum Dots in a Vast Energy Landscape. J Phys Chem Lett 13(10):2404–2417 Zhang Z, Zhang S, Gushchina I, Guo T, Brennan MC, Pavlovetc IM, Grusenmeyer TA, Kuno M (2021) Excitation Energy Dependence of Semiconductor Nanocrystal Emission Quantum Yields. J Phys Chem Lett 12(16):4024–4031 Ghosh S, Mandal S, Mukherjee S, De CK, Samanta T, Mandal M, Roy D, Mandal PK (2021) Near-Unity Photoluminescence Quantum Yield and Highly Suppressed Blinking in a Toxic-Metal-Free Quantum Dot. J Phys Chem Lett 12(5):1426–1431 De CK, Mandal S, Roy D, Ghosh S, Konar A, Mandal PK (2019) Ultrafast Dynamics and Ultrasensitive Single-Particle Intermittency in Small-Sized Toxic Metal Free InP-Based Core/Alloy-Shell/Shell Quantum Dots: Excitation Wavelength Dependency Toward Variation of PLQY. J Phys Chem C 123(46):28502–28510 Grabolle M, Spieles M, Lesnyak V, Gaponik N, Eychmüller A, Resch-Genger U (2009) Determination of the Fluorescence Quantum Yield of Quantum Dots: Suitable Procedures and Achievable Uncertainties. Anal Chem 81(15):6285–6294 Ebenstein Y, Mokari T, Banin U (2002) Fluorescence Quantum Yield of CdSe/ZnS Nanocrystals Investigated by Correlated Atomic-Force and Single-Particle Fluorescence Microscopy. Appl Phys Lett 80(21):4033–4035 Yao J, Larson DR, Vishwasrao HD, Zipfel WR, Webb WW (2005) Blinking and Nonradiant Dark Fraction of Water-Soluble Quantum Dots in Aqueous Solution. Proc. Natl. Acad. Sci. 102 (40), 14284–14289 Durisic N, Wiseman PW, Grütter P, Heyes CD (2009) A Common Mechanism Underlies the Dark Fraction Formation and Fluorescence Blinking of Quantum Dots. ACS Nano 3(5):1167–1175 Pons T, Medintz IL, Farrell D, Wang X, Grimes AF, English DS, Berti L, Mattoussi H (2011) Single-Molecule Colocalization Studies Shed Light on the Idea of Fully Emitting versus Dark Single Quantum Dots. Small 7(14):2101–2108 Tan J, Liang Y, Wang J, Chen J, Sun B, Shao L (2015) Facile Synthesis of CdTe-Based Quantum Dots Promoted by Mercaptosuccinic Acid and Hydrazine. New J Chem 39(6):4488–4493 Würth C, Grabolle M, Pauli J, Spieles M, Resch-Genger U (2013) Relative and Absolute Determination of Fluorescence Quantum Yields of Transparent Samples. Nat Protoc 8(8):1535–1550 Tonti D, van Mourik F, Chergui M (2004) On the Excitation Wavelength Dependence of the Luminescence Yield of Colloidal CdSe Quantum Dots. Nano Lett 4(12):2483–2487 Knappenberger KL, Wong DB, Romanyuk YE, Leone SR (2007) Excitation Wavelength Dependence of Fluorescence Intermittency in CdSe/ZnS Core/Shell Quantum Dots. Nano Lett 7(12):3869–3874 Martynenko IV, Baimuratov AS, Osipova VA, Kuznetsova VA, Purcell-Milton F, Rukhlenko ID, Fedorov AV, Gun’ko YK, Resch-Genger U, Baranov AV (2018) Excitation Energy Dependence of the Photoluminescence Quantum Yield of Core/Shell CdSe/CdS Quantum Dots and Correlation with Circular Dichroism. Chem Mater 30(2):465–471 Sousa JCL, Vivas MG, Ferrari JL, Mendonca CR, Schiavon MA (2014) Determination of Particle Size Distribution of Water-Soluble CdTe Quantum Dots by Optical Spectroscopy. RSC Adv 4(68):36024–36030 Cordero SR, Carson PJ, Estabrook RA, Strouse GF, Buratto SK (2000) Photo-Activated Luminescence of CdSe Quantum Dot Monolayers. J Phys Chem B 104(51):12137–12142 Martínez Maestro L, Jacinto C, Rocha U; Carmen Iglesias-de la Cruz, Sanz-Rodriguez M, Juarranz F, García Solé A, Jaque J (2012) D. Optimum Quantum Dot Size for Highly Efficient Fluorescence Bioimaging. J. Appl. Phys. 111 (2), 023513 Murphy GP, Zhang X, Bradley AL (2016) Temperature-Dependent Luminescent Decay Properties of CdTe Quantum Dot Monolayers: Impact of Concentration on Carrier Trapping. J Phys Chem C 120(46):26490–26497 Dong C, Liu H, Ren J (2014) Assessing the Blinking State of Fluorescent Quantum Dots in Free Solution by Combining Fluorescence Correlation Spectroscopy with Ensemble Spectroscopic Methods. Langmuir 30(43):12969–12976 Goushi K, Yamada T, Otomo A (2009) Excitation Intensity Dependence of Power-Law Blinking Statistics in Nanocrystal Quantum Dots. J Phys Chem C 113(47):20161–20168 Durisic N, Godin AG, Walters D, Grütter P, Wiseman PW, Heyes CD (2011) Probing the Dark Fraction of Core–Shell Quantum Dots by Ensemble and Single Particle PH-Dependent Spectroscopy. ACS Nano 5(11):9062–9073 Additional Declarations There is NO Competing Interest. Supplementary Files ESI29022024.pdf GA.png Graphical Abstract Excitation dependent unusual quantum yield being explained with the help of a greater number of permanently dark CdTe Quantum dots at higher wavelength excitations in comparison to the lower wavelength excitations. Cite Share Download PDF Status: Published Journal Publication published 18 Dec, 2024 Read the published version in Nanoscale → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3999532","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":275644872,"identity":"c1790411-5aad-4871-8988-743e244fcb11","order_by":0,"name":"Chayan Nandi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYDACCTBpw8MGptkg3ANEaEnjYWMjUcthoGqwFiLcxT+7+ZnEzx3nZfjkuxM/F5RZMPC3H2A8XIDPkjvHzCR7z9wGOox3s/SMcxIMEmcSGA7PwKPFQCLBTIK3DaxlgzRvG9CdN4Du5MGrJf2b5N+2c2BbfoO0yBPWkmMGNPwASMs2sC0GhLRI3MgptpZtSwZqyd1mzXNOgsfwTGIDXi38M9I33nzbZmcv33x2822esjo5ueOHD3/GpwUIWCSQeUDFjA34NTAwMH8gpGIUjIJRMApGOAAAERxBCzsIuyMAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-4584-0738","institution":"Indian Institute of Technology Mandi","correspondingAuthor":true,"prefix":"","firstName":"Chayan","middleName":"","lastName":"Nandi","suffix":""},{"id":275644873,"identity":"94e78cd9-ec7e-454e-ae59-87dea46bcd18","order_by":1,"name":"Kush Kaushik","email":"","orcid":"","institution":"Indian Institute of Technology Mandi","correspondingAuthor":false,"prefix":"","firstName":"Kush","middleName":"","lastName":"Kaushik","suffix":""},{"id":275644874,"identity":"bd43b39e-264d-4248-a0cc-12f3fb156268","order_by":2,"name":"Jiban Mondal","email":"","orcid":"","institution":"Indian Institute of Technology Mandi","correspondingAuthor":false,"prefix":"","firstName":"Jiban","middleName":"","lastName":"Mondal","suffix":""},{"id":275644875,"identity":"d4f543bf-6832-440d-b7d9-bd1f8e4563a5","order_by":3,"name":"Ritesh Bag","email":"","orcid":"","institution":"Indian Institute of Technology Mandi","correspondingAuthor":false,"prefix":"","firstName":"Ritesh","middleName":"","lastName":"Bag","suffix":""},{"id":275644876,"identity":"b30a840a-27b7-4810-b603-f3ce834acbcb","order_by":4,"name":"Shagun Sharma","email":"","orcid":"","institution":"Indian Institute of Technology Mandi","correspondingAuthor":false,"prefix":"","firstName":"Shagun","middleName":"","lastName":"Sharma","suffix":""},{"id":275644877,"identity":"02a607aa-9e02-4ad4-a682-9b3d2459f904","order_by":5,"name":"Farhan Anjum","email":"","orcid":"","institution":"Indian Institute of Technology Mandi","correspondingAuthor":false,"prefix":"","firstName":"Farhan","middleName":"","lastName":"Anjum","suffix":""}],"badges":[],"createdAt":"2024-02-29 11:45:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3999532/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3999532/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1039/D4NR04344H","type":"published","date":"2024-12-19T00:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":52302712,"identity":"9c7e1953-7d85-4dda-95fd-263de3b14879","added_by":"auto","created_at":"2024-03-08 18:47:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":124208,"visible":true,"origin":"","legend":"\u003cp\u003eGeneral trend shown by other reported studies suggests the increased number of trap states at higher energy levels are responsible for the decrement in quantum yield at lower wavelength excitations.