Photo-Toxicity
Measurements of exposure time. A digital oscilloscope
(DS1054Z; Rigol, Beijing, China) coupled to a DET36A/M Si Based Detector (Torlabs, Dachau, Germany) was used to measure the total illumination time delivered through a Plan ApoChromat 20× 0.8 NA objective lens on a Zeiss AxioOb- server fully automated inverted microscope (Carl Zeiss, Jena, Germany). An XCite 120LED light source (Excelitas Tech- nologies, Waltham, MA) was used to deliver excitation light via the microscope soſtware (ZEN pro, version 2.6). Te light source was electronically triggered via a USB cable connection between the computer and the light source (scenario 1) or a TTL cable between an Axiocam 506 camera (Carl Zeiss) and the light source (scenarios 2 and 3). In the latter case, the USB cable between the computer and the light source was either connected (scenario 2) or disconnected (scenario 3). A vari- ety of camera exposure times (24–60,000 ms) and acquisition intervals (25–500 ms) were input into the microscope soſtware, and the light output was recorded. Te oscilloscope was also used to measure the total illu-
mination time delivered through an HC PLAN APO 20× 0.7 NA objective lens on a Leica DMI6000B inverted microscope equipped with a Quorum WaveFx-X1 spinning disk confocal system (Quorum Technologies, Guelph, ON). A 561 nm diode laser was used to deliver excitation light via an acousto-optic tunable filter (AOTF) crystal controlled by MetaMorph soſt- ware (version 7.10.2.240; Molecular Devices, Sunnyvale, CA). Te AOTF crystal was controlled by USB triggering. Voltage traces were captured with interval imaging or stream to ran- dom access memory (RAM) acquisition. Camera exposure time was set to 100 or 200 ms. Te acquisition interval was set to 0 ms. Voltage traces obtained from the oscilloscope were ana-
lyzed in MATLAB (version 9.8.0, Rel. 2020a; Te MathWorks, Natick, MA). IO was determined by subtracting the desired (input) exposure time from the actual (output) exposure time. Dividing IO by input exposure time yielded percent IO. Measurements of power. A PM400 Optical Power and
Energy Meter with an S170C Microscope Slide Power Sen- sor (Torlabs) was used to measure incident light intensity through oil immersion objective lenses: Zeiss PlanApo 63× 1.4 NA on the AxioObserver and Leica HCX PL APO 63× 1.4 NA on the spinning disk confocal microscope. Measurements were performed on three separate days and averaged. ROS production in response to photo-bleaching. CHO-
K1 cells expressing paxillin-EGFP were seeded onto μ-slide 8-well plates (cat. no. 80821, IBIDI, Fitchburg, WI) coated with 0.21 µg/cm2
fibronectin (cat. no. F-0895, Sigma-Aldrich)
diluted in 1x phosphate-buffered saline (PBS). Cells were allowed to adhere and grow under exponential conditions for at least 12 h prior to experimentation. Cells were then stained with 0.83 µM CellROX™ Deep Red (cat. no. C10422, Termo Fisher Scientific). Images were acquired on the Zeiss AxioOb- server with a PlanApo 63×1.4 NA oil immersion objective lens and Chamlide TC-L-Z003 stage top environmental con- trol incubator (Live Cell Instrument, Seoul, South Korea). An XCite 120LED was used to deliver excitation light through an EGFP filter cube (filter set 10; 450-490 nm excitation, 515- 565 nm emission; Carl Zeiss) at
three different intensities:
21.3 mW, 10.8 mW and 0.476 mW. Image acquisition settings 32
were adjusted to maintain a constant number of photons impacting the sample during camera exposure time without taking IO into consideration (21.3 mW x 24 ms, 10.8 mW x 48 ms, 0.476 mW x 1060 ms). Te light source was USB trig- gered through the microscope soſtware. CellROX™ was imaged before and aſter paxillin-EGFP photo-bleaching (400 frames) with a total
light dose of 1030 mW× ms delivered
through a Cy5 filter cube (filter set 49006; 590–650 nm exci- tation, 662–737 nm emission; Chroma Technologies, Bellows Falls, VT). Exposure time was set to 1000 ms. Paxillin-EGFP and CellROX™ images were pseudo-colored (Rainbow RGB) in ImageJ (NIH, Bethesda, MD) to emphasize changes in fluo- rescence intensity. Te intensity scale is the same for all image panels for each fluorophore (Figure 1). Lysosomal dynamics. MCF7 cells were seeded onto
μ-dish 35 mm high glass bottom dishes (cat. no. 81158, IBIDI) coated with 5 µg/cm2
fibronectin (cat. no. FC010, EMD Milli-
pore) and stained with 200 nM LysoTracker™ Green DND-26 (cat. no. L7526, Termo Fisher Scientific). Cells were imaged on the spinning disk confocal microscope with a Leica HCX PL APO 63× 1.40 NA oil immersion DIC objective lens, Prime BSI sCMOS camera (Photometrics, Tuscon, AZ), and CU-501 stage-top incubator system (Live Cell Instrument, Seoul, South Korea). Each cell was illuminated with a 491 nm diode laser set to ∼0.02 mW. Stream to RAM acquisition in Meta- Morph was used to acquire images continuously for over 30 seconds. Camera exposure time was set to 200 ms with 2 × 2 pixel binning (1 pixel = 0.1172 μm × 0.1172 µm). IO was found to contribute an additional 17 ms delay resulting in a time resolution of 217 ms. Te pinhole size of the spinning disk was fixed at 50 µm.
Figure 1: CHO-K1 cells expressing paxillin-EGFP were seeded onto fibronec- tin-coated dishes and stained with CellROX™ Deep Red Reagent. Paxillin-EGFP was repeatedly imaged for 400 frames with USB triggering of the light source. Three different light doses were chosen: 21.3mW× 24ms, 10.8mW× 48ms, and 0.476mW×1060ms. Light power and exposure time were adjusted such that the total light dose per frame remained constant (∼250 W×ms × cm−2
); in other words,
the amount of light on the sample during the camera image acquisition time was constant between conditions. CellROX™ was imaged before and after paxillin- EGFP acquisition to measure ROS production. Images were pseudo-colored to highlight changes in paxillin-EGFP and CellROX™ intensity. Scale bar is 10 µm.
www.microscopy-today.com • 2020 July
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