Yes, fluorescing particles can be measured with dynamic light scattering (DLS). However, the quality of the data collected and the subsequent confidence in the size results will be dependent upon both the absorption and emission spectra, and whether or not narrow band wavelength filters are employed.
In order for a sample to fluoresce light, it must first absorb light. As a consequence of vibrational relaxation, manifested as thermal energy release, fluoresced photons are emitted at a lower energy or longer wavelength than that of the absorbed photons. Scattered photons on the other hand, have roughly the same wavelength as the incident photons. So if one were detecting only the photons with the same frequency as the incident light, then fluorescence would have negligible influence on light scattering measurements. In practice however, DLS systems typically utilize APD detectors to detect the scattering light, and APDs exhibit significant sensitivity to photons across a broad range of wavelengths.
As an example, consider the figure below, which shows the representative absorbance and emission spectra of the fluorophore pyrene, along with the wavelength dependent photon detection efficiency for a typical APD. The pyrene absorbance spectrum, shown in blue, exhibits a maximum absorbance at circa 350 nm. The emission spectrum, shown in green, exhibits the classical mirror image profile, with an emission maximum at circa 370 nm. While the photon detection efficiency for the APD is low in the 350 to 400 nm range, the APD would still be capable of measuring light scattered from a 350 nm incident source. However, if the particle being sized with the 350 nm source contained pyrene, the > 350 nm fluoresced light detected by the APD would be significant. Since the fluoresced light would be non-coherent, it would manifest itself as baseline noise in the DLS measured correlogram, resulting in a decreased signal to noise ratio and reduced confidence in the size results.
Also shown in the figure above, is the wavelength of the typical lasers employed in dynamic light scattering systems, e.g. the green 532 nm Argon laser and the red 633 nm HeNe laser. As evident in the above, use of either of these incident light sources would ensure the absence of absorbance by pyrene, thereby eliminating any fluorescence. The availability of fluorophores absorbing in the 500 to 650 nm range however, has increased significantly in the past decade, particularly within the quantum dot field. In fact, the wavelength at which a quantum dot fluoresces is very strongly dependent upon the particle size; and since these sizes are typically sub-micron, the question of fluorescence effects on DLS results has taken on new importance.
The two principle issues that need to be considered on the subject of fluorescence effects on DLS results are noise and sensitivity. In DLS, "sensitivity" is defined as the minimum concentration at which at particle of a defined size can be measured. For a fixed optical geometry, the sensitivity of a DLS system is strongly correlated to the number of photons being scattered. Photons absorbed in the fluorescence event will not scatter. So if the particles being measured in a DLS experiment absorb some of the incident light, an inherent loss of sensitivity will be observed. The magnitude of this loss will depend upon the quantum efficiency of the absorbance mechanism. As mention above, fluoresced light is non-coherent. As such, fluoresced light detected in a DLS experiment will be manifested as baseline noise in the measured correlogram.
The upside to the issues of sensitivity and noise is that the reduced data quality arising from both of these issues can be attenuated by increasing the sample concentration (increases the number of scattered photons) and/or increasing the number of runs or correlogram acquisitions (increases the signal to noise ratio) collected during the measurement. The downside is that for samples such as quantum dots, increasing the concentration can sometimes be challenging.
As a general rule of thumb then, the sizing measurement of fluorescing samples by DLS, while challenging, is certainly achievable. As is typically the case however, there are always exceptions to a rule of thumb. An example of a likely exception would be a small particle, which absorbs strongly at the wavelength of the incident laser and fluoresces (emits) strongly in the wavelength range of high photon detection efficiency for the APD, e.g. a sample that absorbs at circa 630 nm and fluoresces in the 650 to 850 nm range. The figure below shows the correlogram measured with a Zetasizer Nano (633 nm laser) for a 20 nm diameter quantum dot sample exhibiting strong absorbance in the 630 nm range and strong fluorescence in the 800 nm range. For high quality DLS measurements, one would target an amplitude (Y intercept value) of > 0.8. As noted in the figure below however, the combination of a reduced scattering intensity (due to sample absorbance) and an increase in signal noise (arising from the non-coherent fluoresced photons) leads to a measured correlogram with a very low amplitude (0.016) and a noisy baseline. While it is possible to deconvolute a particle size distribution from the correlogram shown below, confidence in the results would be low.
For scenarios such as that described and shown above, insertion of a narrow band wavelength filter between the scattering sample and the APD can often improve data quality. The narrow band filter functions to remove much of the non-coherent fluoresced light, allowing only scattered photons with a wavelength similar to that of the incident source to reach the detector – thereby reducing the level of noise in the detected signal. The figures below show overlays of the results for the exception quantum dot sample discussed above, measured in the presence and absence of a narrow band wavelength filter. As noted in the first figure, the presence of the filter significantly enhances the quality of the measured correlogram, which shows negligible baseline noise and an amplitude (0.85) well within the acceptable range for high confidence results. The calculated particle size distributions for the two measurements are shown in the latter figure, where the presence of the narrow band filter generates a distribution of much lower polydispersity (distribution width) and a size consistent with the expected 20 nm diameter reported for the quantum dot sample.
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