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3999532/v1/4cebee868b6c09ea534fbef6.png"},{"id":52302713,"identity":"e1a7092c-4098-4225-a367-dcf72917adc6","added_by":"auto","created_at":"2024-03-08 18:47:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":251564,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFluorescence lifetime and quantum yield for CQDs:\u003c/strong\u003e (a) shows the fluorescence lifetime spectrums data along with three exponential fits for CQDs excited with 389, 454, and 574 nm excitations. There exists no visible difference in all of these spectrums. (b) Shows the relation between fitted parameters, i.e., lifetime values and amplitude values, and excitation wavelengths. All curves are independent of the excitation wavelengths. (c) Fluorescence lifetime and quantum yield values are shown with respect to excitation wavelengths. Quantum yield is found to decrease with an increase in excitation wavelength, and lifetime showed no significant variation. Lifetime values at 488 nm and 532 nm excitations are extrapolated values. (d) Shows the variation of radiative and non-radiative rate with respect to excitation wavelength. Radiative rate, k\u003csub\u003er\u003c/sub\u003e is found to decrease with an increase in excitation wavelength and non-radiative rate, k\u003csub\u003enr\u003c/sub\u003e is found to decrease with increasing excitation wavelength.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3999532/v1/1468c8f18e5edb0e1d58c53a.png"},{"id":52302715,"identity":"cde22a4d-52a7-42c9-9bcd-5ec6fd437daa","added_by":"auto","created_at":"2024-03-08 18:47:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":303339,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSingle particle level fluorescence spectroscopy analysis at 488 nm and 532 nm excitations: \u003c/strong\u003e(a) (i \u0026amp; ii) are the representative single particle fluorescence intensity-time traces at both excitations. similar type of blinking behaviour can be seen in both. (b) (i \u0026amp; ii) shows the photon counting histogram with the total photons emitted by all CQDs under both excitations. No significant difference was observed. (c) (i \u0026amp; ii) shows the probability density distribution histogram of ON dwell time and OFF dwell time for all the CQDs excited with both lasers. ON times were found to be following truncated power law type behaviour with similar m\u003csub\u003eon\u003c/sub\u003e, t\u003csub\u003ec\u003c/sub\u003e values. OFF dwell time histogram is found to be following inverse power law type behaviour under both excitations with similar m\u003csub\u003eoff\u003c/sub\u003e values. Approximately 400 bright CQDs in 488 nm excitation and ~250 bright CQDs in 532 nm excitation were analysed with time traces, photon counting statistics, and probability density histograms. (d, e) shows the experimental localisation precision determination at both excitations. The scatter plot (d, e) (iii) is obtained by the localisation of ~8 QDs keeping the centre of mass of localisations at origin. (d, e) (i \u0026amp; iv) shows distributions of these localisations along x and y-direction. (iv) shows distributions of these localisations in y direction. (d, e) (ii) shows representative individual localisations obtained from ThunderSTORM used for localisation precision study. Fit line in (i \u0026amp; iv) shows Gaussian distribution with standard deviation values ~50 nm. Scale bar in single localisations (ii) is 100 nm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3999532/v1/61d23c9f0fb59d84df4f0bba.png"},{"id":52302723,"identity":"9e017883-eeea-4369-ae63-8a0755a9bf8c","added_by":"auto","created_at":"2024-03-08 18:47:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":603813,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNumber-based analysis of single particle videos recorded under both 488 nm and 532 nm excitations\u003c/strong\u003e (a) Representable maximum intensity projection images obtained by 488 nm, and 532 nm excitations showing a higher number of bright particles (~50) under 488 nm excitation in comparison to the number of bright particles (~33) under 532 nm excitation. (c) showing the difference in the number of bright particles via a box chart. Lower wavelength excitation laser shows a greater number of bright particles than the higher wavelength excited CQDs. Image area in (a) is 20.48 µm x 20.48 µm. The power density for both excitations was kept same at 0.0107 kW/cm\u003csup\u003e2\u003c/sup\u003e. The number analysis was done for the same glass coverslip to avoid any anomalies from laser intensity variation and concentration variation. The scale bar is 5 µm. All analysed video’s max intensity image can be seen in \u003cstrong\u003eFig. S6\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3999532/v1/b684607877f969f83cfba0ea.png"},{"id":52302717,"identity":"cddb7313-1da8-4acf-aa8e-9b6a8500e65a","added_by":"auto","created_at":"2024-03-08 18:47:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":213000,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic explaining the unusual behaviour of quantum yield in CdTe quantum dots due to the presence of permanent dark particles with different populations at different energy levels.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3999532/v1/3468c1499e09c9834cce74ff.png"},{"id":52303128,"identity":"7143c5bf-971d-4943-8cb0-720c0a289127","added_by":"auto","created_at":"2024-03-08 18:55:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":313150,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFCS and fluorescence antibunching study of CQDs:\u003c/strong\u003e (a) Total correlation FCS at ns timescale show no correlation and (b) fluorescence antibunching exhibits G\u003csup\u003e2\u003c/sup\u003e(0) ~0.27 which is less than 0.5 confirming single emitter emission from CQDs. (c) shows the FCS data (scatter points) and fitted line (solid line) for same CQD solution excited with 400, 488, and 561 nm lasers. (d) Number of bright particles obtained after fitting the FCS data gives decreasing number of bright particles in confocal volume on higher wavelength excitations. Laser power was fixed at 2.6 µW for all lasers.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3999532/v1/7b3b08491a5de87bae02ec81.png"},{"id":52302719,"identity":"275f73a0-7031-46ff-99b0-776a3bf17635","added_by":"auto","created_at":"2024-03-08 18:47:40","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":133819,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of H+ ion over the fluorescence intensity and lifetime of CQDs: \u003c/strong\u003eFluorescence kinetics mode measurement of 10 µL of as synthesised CdTe QDs dispersed in\u003cstrong\u003e \u003c/strong\u003e(a) pH-3 solution (water and HCl) (b) pH-7.4 solution and (c) pH 11 solution. (a-c) Intensity maxima monitored for 10 mins with 5 s time interval. (d) Intensity maxima and fluorescence lifetime measured over ~3 hours for pH-3. Excitation wavelength: 390 nm.