Photo-Toxicity
light levels and stream acquisition allowed for the simultane- ous capture of images of EB3-mEmerald and H2B-mCherry with high time resolution (200 ms camera exposure time plus 17 ms IO) with no apparent photo-bleaching [2]. Tese parameters could also be used to quantify the speed and per- sistence of lysosomes using LysoTracker™ Green staining (Figure 6).
Discussion and Conclusions Overall, our results indicate that IO is a highly complex
issue that appears to be caused by hardware and soſtware delays. Tese hardware and soſtware delays are likely unique to every microscope [5]. Tus, there are many factors that contribute to additional sample illumination (as seen here with USB triggering) and extended image acquisition inter- vals with TTL triggering. Camera soſtware drivers, computer processing speeds, computer components (video cards, RAM, CPU), soſtware versions, and connections between the com- puter and microscope components all likely affect IO and image acquisition intervals. Te complexity is clear. Indeed, microscopists have previously noted that faster image acqui- sition can be achieved by imaging a smaller region of inter- est (ROI) on the camera chip, and the acquisition speed can vary depending on the physical location of the ROI on the chip (
https://andor.oxinst.com/learning/view/article/what- is-cropped-sensor-mode) [6]. Efforts are currently underway to design hardware and soſtware for improved high-speed synchronization of multiple devices (https://micro-manager. org/wiki/Hardware-based_Synchronization_in_Micro- Manager) [7,8]. In the meantime, microscopists should mea- sure imaging parameters with an oscilloscope to determine the impact of IO and adapt image acquisition parameters for healthy live cell imaging conditions while maintaining time resolution and accurate time intervals for time lapse imaging.
Acknowledgements We thank Dr. Marco Biondini and Leeanna El-Houjeiri
(McGill University, Montreal, QC) for preparing MCF7 cells stained with LysoTracker™ Green. Work performed in the author’s laboratory (CMB) was supported by grants from the National Sciences and Engineering Research Council (NSERC) (Grant #493616-16, #386084-12) and CIHR (Grant #CPG- 146475). AK acknowledges support in the form of a Fonds de Recherche Santé (FRQS) doctoral studentship. Imaging experi- ments were performed at the McGill University Advanced Bio- Imaging Facility (ABIF).
References [1] F Mubaid and CM Brown, Microscopy Today 25(6) (2017) 26–35.
[2] A Kiepas et al., J Cell Sci 133(4) jcs.242834.
(2020) doi:10.1242/
[3] JB Bosse et al., PLOS One 10(11) (2015) e0143547. [4] T Nishigaki et al., Biotechniques 41(2) (2006) 191–97. [5] MA Davis, Nat Methods 14(12) (2017) 1223–25. [6] P Almada et al., Methods 88 (2015) 109–21. [7] MJ Colville et al., Sci Rep 9(1) (2019) 12188–12200. [8] A Edelstein et al., Curr Protoc Mol Biol 92(1) (2010) 14.20.1–14.20.17.
36 CELESTA
THE NEXT GENERATION COMES TO LIGHT
®
• Spectral breadth: 405, 446, 477, 520, 546, 638, 749nm
• Brightness: Seven lasers ~1.1 W/mm2
• Power: ~1000mW/ line at distal end of 1.5 mm diameter fiber, ~7W total optical power
• Control: Active power stabilization
• Stability: Exceptional reproducibility, ideal for quantitation
• Ease of use: Pre-aligned, independent lasers
• Applications: Confocal, MERFISH, super-resolution microscopy, photoactivation, optogenetics, FRAP, DNA-PAINT and more
• Customization: Off-the-shelf and OEM configurations upon request
www.lumencor.com
www.microscopy-today.com • 2020 July sr
at the distal end of a 1.5 mm diameter fiber
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84
