Microscope Hardware and Software Delays Cause Photo-Toxicity
Alex Kiepas1,2 and Claire M. Brown1,3 *
1Department of Physiology, McGill University, Montreal, QC, H3G 0B1 2Goodman Cancer Research Centre, McGill University, Montreal, QC, H3G 1A1
3Advanced BioImaging Facility (ABIF), 3649 Promenade Sir William Osler, McGill University, Montreal, QC, H3G 0B1 *
claire.brown@
mcgill.ca
Abstract: Technological advancements in the areas of sample illu- mination, image acquisition, and image processing have significantly improved the speed and sensitivity of fluorescence microscopy. In particular, light emitting diodes (LEDs) coupled to transistor-transistor logic (TTL) circuits have reduced photo-bleaching and photo-toxicity by limiting sample illumination to the camera exposure time. Unfortu- nately, many microscopes still rely on bulb-based light sources that cannot be configured with TTL. Moreover, even when TTL is enabled in conventional microscope software, hardware and software delays can still contribute to photo-toxicity and lead to additional delays between subsequent images, introducing errors in time lapse image recordings. The goal of the present article is to highlight the signifi- cance of these issues.
Keywords: fluorescence microscopy, photo-toxicity, photo-bleach- ing, illumination overhead, lysosomal dynamics
Introduction In the November 2017 issue of Microscopy Today, our
laboratory published an article indicating that longer expo- sure times with lower light intensities reduced photo-toxicity to live cells [1]. In particular, fluorescence wide-field imaging experiments showed that employing longer exposure times with lower light powers drastically reduced photo-bleaching and increased cell migration and cell protrusion speeds with- out impacting image quality. Further investigation into this phenomenon pointed us to the issue of illumination overhead (IO). IO is the time fluorescent samples are exposed to incident light, but fluorescence emission is not being collected by the detector [2]. IO leads to excessive light exposure to the sample with no improvement in image quality. Te authors were aware of IO when the Microscopy Today article was published in 2017, but not the extent of the problem. Since then additional experi- ments have been conducted to further investigate the impact of IO. Importantly, photo-bleaching and photo-toxicity were not due to increased incident light power. Modern light sources for fluorescence microscopy, such as
light-emitting diodes (LEDs), can be electronically switched on and off within a few milliseconds [3,4]. Tis technologi- cal advancement has significantly reduced photo-bleaching and photo-toxicity during fluorescence microscopy by chang- ing the way sample illumination is controlled. In the past, mechanical shutters were required to regulate sample exposure time for bulb-based light sources. When using shutters, the microscope soſtware synchronizes shutter opening with cam- era acquisition time. Image acquisition soſtware introduces a delay so that camera acquisition does not begin until the shut- ter is fully open (
https://www.microscopyu.com/applications/ live-cell-imaging/the-automatic-microscope). Tis ensures uniform illumination across the entire field of view; however,
30 doi:10.1017/S1551929520001145
samples are exposed to extra illumination (IO) beyond the camera exposure time. Te amount of IO samples experience is a function of shutter speed. In contrast, most newer micro- scopes are equipped with LED-based light sources, and sam- ple illumination is largely controlled by electronic triggering. Electronic activation of light sources is significantly faster than physical shutter speeds, resulting in a dramatic reduction in IO [2]. Triggering can be achieved in two ways: (1) the micro- scope soſtware directly triggers the light source through a USB connection to the device; or (2) the microscope soſtware initi- ates camera acquisition, which in turn triggers the light source through a transistor-transistor logic (TTL) circuit. In this follow-up study, the complexity of IO and inter-
val imaging with USB and TTL triggering was explored. IO generated more reactive oxygen species (ROS) when shorter exposure times were used, complementing the results of our original work. Te percent of IO decreased with longer expo- sure times; however, the amount of IO samples experienced was not constant across all exposure times. Additionally, results showed that the light source had to be disconnected from the microscope soſtware for TTL light triggering to function prop- erly; otherwise, hardware and soſtware delays continued to contribute significantly to IO. Finally, although TTL success- fully eliminated IO, hardware and soſtware delays continued to impact the acquisition interval resulting in inaccurate time resolution of experiments.
Materials and Methods Cell culture. CHO-K1 cells stably expressing paxillin-
EGFP were obtained from the lab of Dr. Rick Horwitz (Uni- versity of Virginia, Charlottesville, VA). Cells were grown in low glucose (1.0 g/L) Dulbecco’s modified Eagle’s medium (DMEM; cat. no. 11885-084, Termo Fisher Scientific), sup- plemented with 10% fetal bovine serum (FBS; cat. no. 10082- 147, Termo Fisher Scientific), 1% non-essential amino acids (NEAA; cat. no. 11140-050, Termo Fisher Scientific), 25 nM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; cat. no. 15630-080, Termo Fisher Scientific), and 1% penicil- lin-streptomycin (cat. no. 10378-016, Termo Fisher Scientific). Cells were maintained in 0.5 mg/mL Geneticin-418 (G418; cat. no. 11811-031, Termo Fisher Scientific) antibody selection to maintain paxillin-EGFP expression. MCF7 cells were obtained from the American Type Cul-
ture Collection (ATCC; cat. no. HTB-22). Cells were grown in high glucose (4.5 g/L) DMEM (cat. no. 319-005-CL, Wisent Bioproducts), supplemented with 10% FBS (cat. no. 12483-020, Gibco) and 1% penicillin-streptomycin (cat. no. 450-201-EL, Wisent Bioproducts).
www.microscopy-today.com • 2020 July
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