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3999532/v1/9cb74faeedb451408b70b67a.png"},{"id":72024758,"identity":"987c2f81-97d1-44ae-a1a4-156c90ac23d5","added_by":"auto","created_at":"2024-12-20 18:15:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2468146,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3999532/v1/7133226e-06e9-4626-9c23-31fa47f0b54e.pdf"},{"id":52304353,"identity":"c4e3fcb8-bfd9-48fe-9fa0-eabf94926566","added_by":"auto","created_at":"2024-03-08 19:03:40","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1255287,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"ESI29022024.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3999532/v1/27ae24b7a2cda98e80d9a455.pdf"},{"id":52302714,"identity":"e6031bd9-efd8-4903-a856-8476d511a941","added_by":"auto","created_at":"2024-03-08 18:47:40","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":235337,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExcitation dependent unusual quantum yield being explained with the help of a greater number of permanently dark CdTe Quantum dots at higher wavelength excitations in comparison to the lower wavelength excitations.\u003c/p\u003e","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-3999532/v1/a3af4e33199d89bd99760135.png"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Unusual Excitation Wavelength Dependency of Quantum Yield in Water Soluble CdTe Quantum Dots","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSemiconductor QDs are highly fluorescent nanostructures with unique optoelectronic properties. Due to their high QY\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, high photostability\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, broad absorption spectrum\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e with very narrow and tunable emission spectrum\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, QDs have been employed in numerous applications such as light emitting diodes (LEDs), photonics, Solar cells, LASER, sensing and bioimaging agents\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Unfortunately, QY of the QDs is severely hampered by the inherent fluorescence intermittency (called blinking), which is the phenomenon of random switching between ON (bright) and OFF (dark) states of the emitter upon continuous excitation. Efforts have been made to understand the mechanism of such intermittency both at the ensemble and single particle level\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9 CR10 CR11\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The unusual feature in this blinking phenomenon is the power law statistics of the ON and OFF time distributions\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Due to this fact, the time-averaged intensity of a single emitter is not reproducible in different experiments and implies a complex dynamic of the involved process and generally hampers the QY in almost all the QDs.\u003c/p\u003e \u003cp\u003eA substantial effort has been made to manipulate the QY of QDs by modifying the surrounding environment, surface functionalization, change in pH, ligand length, electron-donating and withdrawing agents\u003csup\u003e\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Recent reports suggest that excitation wavelength plays a very significant role in affecting the QY\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan additionalcitationids=\"CR16 CR17 CR18\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. A general observation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e is that with the increase in excitation energy (shorter wavelength) the absorption of light by the QDs increases substantially, however, the emission intensity decreases monotonically in the reverse order from blue to the band edge emission. This is attributed to (a) exciting particles into continuum states, distinct from confined states\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e and (b) the charge carriers trapping in a dark state or a very stable trapped state that decreases the radiative recombination and thus decreases the QY of the material. Hoy et al. suggested that the QY in CdSe and CdSe/ZnS QDs of different sizes exhibited a proportional relationship with excitation energy\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Roy et al. performed excitation wavelength dependent single particle study on InP and CdSe\u0026rsquo;s core-shell and core alloy shell quantum dots. They observed that at longer wavelength, PLQY decreases with increment in ON truncation time and decrement in OFF truncation times. It was also shown that the core-alloy shell exhibited more ON fraction than the core-shell QD structures\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Often, these changes in QY, ON fraction, ON-OFF truncation times are attributed to high density of trap states at higher energy levels. De et al. performed the excitation-dependent ultra-fast dynamics in small-sized InP-based core/alloy-shell/shell QDs\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. They showed that the excitons took a longer time to recombine for lower wavelength excitations in comparison to higher wavelength excitation and proposed that the high degree of trapping yielded lower QY values at low wavelength excitations. Surprisingly, the excitation wavelength-dependent emission was conflicted in describing the probable measurement error or artifacts due to the presence of QDs aggregates as well as excess surface ligands present in the solution that leads to scattering losses in QY measurement at shorter excitation wavelength\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The mechanism of photoluminescence intermittency and the relationship between ensemble quantum yields and the blinking behavior in the QDs were found to be deeply related to the surface of QDs\u003csup\u003e\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Most of the above studies found the dependence of QY on single particle fluorescence ON-OFF time fraction variations that occurred due to the surface state modification.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInterestingly, on the contrary, a few interesting reports showed that QY in QDs is hardly dependent on ON-OFF fractions. In the seminal paper by Ebenstein et al., complete dark particles were proposed using correlative light and atomic force microscopy for the first time. They revealed the presence of several particles which do not turn ON or emit light at all at any interval of time. The bright-to-dark fraction ratio in such QD samples was found to be directly proportional to ensemble QY\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Yao et al. discovered the existence of such particles in the solution phase with the help of fluorescence correlation spectroscopy and confocal fluorescence coincidence analysis. They proposed that the QY was majorly dependent upon the complete dark fractions across different QDs samples, whereas the individual QD brightness was almost constant. They observed insignificant differences in ON-OFF time fractions for individual QDs, thus suggesting the importance of complete dark fractions for the ensemble QY\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In another work, Durisic et al. were shown to form a completely dark state upon decreasing the pH, which is mainly responsible for the decrease in fluorescent ON-fraction as well as bright fraction, ultimately reducing the fluorescent QY\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. On the other hand, Pons \u003cem\u003eet al\u003c/em\u003e. showed the presence of completely dark particles that were mainly responsible