A goal of this work was to elucidate the relative scattering efficiencies of specific cellular organelles. The results clearly show that objects stained with LysoSensor Green are as efficient or more efficient than other cellular structures. We have assumed that LysoSensor Green only stains lysosomes, however, it should be noted that Lyosensor Green may not be specific to only lysosomes. LysoSensor Green DND-189 has a pKa of ~ 5.2 and does not fluoresce at neutral pH. The fluorescence intensity increases with acidification. Several cellular organelles are acidic including the Golgi apparatus and lysosomes. The trans-Golgi network has a pH of ~6.0 and recycling endosomes have a pH of less than 6.0. Secretory vesicles have a pH of ~5.5 and lysosomes have a pH of ~5.5 [23
]. Therefore, while lysosomes are expected to exhibit the strongest fluorescence, secretory vesicles and recycling endosomes may have contributed to the fluorescence of LysoSensor Green.
Estimates of the percent of side scattering originating from each type of structure. The results are offset slightly for MR1 and SiHa cells for clarity.
For each experiment, there was a wide distribution of scattering efficiencies (see for example ) as discussed in Section 3.4. The wide width of the distributions likely has at least two fundamental causes, scattering from different cellular structures and speckle. The 785 nm laser used in this work is coherent and consequently generates speckle. This speckle may be the cause of some of the structure seen in the side scatter image and makes the interpretation of individual images difficult. However, by imaging and averaging results of thousands of cells in each experiment the intensity variations due to speckle should average out. A further advantage of averaging thousands of cells is that any effects on cell scattering due to cell orientation [24
] are averaged out.
Throughout the paper, the assumption was made that scattering from regions with only a specific fluorescence was due only to that particular organelle. For example, scattering from regions with only MitoTracker fluorescence was assumed to be only due to mitochondria. In addition, however, there could have been a contribution to the scattering from nonfluorescent objects. This contribution can be estimated from , in which scattering from the nonfluorescent particles was different for MR1 and SiHa cells. We assume that lysosomes and mitochondria have the same scattering efficiency in both cell types and that the contributions of nonfluorescent particles are the same in MitoTracker and LysoSensor fluorescing regions of both cell lines. The result is that nonfluorescent particles are ~6 – 20% of the scattering efficiency from lysosomal and mitochondrial regions when the incident light is perpendicular to the scattering plane. Since, the nonfluorescent particles are more scattering than mitochondria, but less scattering than lysosomes, the difference between lysosomal and mitochondrial scattering efficiencies is probably greater than shown in .
Presented data support previous results that the nucleus is an efficient side scatterer. The nucleus is not homogeneous, but has many inhomogeneities [17
] with a maximum chromatin clump size of about 1 μ
]. FDTD simulations demonstrate that the greater the index of refraction variations within the nucleus, the more they scatter light at a wide variety of angles including 90° [25
]. Additionally, in our own work modeling the angular distribution of scattering from nuclei, we found that homogeneous nuclei could not be used to explain the scattering from isolated nuclei - the scattering at 90° was significantly low (fig. 12 of [17
]) and smaller scattering centers were needed to model the measured light scattering.
Wilson and Foster previously reported that lysosomes scatter approximately 14–15% of the light from EMT6 cells at 633 nm [26
] while we estimate that lysosomes are 20–30% of the side scattering at 785 nm. Their results were based on measurements of angularly light scattering from ~7° to 83° before and after the ablation of lysosomes. The contribution of lysosomal scattering to side scatter was found to be much greater in our work, between 20% and 30% depending on polarization and cell type. Our results may slightly overestimate the contribution of lysosomes due to the contribution of nonfluorescent particles (as discussed above). In comparing our work with Wilson and Foster, the facts that the wavelengths are different and that our work only looks at side scatter must be considered. Our data was taken at 785 nm versus Wilson and Foster’s data at 633 nm. Generally, the contribution of smaller particles to scattering decreases as wavelength is increased and our data demonstrate that the scattering centers in lysosomes are small. Based on this comparison, our results would be expected to give a lower contribution of lysosomes. On the other hand, from of Wilson and Foster [26
], it can be inferred that lysosomes contribute more of the scattering at 90° than at angles less than 30°. Our results would, therefore be expected to show a higher contribution of lysosomes to scatter than was found by Wilson and Foster. Furthermore, our stain may have stained some low pH vesicles in addition to lysosomes. In conclusion, our results indicate a higher contribution of lysosomes to side scatter than the contribution to total scatter reported by Wilson and Foster.
A more profound difference between our results and previously reported results is the relative
contribution of mitochondria and lysosomes to scattering. We found that not only are lysosomes much more efficient scatterers than mitochondria, lysosomes contribute at least as much and possibly more to side scatter in both MR1 and SiHa cells especially when the polarization is perpendicular to the scattering plane. Wilson et al., however, reported about a 5 times greater contribution from mitochondria than from lysosomes to the total scattering cross section [13
]. Potentially, this discrepancy is due to the fact that Wilson et al. assumed that their light scattering signal was due to objects in the cytoplasm. Some of the scattering from particles that were assigned to mitochondria by Wilson et al. might be from the nucleus. We found that ~ 30% of the side scattering from MR1 cells and ~45% of the side scattering from SiHa cells was due to the nucleus.
The discrepancy between the result of Wilson et al. that mitochondria have a factor of 5 greater contribution to total scatter than total scatter than lyosomes and our result that lysosomes contribute similarly or slightly more to “side scatter” is unlikely to be primarily due to the fact that we measured side scatter while Wilson et al. [26
] were measuring total scatter. Our measurements used a microscope objective with an NA of 0.75. Therefore we were collecting light over an angle range of ~97° centered at 90°. Consequently, for the ratios of total scatter, and side scatter to vary greatly, there must be a huge difference in the light scattering from mitochondria and lysosomes in the near forward or near backscattering directions. These differences are unlikely since mitochondria and lysosomes are similarly sized particles. Mitochondria are elongated particles which can vary in size with the cell cycle. Based on the results of Kennady et al. [27
], the mitochondria in our MR1cells were roughly 0.7 to 3 μ
m long. Lysosomes are generally spheric or ovoid and vary in size from ~1μ
m to a little over a micron in many cell types including CHO (Chinese hamster ovary) cells [28
]. In conclusion, the difference in some of the measurement angles between our work and that of Foster and coworkers may be the cause of some of the differences in results for the relative contribution of mitochondria and lysosomes. However, the inclusion of the nuclear contribution to side scattering in our work and possibly the inclusion of more low pH organelles (not just lysosomes) in the lysosomal contribution in our work likely contributed more to the differences in results.
In addition to providing insight into the fundamental question of what is scattering light, this paper also demonstrates some of the intrinsic advantages of measuring unstained/unprocessed samples over measuring stained samples. Images of stained samples can be used for localization of organelles and can provide beautiful pictures for insight into cellular phenomena. However, applications requiring the assumption that the dyes do not alter the cell should be undertaken with caution. In our case we found that Hoechst staining increases side scattering from the cells. Additionally, the size of SiHa cells increased when they were stained with all three of the dyes used. While it is beyond the scope of this paper to understand the biochemical interactions causing the changes in scattering or in cell size, the results demonstrate that frequently used stains have marked affects on the cells and that care must be used when interpreting results of experiments using fluorescent dyes.