Photo-Toxicity
Image stacks were imported into Imaris (version 9.2.0;
Bitplane, AG, Zurich, Switzerland) to track the position of lysosomes over time. Lysosomes were masked with the Spots function using an estimated diameter of 0.5 μm and local background subtraction. An autoregressive motion algorithm with a maximum distance of 1 μm and gap size of 3 frames was used to follow lysosomes. Data were then exported to MATLAB to quantify speed and persistence. Tracks less than 15 frames (3.255 s) were removed by filtering. Average speed was calculated from mean change in x,y position between each time point. Persistence was calculated by dividing the net dis- placement of each vesicle track aſter 15 frames by the total dis- tance traveled. Data from three cells were then pooled together and plotted as frequency distributions using Prism 8 (version 8.4.2; GraphPad Soſtware, San Diego, CA).
Results Sample illumination with LED light sources can be con-
trolled through the microscope soſtware by (1) opening and closing a mechanical shutter, (2) electronically triggering the light source via a USB cable connection, or (3) electronically triggering the light source via a TTL cable from the camera to the light source. We previously observed that electronic activa- tion of the light source is ∼20-fold faster than opening/clos- ing mechanical shutters [2]. Terefore, we focused on USB and TTL triggering of the light source in the present article. USB and TTL turn the light source on and off at approximately the same rate [2]. A notable advantage of USB triggering is that it allows the microscope soſtware to modify the properties of the light source (for example, light power; for multiple LEDs they can be turned on and off selectively, programmed for different exposure times). In contrast, TTL triggering relies on an elec- tronic circuit between the camera and the light source; when the camera begins acquiring an image, a current is sent to the light source to turn it on. Tis is an all-or-nothing event, mean- ing that the light source turns on to a preset user-defined inten- sity. However, if both USB and TTL cables are connected to the light source, light intensity can be adjusted in the microscope soſtware prior to TTL triggering. Tus, it may be useful to have both connections to the light source, especially if performing multi-color and multi-dimensional acquisition. Based on this information, we tested three different set-
tings on a Zeiss AxioObserver equipped with an X-Cite 120LED: TTL triggering of the light source with the USB cable (1) disconnected or (2) connected, and (3) USB triggering of the light source with the TTL cable disconnected. TTL effectively synchronized and limited light exposure to the camera image acquisition time within a millisecond or so (Figure 2, top panel). Although USB triggering is equally effective in turning the light source on and off [2], light exposure time was almost six times longer with USB triggering when compared to TTL (Figure 2, bottom panel). Consequently, USB and TTL trigger- ing of the light source are not equivalent. Interestingly, the USB cable had to be disconnected from the microscope soſtware for TTL triggering to work properly; even if TTL override was enabled, the exposure time was three times longer when the microscope soſtware detected the light source via the USB con- nection (Figure 2, middle panel). Tis suggests that determina- tion of IO time is complex, as it can arise from both hardware
2020 July •
www.microscopy-today.com
and soſtware delays. IO is an especially important issue for live cell microscopy, as the extra illumination time with USB trig- gering results in a significant amount of photo-bleaching and ROS production [2]. Te impact of IO is more drastic when shorter exposure times with higher light powers are used as the relative contribution of IO to photo-bleaching and photo- toxicity is greater. For example, when 400 images of paxillin- EGFP were collected with high-power illumination light and a 24 ms exposure time, the sample was illuminated almost six- fold longer (138 ms) for each of the 400 images collected. Tis resulted in significant ROS production (Figure 1). However, when low-power light was used with 1060 ms exposure times, the contribution of IO was only 10–20%, so there was minimal ROS accumulation even aſter collecting 400 images (Figure 1). Further in-depth experiments exploring the issue of IO
revealed that the amount of additional sample illumination was not constant but varied with changes in exposure time. Indeed, longer exposure times exhibited longer IO when USB trigger- ing was used, with some variability in the times (Figure 3). Nevertheless, the percentage contribution of IO decreased with longer exposure times, resulting in healthier live cell imaging conditions [2]. Tus, the conclusion of our Microscopy Today article (that is, longer exposure times with lower light power are less photo-toxic) still stands and is especially important if IO is significant. Tis is especially true for mercury or metal- halide light sources that cannot be electronically switched on and off but rely on mechanical shuttering. If fluorescence light sources, such as LEDs, can be directly triggered by TTL to limit light exposure of the sample to the camera exposure time, then higher light powers and shorter exposure times are compat- ible with live cell imaging experiments. It should be noted that increased photo-bleaching was not caused by increased inci- dent light power in our set of experiments [2]. It is possible that the production of ROS over shorter periods of time with high light power could overwhelm cellular systems designed to remove ROS and cause photo-toxicity in live samples; however,
Figure 2: Oscilloscope measurements showing sample illumination time. Experiments were conducted on a Zeiss AxioObserver microscope running ZEN pro software (version 2.6). An X-Cite 120LED light source was triggered with TTL while the USB connection between the microscope computer/soft- ware and light source was disconnected (green trace) or connected (yellow trace), and USB directly while the TTL cable was disconnected (red trace). Exposure time was set to 24ms in the microscope software; imaging interval was set to 500ms.
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