for the QY change in the quantum dots\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere, we present completely unusual behavior of the excitation wavelength dependent QY in marcaptosuccinic acid (MSA) surface functionalized CdTe QDs. We observed a decrease in the QY with the increase of the excitation wavelength and the QY near the band edge is much smaller in comparison to shorter wavelength excitation. Surprisingly, we didn\u0026rsquo;t observe any change in the ON-OFF time histogram, or mean-photon counts under different excitation wavelengths, rather, we demonstrated that some of the particles are in completely dark states, and don\u0026rsquo;t show any emission at the experimental time scale. We proposed that the more the excitation energy in the shorter wavelength lesser are dark particles, thus increasing the QY. We also confirmed the presence of a larger number of dark particles in solution phase at longer wavelength using fluorescence correlation spectroscopy (FCS), which is closely a single molecule solution-based technique, provides very insightful information on the exact number of \"only emitted fluorescent particles\" in the confocal volume. We validated our observation upon internalization of the QDs in HeLa cell. We observed that the cellular images are brighter at shorter wavelengths than the longer wavelength under the same experimental condition.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eWater soluble mercaptosuccinic acid (MSA) capped QDs were synthesized following slightly modified protocol of Tan et al.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The detailed synthesis protocol can be found in the supporting information. The formation of CQDs is confirmed by several characterization techniques. The UV-visible spectrum shows band edge absorption at 587 nm with a steady increase in absorption intensity towards the blue region and is well matched with the reported absorption spectrum (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea\u003c/b\u003e). The fluorescence emission spectrum showed maxima at 604 nm (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb\u003c/b\u003e) and closely matched the reported result. The CQDs solution can be seen as red-color under white light illumination and orange-red color under UV light (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb inset\u003c/b\u003e). XRD spectrum (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ec\u003c/b\u003e) showed CQDs peak at 24.08, 39.7 and 46.97 degrees (2θ) representing (111), (220), and (311), thus confirming the Zinc blended structure of CQDs (JCPDS 75-2086). TEM micrograph (\u003cb\u003eFig. S2)\u003c/b\u003e shows a uniform size distribution of CQDs with a mean particle size of ~\u0026thinsp;3.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 nm. AFM image (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ed\u003c/b\u003e) shows uniform height profiles of individual CQDs\u0026thinsp;~\u0026thinsp;3.5 nm.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExcitation wavelength dependent ensemble photophysical properties\u003c/h2\u003e \u003cp\u003eWe performed fluorescence QY, fluorescence lifetime, and single-particle level fluorescence studies at various excitation wavelengths to understand the effect of excitation wavelength dependency of the QY in CQDs at the ensemble and single-particle level. Fluorescence lifetime studies were performed, keeping the emission maxima fixed at 604 nm. Pulsed diode LEDs of wavelength 389 nm, 454 nm, and 574 nm were used to excite the CQDs sample. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea represents the fluorescence decay curve of the CQDs. Interestingly, no significant difference in lifetime was observed when the excitation wavelength changed from 389 nm to 454 nm, and finally to 574 nm. A similar trend in fluorescence lifetime was also observed in CdSe QDs\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The emission decay profiles were fitted with tri-exponential model. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb \u003cb\u003eand\u003c/b\u003e Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e show the fitted lifetime components along with their fractions. We measured the QY at 389, 454, 488, and 532 nm wavelengths. We have performed the absolute QY measurements at above mentioned wavelengths using integrating sphere-based method. The detail of the measurement can be found in the supporting information (\u003cb\u003eFig. S3 and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Absolute QY methods is best suited here for eliminating any unwanted error that may arise due to change in the wavelength. To our surprise, the QY was found to be a maximum of ~\u0026thinsp;81% when excited at 389 nm, ~\u0026thinsp;50% at 454 nm, ~\u0026thinsp;32% at 488 nm and ~\u0026thinsp;22% at 532 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec \u003cb\u003eand\u003c/b\u003e Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We also have performed relative QY measurements following the standard methods and procedures for performing relative QY measurements\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, as described in detail in supporting information (\u003cb\u003eFig. S4\u003c/b\u003e). Interestingly, the QY was found to be a very closely similar to the measured absolute QY with maximum of 78% when excited at 389 nm, 57% at 454 nm, 33% at 488 nm and 22% at 532 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec \u003cb\u003eand\u003c/b\u003e Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The comparable data can be seen in the Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. We acknowledge that a comprehensive understanding of the surface properties, particularly the ligand density that varies with particle size, may mislead such observation of wavelength dependent QY. However, it is essential to note that our investigation is confined to a homogeneous particle size distribution within the acceptable range of a standard deviation of ~\u0026thinsp;14% of around 175 particles. The observed excitation independent emission spectral signature along with narrow full width half maximum (FWHM) confirms the CQDs homogeneity in the sample (\u003cb\u003eFig. S5\u003c/b\u003e). For our curiosity, the QY at each wavelength was measured in triplicate, and we got similar results with a 5% standard deviation in the measurement every time. The trend in QY values is quite unusual, as per the reported literature for other QDs\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. However, it closely matched with the QY 36% when excited at 479 nm reported by Grabolle et al.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In addition, as observed in our study, the decrease in QY was also observed by them, i.e., the QY decreased from 36\u0026ndash;31% when the excitation wavelength changed from 479 nm to 502 nm. We calculated the radiative and non-radiative rates for each of the excitation wavelengths. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed shows that the radiative decay rate is decreasing, while the non-radiative rate increases with the increase in the excitation wavelength. The calculated radiative and non-radiative decay rates at the ensemble level of the CQDs at different excitation wavelengths seem to be quite supportive to the observed change in the QY. However, the opposite trend of the change in the QY as per the vast literature\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e led us to carry out a detailed analysis of the observed phenomena.