Letter to the Editor
Figure 5 (revised): Average cell migration speeds compared to bright-fi eld controls.
and, in turn, photo-toxicity is the power of the light. In the paper, the camera exposure time was adjusted so that the light dose would be similar for each image with diff erent incident light powers. In writing the article, we chose the “temporal light dose” because it is very straightforward and accessible to a wide array of microscopy users. Microscopists can easily measure incident light power with a 10× lens as was done in the paper and then divide that number by the exposure time to calculate the “temporal light dose.” As seen in Table 1 of the paper, the smaller the temporal light dose value is, the better is the imaging condition. Aſt er reexamining this calculation following the Letter to the Editor above, we realized that if power is leſt constant and the exposure time is increased then the temporal light dose would decrease. However, a longer exposure time would not result in reduced photo-toxicity and would not improve cellular health. T us, instead of the “temporal light dose” parameter we would like to recommend that researchers keep the light power at a minimum and increase exposure time as much as required to generate a good signal-to-noise image. However, this may not always be possible as shorter exposure times may be required to image rapid biological processes. In this case, keeping the light power at a minimum is still essential. An interesting observation made in the above Letter is the exponential correlation between the decay rate of fl uorescence and the power. T is is in line with the two-step photolysis
mechanism of photo-bleaching, which strongly depends on light power intensity that has been previously published [ 1 ]. T is is consistent with our data and the thought that the photo- bleaching and photo-toxicity are mainly linked to photo-lysis of triplet state fl uorescent molecules. In fact, if the data were collected so that the camera exposure time was kept constant and only the incident light power was changed, the exponential trend of the photo-bleaching rate with increasing power would be even more prominent. T e authors of the Letter also asked about the temporal variation of the incident light intensity. We have in fact measured that for many LED-based light sources, and it is well below 0.5% on all time scales we measured (ms, seconds, hours, days). We have reproduced key fi gures from the article, related to live cell imaging, in terms of power rather than temporal light dose. Both cell migration speeds ( Figure 5 (revised) ) and cell protrusion speeds ( Figure 7 (revised) ) are signifi cantly reduced at light power of 12 mW compared to 1.64 mW. In our original article conditions of high light dose and low light dose were 12 mW for 35 ms and 1.64 mW for 350 ms, respectively.
In conclusion, we would like to thank the authors of the Letter to the Editor for helping us clarify our parameters. We no longer recommend using the “temporal light dose” parameter but minimal light power and longer exposure times to minimize photo-toxicity.
References [1] LA Deschenes and DA Vanden Bout , Chem Phys Lett 365 ( 5–6 ) ( 2002 ) 387 – 95 .
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1-800-538-3672 Figure 7 (revised): Average cell protrusion speeds versus increasing light power. 2018 March •
www.microscopy-today.com doi: 10.1017/S1551929518000275 59
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