Photo-Toxicity
Figure 3: Oscilloscope measurements of sample illumination time compared to input exposure time. An X-Cite 120LED light source was USB triggered through ZEN pro software (version 2.6). The amount of IO changed with exposure time.
further experimentation is required to determine if this is indeed the case. Another important observation from our studies was
that hardware and soſtware delays can also negatively impact the image acquisition interval. When a sufficiently long imag- ing interval was set in the microscope soſtware, TTL trigger- ing was able to capture images at the desired time resolution (Figure 4A, top panels in black). Unfortunately, imaging intervals below a certain value could not be realized, even though the time resolution was theoretically possible (Figure 4A, bottom panels in red). Moreover, the minimum imag- ing interval appeared to vary with exposure time and did not change in a systematic way (Figure 4B). Tis is especially problematic when imaging dynamic cellular processes that
occur on the millisecond to second time scale. As a result, it is recommended that users verify the time resolution of their experimental system using an oscilloscope or accurate soſt- ware time stamps indicating exactly when the images were saved. Some hardware and soſtware delays can be mitigated with stream acquisition if it is available in the image acquisi- tion soſtware. In this case, stream acquisition with tempo- rary data storage on random access memory (RAM) allowed rapid imaging with a 5.005 ms delay between images (data not shown). Similar results were obtained on a spinning disk confocal system; stream acquisition reduced IO from ∼42 to ∼17 ms [2]. Furthermore, stream acquisition allowed sub- sequent images to be captured with no time delay beyond ∼17 ms of IO (Figure 5). Longer exposure times with lower
Figure 4: (A) Time lapse experiments were performed with TTL triggering of the X-Cite 120LED light source. Several camera acquisition intervals were chosen: 500ms, 100ms, 50ms, and 25ms. Camera exposure time was set to 24ms. (B) Time lapse experiments in (A) were repeated with various camera exposure times: 48ms and 100ms. The image acquisition interval was set to 100ms.
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