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eExperimentally measured data of QY via relative and absolute method, Average lifetime, lifetime components, amplitudes, and derived values of k\u003csub\u003er\u003c/sub\u003e and k\u003csub\u003enr\u003c/sub\u003e. Lifetime values at 488 nm and 532 nm (red coloured) are extrapolated values. Steps of extrapolation are available in supplementary information. Lifetime components are written in ns, amplitude values are written in percentage (%), and radiative and non-radiative rates are reported in ns\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eExcitation wavelength (nm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eQ.Y (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u0026#120591;\u003csub\u003eavg\u003c/sub\u003e (ns)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u0026#120591;\u003csub\u003e1\u003c/sub\u003e (A)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u0026#120591;\u003csub\u003e2\u003c/sub\u003e (A)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u0026#120591;\u003csub\u003e3\u003c/sub\u003e (A)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eK\u003csub\u003er\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eK\u003csub\u003enr\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eAbs\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eRel\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e\u003cb\u003e[\u0026times;10\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;2\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e] ns\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e389\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e23.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.34 (2.55)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24.84 (83.37)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e51.46 (14.08)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e454\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e23.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.76 (2.56)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24.54 (80.84)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e60.41 (16.60)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e488\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e24.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e532\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e24.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e574\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e24.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.32 (3.84)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e25.94 (84.63)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e57.35 (11.52)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eExcitation wavelength dependent Single particle analysis\u003c/h2\u003e \u003cp\u003eHence, next, we performed single particle level fluorescence spectroscopy using a very diluted solution of CQDs that were spin-coated over a glass coverslip and excited at two different wavelengths (488 nm and 532 nm). To better compare all the necessary single molecular parameters, the power density for both lasers was kept at 0.016 kW/cm\u003csup\u003e2\u003c/sup\u003e, and all the measurements were carried out on the same glass coverslip spin-coated samples. Since QDs blink at a single-particle level, it was technically not possible for us to visualize the same area under both excitations at this stage of time. Hence, we captured approximately seven videos of random areas with 5000 frames (250 s, 20 Hz), 20.48 \u0026micro;m x 20.48 \u0026micro;m area under both the excitation sources. After the analysis of single particle data, interesting observations were made. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea \u003cb\u003e(i and ii)\u003c/b\u003e showed representative real-time single particle intensity-time traces upon excitation at 488 nm and 532 nm, respectively. A substantial blinking phenomenon was observed in both cases. On contrary to the ensemble observation of QY, no significant difference in total photon counts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb \u003cb\u003ei and ii\u003c/b\u003e) were observed. CQDs excited at 488 nm laser emitted 7554\u0026thinsp;\u0026plusmn;\u0026thinsp;495 photons, while emitted 6475\u0026thinsp;\u0026plusmn;\u0026thinsp;600 photons when excited at 532 nm laser. This little difference cannot explain the decrease in the QY observed and may exist due to lesser absorbance cross section of 532 in comparison to that of 488 nm. Experimental single particle localization precision was also measured with 488 nm and 561 nm excitations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed \u003cb\u003eand e\u003c/b\u003e), and no difference was observed in localization precision value. The data was also similar to one of our previously reported localization precision for a slightly different-sized CQDs\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. This can be explained as the number of photons are comparable and the localisation precision is heavily dependent on number of photons. We then plotted the probability density distribution curves of ON and OFF times (combined) under 488 nm and 532 nm excitations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec \u003cb\u003ei and ii\u003c/b\u003e). Approximately 400 bright CQDs in 488 nm and ~\u0026thinsp;250 bright CQDs in 532 nm excitation were analysed with time traces, photon counting statistics, and probability density histograms for ON-OFF dwell times. ON times followed truncated power law behaviour under both excitations with similar m\u003csub\u003eon\u003c/sub\u003e, t\u003csub\u003ec\u003c/sub\u003e values. The OFF-dwell time histogram follows inverse power law behaviour under both excitations with similar m\u003csub\u003eoff\u003c/sub\u003e values. Surprisingly, we observed very close ON and OFF time distribution for both laser excitations. This observation is quite astonishing compared to what was observed earlier in reported QDs. This is also quite contradictory to the ensemble results, where the radiative rate was found to decrease with an increase in excitation wavelength.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIt is to be pointed out here that the change in QY in CQDs in earlier reports proposed several hypotheses. For example, GSH-capped CQDs showed an increase in QY with the concomitant increase in fluorescence lifetime when the average particle size increased during the course of reactions\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Thioglycolic acid (TGA) capped CQDs showed an increased ON (bright) state, while the OFF state remained constant with decreased excitation intensity. TGA-capped CQDs showed its improved QY by amine functionalization due to the increase in ON time by improving surface passivation\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The surrounding water molecules were found both to increase or decrease the QY depending on the surface passivation and defects state creation \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. On the other hand, an optimum size of CQDs was proposed to have maximum QY, below and above which the QY was found to decrease substantially in considering the amount of surface states and its density\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The above observation on size\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, surface passivation\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, excitation intensity-dependent and concentration-dependent QY\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e was attributed to the trap states and the change in the ON-OFF time (bright fraction) at the single-particle level. On the contrary, our study found that ON and OFF states do not significantly change with the change in excitation wavelength. Instead, we proposed the coexistence of two optically distinct populations of CQDs: one type of particles that are permanently dark (non-emitting) particles and the other one is the intermittently luminescent particles (emissive) that showed inherent ON-OFF blinking statistics. A similar behaviour was observed in mercaptopropionic acid (MPA) capped CQDs when studied by fluorescence correlation spectroscopy (FCS)\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The apparent concentration of QDs using FCS technique was found to be far less than the actual concentration measured by the ensemble spectroscopic method and hence the observed reduced QY was proposed due to the presence of available dark particles in the solution but not the ON-OFF state time change\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. On the other hand, correlative light, and atomic force microscopy in CdSe/ZnS QDs showed the absence of emission properties in optical microscope for some of the particles that were observed in atomic force microscopy\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSingle particle number analysis using intensity projection\u003c/h2\u003e \u003cp\u003eAlthough we are not able to measure the number of single permanent dark CQDs directly onto the glass coverslip with the current experimental fluorescence microscopy setup, we speculated that they must have shown the same number of bright particles in a known area if there is no difference in dark/bright fraction under different laser excitations. Hence, we carried out an alternative approach to measure the number of particles in maximum intensity projection from the observed single-particle fluorescence videos of 5000 frames. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea \u003cb\u003eand b\u003c/b\u003e shows the maximum intensity projection of one video recorded for both excitation lasers at 488 nm and 532 nm. Surprisingly, we observed a quite significantly different number of bright CQDs under these excitations. We took 7\u0026ndash;8 videos (\u003cb\u003eFig. S6\u003c/b\u003e) for this measurement. We plotted the number of bright particles observed in each video as a box chart in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, showing lesser number of bright particles in all videos of 532 nm excitations in comparison of the 488 excitation videos. The average number of bright particles found in 488 nm excitation was ~\u0026thinsp;43, and 532 nm excitation showed\u0026thinsp;~\u0026thinsp;33 bright particles. A different number of bright particles on the same glass coverslip, keeping the same power density for 488 nm and 532 nm excitations, advocates the presence of a greater number of dark particles of CQDs at 532 nm excitation in comparison to 488 nm excitation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eFluorescence antibunching, and 2nd order correlation analysis\u003c/h2\u003e \u003cp\u003eTo ensure that the CQDs are emitting as a single emissive species, fluorescence antibunching studies were performed. We have performed total correlation FCS and fluorescence antibunching (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003ea \u003cb\u003eand b)\u003c/b\u003e with 405, 488 and 532 nm CW laser excitations for a very diluted solution (\u0026lt;\u0026thinsp;0.6 average CQDs in confocal volume). There exists no correlation at the ~\u0026thinsp;ns time scale in all of the total correlation FCS traces confirming the presence of single emitter in solution. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eb also confirms the CQDs to be single emitter as all G\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e(0) values are \u0026le;\u0026thinsp;0.27, less than 0.5. Also, the two-level distribution of ON-OFF intensity time traces in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea \u003cb\u003e(i and ii)\u003c/b\u003e supports the presence of single CQDs under observation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMean total photons emitted by single CQDs and probability distribution of ON and OFF times at both excitation wavelengths show that the single CQDs from the bright fraction of CQDs emit a similar number of photons under different excitations. Whereas the bulk QY data suggests higher excitations showing lower QY, like the bright fraction of CQDs observed under single particle microscopy. This led us to believe that the major reason for excitation dependency for CQDs is the dark fraction of CQDs. Considering this, we have formulated the relation between QY\u003csup\u003eBulk\u003c/sup\u003e and bright fraction as follows:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$${QY}^{Bulk}\\propto Bright fraction\\propto no of bright particles$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eTherefore, we can use this relation between QY and the number of bright particles.\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$at 488 nm excitation, 46.5 bright CQDs\\gg 33.28\\text{% }QY$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$$at 532 nm excitation,32.8 bright CQDs\\gg \\frac{33.28}{46.5}\\times 32.8=23.5\\text{%}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eThe QY at 532 nm calculated with the help of dark fraction matches with the bulk QY observed at 532 nm. This observation of obtaining QY at 532 nm excitation only with the help of the number of bright particles at 532 nm, 488 nm excitations and with QY at 488 nm excitations reaffirms our speculation of dark fraction responsible for QY decrement at ensemble/bulk level for CQDs. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e represents a detailed understanding of the fact observed in our case on excitation-dependent measurements in CQDs, where the decrease in the QY directly correlates with the number of permanently dark states at higher wavelengths.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eDiscussion on dark fraction studies\u003c/h2\u003e \u003cp\u003eIt is to be emphasized that the QY of QDs may vary due to three main reasons at the single particle level: (a) A decrease in the ON time fraction, which has been commonly observed\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan additionalcitationids=\"CR16 CR17 CR18\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e; (b) an increase in the dark fraction either partial or complete dark particles\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e; and (c) a combination of both\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e above. In most cases, the change in QY is due to factors a and c, which can lead to a change in the radiative rate and non-radiative rate of fluorescent bright fraction. In our present study, the change in QY is mainly due to factor b alone with the formation of completely dark particles. In this case, the QY-lifetime relation \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\left(QY={k}_{r}\\times \\tau \\right)\\)\u003c/span\u003e\u003c/span\u003e can give a false sense of decreased radiative and increased non-radiative rates. Here, the bright fraction\u0026rsquo;s fluorescence lifetime remained constant, and their behaviour was also found to be same under various excitations at single particle level. This implies that the radiative and non-radiative rates of the bright fraction are constant across all settings in our case. Therefore, the change in QY is not due to a change in the radiative rate or non-radiative rate of bright fraction but rather to a change in the dark fraction (permanently dark particles) only. Also, since the complete CQDs solution contains both fractions, the overall rates (ensemble averaged) will vary from the bright and dark fractions\u0026rsquo; radiative and non-radiative rates.\u003c/p\u003e \u003cp\u003eThis also signifies that the radiative and non-radiative rates determined in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed are actually apparent rates or (ensemble averaged). To address these issues, we have introduced the concept of apparent rates (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({K}_{r}^{App} and {K}_{nr}^{App}\\)\u003c/span\u003e\u003c/span\u003e), and shown their relationship with the intrinsic rates of bright fraction (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({k}_{r}^{B} and {k}_{nr}^{B}\\)\u003c/span\u003e\u003c/span\u003e) in the supplementary information. The relationships between the real and apparent rates are derived as follows:\u003cdiv id=\"Equd\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equd\" name=\"EquationSource\"\u003e\n$${k}_{r}^{App}=\\left(1-D\\right)\\times {k}_{r}^{B}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eand\u003cdiv id=\"Eque\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Eque\" name=\"EquationSource\"\u003e\n$${k}_{nr}^{App}=\\frac{D}{\\tau }+\\left(1-D\\right)\\times {k}_{nr}^{B}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere,\u003cdiv id=\"Equf\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equf\" name=\"EquationSource\"\u003e\n$$D is the fluorescent dark fraction, \\tau is the fluorescence lifetime$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equg\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equg\" name=\"EquationSource\"\u003e\n$${k}_{r}^{App}, {k}_{r}^{B} are the radiative rate for ensemble solution and Q{D}^{{\\prime }}s bright fraction respectively$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equh\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equh\" name=\"EquationSource\"\u003e\n$${k}_{nr}^{App}, {k}_{nr}^{B} are the non-radiative rate for ensemble solution and Q{D}^{{\\prime }}s bright fraction respectively$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eThe above equation suggests that both apparent rates (radiative and non-radiative) are directly dependent upon the dark (or bright) fraction of the CQDs solution only. Also, when there is no dark fraction, the intrinsic radiative rates and non-radiative rates of bright fraction represent the apparent rates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eExcitation wavelength dependent FCS studies and H\u003csup\u003e+\u003c/sup\u003e ion addition study\u003c/h2\u003e \u003cp\u003eAll of the single particle analysis performed above was for the immobilized CQDs over glass coverslip. To understand that the formation of permanently dark particle is also occurring in solution phase, we calculated the number of fluorophores diffusing through the confocal volume in FCS measurement. FCS is a solution-based technique, which provides valuable information at closely single molecule level by averaging out a fewer number of particles (molecules) diffusing through the confocal volume. It provides quantitative information in the triplet state contribution, its lifetime and the number of fluorescent particles diffusing out through the confocal volume. Experimental details are given in the supplementary information.A drop of highly diluted CQDs sample was excited with 561 nm laser and recorded the FCS spectra. We then repeated the procedure with 488 nm and 405 nm excitations, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). All these lasers were ensured to have the same laser power (2.6 \u0026micro;W). Fitting the FCS data with the triplet model revealed that the number of emissive species inside the confocal volume (~\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;15\u003c/sup\u003e litre) increased from ~\u0026thinsp;3 at 561 nm excitation to ~\u0026thinsp;9 at 405 nm excitation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). Also, it must be considered that the higher wavelength excitation makes a larger confocal volume, as the size of spot is directly proportional to the wavelength of incident light \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\left[diffraction limit=\\frac{Wavelength of light}{2 \\times Numerical Aperture}\\right]\\)\u003c/span\u003e\u003c/span\u003e. Therefore, if there is no dark fraction, then the number of particles must be higher in higher excitations as more particles can now diffuse through confocal volume due to its increased size. Hence, the observation clearly supports the existence of higher dark particles of CQDs at longer wavelength in the solution, as was observed in ensemble QY and at single particle level data.\u003c/p\u003e \u003cp\u003enext, we elucidated the reason for excitation wavelength dependent dark fraction formation. It has been reported by several studies that the H\u003csup\u003e+\u003c/sup\u003e ion plays an important role in QDs photophysical properties and can force the QD into permanently dark states \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. We carried out a pH dependent emission studies for the CQDs at three different pH. We incubated the CQDs solution into pH-3, pH 7.4 and pH 11 and measured the fluorescence intensity initially for 10 minutes with a 5 second time interval at the fluorescence maxima (λ\u003csub\u003emax\u003c/sub\u003e) and then measured the intensity for a longer period of about 3 hours. It is observed that at lower pH (~\u0026thinsp;3), the fluorescence intensity dropped down (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e. Interestingly, no such behavior was observed either at pH 7.4 or pH 11. To our surprize, the fluorescence lifetime did not change iat any of the pH solution. Here we speculate that the H\u003csup\u003e+\u003c/sup\u003e ions are adsorbed over the CQDs surface forcing it to go in permanently dark state, and since different energy excitation may influence the CQDs-H\u003csup\u003e+\u003c/sup\u003e complex differently, the amount of dark fraction will also be varied. It is to be noted that, this complex is formed in the ground state and hence it is not influencing the fluorescence lifetime. Higher energy excitations can break this complex easily forcing darker fraction particle to go in the bright fraction causing more QY in the lower wavelength excitations in comparison to the higher wavelength excitations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eWavelength dependent Bioimaging of the QDs\u003c/h2\u003e \u003cp\u003eWe finally ratified our hypothesis in a real bioimaging platform. We measured the brightness of the image of human cancer cells upon being treated with the CQDs. The detail of the cell imaging protocol and the experimental condition is presented in the supporting information. We have shown a confocal microscopy image of CQDs stained HEK cells excited at 488 nm and 561 nm laser (\u003cb\u003eFig. S7)\u003c/b\u003e. It is clearly visible that the image obtained under 488 nm excitation is brighter and more intense than the intensity upon 561 nm excitation under the same excitation laser power and all other similar experimental condition. This result suggested that the number of particles at shorter wavelengths are more emissive in nature than at longer wavelengths. QDs are widely used in various modern technological applications, such as the development of quantum computers as single photon sources. However, if a QD has a large number of dark fractions, then the single photon source efficiency will be reduced, as more bright fraction is needed.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe bulk fluorescence measurements for CQDs suggested QY to decrease with an increase in excitation wavelength. This trend is unusual than the trend observed for different reported QDs. Single-particle level fluorescence studies showed fewer fluorescent CQDs on 532 nm excitation compared to 488 nm excitation. In contrast, the total photon emitted, the probability distribution of ON and OFF times of CQDs, and the experimental localization precision value remain similar at both excitations. This finding suggests that the CQDs which are emissive in both excitations exhibit the same behaviour, but the number of bright or emissive CQDs are different for these excitations. Also, the FCS analysis revealed fewer bright particles at longer wavelength in comparison to shorter wavelength in solution. This anomaly signifies that there is a fraction of CQDs which has turned completely dark at longer wavelength, causing the unusual excitation dependent QY decrement. These dark fraction formation in this report is a ground state phenomenon and may exist due to the presence of H\u003csup\u003e+\u003c/sup\u003e ions in the water. The different energy excitations alter the CQDs-H\u003csup\u003e+\u003c/sup\u003e complex differently and cause less particle to convert from permanently dark to bright at higher wavelength excitations. The dependence of QY on the excitation wavelength, due to the presence of a permanent dark fraction, may affect the selection of QDs for specific applications, as this can compromise the device functionality. Therefore, proper characterization of the fluorescence dark fraction is necessary, and the influence of other factors on the QY of different QDs must be thoroughly investigated.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eKK conceptualized and designed all the experiments. JM, and RKB performed all the bulk-level measurements with the help of KK. KK performed single particle measurements with the help of RKB and JM. KK performed FCS experiment. KK analysed the data with the help of JM, RKB, and SS. SS helped in several experiments. FA provided HEK cells for the imaging study. CKN guided the complete project. KK wrote the manuscript with the help of CKN.\u003c/p\u003e\u003ch2\u003eAcknowledgment\u003c/h2\u003e \u003cp\u003eCKN is thankful to Science and Engineering Research Board (SERB) core research grant (CRG) India for the project number CRG/2020/000268. CKN is thankful to the facilities of the AMRC center of IIT Mandi, India. KK and CKN acknowledge the Sophisticated Analytical and Technical Help Institutes (SATHI), IIT Delhi and Indian Science Technology and Engineering facilities Map (I-STEM) for absolute quantum yield, total correlation FCS and fluorescence antibunching measurements. KK, and SS, thank the Ministry of Education (MoE), India, for research scholarship. FA acknowledges the SERB for scholarship. KK also acknowledge fruitful discussions with Mr. Abdul, Ms. Richa, Dr. Aditya, Dr. Chethana, and Dr. Monika.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSaha A, Chellappan KV, Narayan KS, Ghatak J, Datta R, Viswanatha R (2013) Near-Unity Quantum Yield in Semiconducting Nanostructures: Structural Understanding Leading to Energy Efficient Applications. J Phys Chem Lett 4(20):3544\u0026ndash;3549\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee LY, Ong SL, Hu JY, Ng WJ, Feng Y, Tan X, Wong SW (2004) Use of Semiconductor Quantum Dots for Photostable Immunofluorescence Labeling of Cryptosporidium Parvum. Appl Environ Microbiol 70(10):5732\u0026ndash;5736\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHeyes CD (2019) Quantum Dots in Single Molecule Spectroscopy. Spectroscopy and Dynamics of Single Molecules. 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ACS Nano 5(11):9062\u0026ndash;9073\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"CdTe quantum dots, quantum yield, Dark fraction, excitation dependency, single particle, fluorescence correlation spectroscopy","lastPublishedDoi":"10.21203/rs.3.rs-3999532/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3999532/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe Quantum yield (QY) of the semiconductor quantum dots (QDs) severely hampered by the inherent fluorescence intermittency. The QY of QDs usually increases with the increase in excitation wavelength. Here, we present an unusual behaviour where QY is found to decrease with increase in excitation wavelength in water soluble CdTe QDs (CQDs). Single particle measurement highlights the increase in permanent single dark particles at longer wavelength that comprehend the overall QY of the QDs in bulk solution. Fluorescence correlation spectroscopy further revealed an increase in number of dark particles at longer wavelength. The presence of H\u003csup\u003e+\u003c/sup\u003e ion in the water plays an important role in creating the permanently dark states in the CQDs. This observation was further supported by the cell internalization study of the QDs where much brighter images at shorter wavelength than longer wavelength were observed. A study of the excitation wavelength-dependent QY in QDs may reveal new insights into the applicability of QDs in different device fabrication cases.\u003c/p\u003e","manuscriptTitle":"Unusual Excitation Wavelength Dependency of Quantum Yield in Water Soluble CdTe Quantum Dots","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-08 18:47:35","doi":"10.21203/rs.3.rs-3999532/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0377df50-7888-4d0f-b83f-8d9714818c17","owner":[],"postedDate":"March 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":29060159,"name":"Physical sciences/Materials science/Nanoscale materials/Quantum dots"},{"id":29060160,"name":"Physical sciences/Nanoscience and technology/Nanoscale materials/Nanoparticles"}],"tags":[],"updatedAt":"2024-12-20T18:15:44+00:00","versionOfRecord":{"articleIdentity":"rs-3999532","link":"https://doi.org/10.1039/D4NR04344H","journal":{"identity":"nanoscale","isVorOnly":true,"title":"Nanoscale"},"publishedOn":"2024-12-19 00:00:00","publishedOnDateReadable":"December 19th, 2024"},"versionCreatedAt":"2024-03-08 18:47:35","video":"","vorDoi":"10.1039/D4NR04344H","vorDoiUrl":"https://doi.org/10.1039/D4NR04344H","workflowStages":[]},"version":"v1","identity":"rs-3999532","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3999532","identity":"rs-3999532","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}