McTips 2018 - 20181217 George McNamara, Ross Fluorescence Imaging Center - light microscopy.pdfMcTips (2018)
McTips 2018 is my 100+ page pdf of interesting light microscopy stuff I wanted to make my colleagues at JHU/JHMI and the imaging community aware of. Mostly information from the literature (ex. PubMed abstracts of cool publications) with some ofmy thoughts bullet pointed in.
McTips 2017 at https://works.bepress.com/gmcnamara/81
MpMicro at https://works.bepress.com/gmcnamara/2 = Multi-Probe Microsopy
==> Fast FLIM simplified for now to "Fast Photon Counting" (FPC):
The McTips 2018 includes some of my thoughts with respect to Fast Photon Counting (FPC) to make fluorescence confocal microscopy both faster and more quantitative than is now practiced by most biomedical researchers (i.e. twiddle the HV gain and offset values until someone proves their boss' hypothesis ... especially when they are using 'Santa Crap' antibodies and don't bother with controls).
FLIM is Fluorescence Lifetime Imaging Microscopy.
Fast FLIM is fast TCSPC FLIM (TCSPC = Time correlated Single Photon Counting, timing is related to excitation pulse for multiphoton laser, or 'appropriate pulsed' visible lasers availablefrom several manufacturers ... such as White light Lasers = Super-Continuum lasers, from Fianium/NKT Photonics, https://www.nktphotonics.com/lasers-fibers/product/fianium-whitelase-micro/ ).
Multi-photon excitation power: typically 80 MHz Ti:Sapphire laser, such as Coherent Laser Chameleon Ultra II or Spectra-Physics MaiTai DeepSee ... 80 MHz is every 12.5 nanoseconds, pulse duration is typically 100 femtoseconds = 0.1 picoseconds = 0.0001 nanoseconds, so a 4 Watt average power laser is 4 / ~0.000008 = 4 * 125,000 = 500,000 W when on). Upshot is that an MP laser works nicely (albeit is expensive).
The lowest price Fianium super-continuum lasers are pulsed and acts as a "white light laser" -- see https://www.nktphotonics.com/lasers-fibers/product/fianium-whitelase-micro/
- Collimated output
- Cut-off wavelength up to 2500 nm
- Cut-in wavelength at 400 nm
Total Power (total spectrum) >200 mW
Visible power (400-750nM) >25 mW
Average Spectral Power Density >100μW/nm
This "WLL" laser would typically use interference filter(s) to select emission wavelength (for speed, could use an AOTF, faster changing, more expensive). If 10 nm excitation filter, then ~1 mW "in band", which would be focused to a point on the specimen by the confocal microscope optics. For a modest investment, a single 12 position excitation wheel could cover the 'useful' visible to near-infrared range, i.e. 400-800 nm, very nicely with single bandpass filters (there might also be benefit to having some multi-band exciter filters).
"Fast FLIM" data deluge: ~125 datapoints per voxel, (12.5 nanoseconds / 100 picosecond bins), 16-bit accumulation, so a 1 million pixel (1000x1000 pixels) single plane confocal slice would be 1 million * 125 * 2 bytes (16-bit) = 250 megabytes per image plane ... a "typical" 40 plane Z-series would be 10 Gigabytes (single time point!).
FPC: 1 data point per voxel --> much more manageable dataset, especially if live cells Z-series * time series.
see McTips 2018 for more information. I submitted sa proposal to Chan-Zuckerberg Initiative CZI imaging scientist program) in 10/2018, asking for money. Because parts of the budget are interspersed in the narrative, I am uncomfortable sharing the proposal.I hope my FPC section of McTips 2018 and summary above isuseful to you. Long term I see FastFLIM as being useful, but with 2018 PC's and networks (and price of local and/or "The Cloud" data storage) -- and most confocal microscope users barely understand confocal microscopy, so FLIM, TCSPC, etc are "several acronyms too far" -- I think FPC is a "sweet spot" for making confocal microscopy better. Writing at end of 2018, I assume the CZI proposal will not be funded (haven't heard from them, start date is Feb 1, 2019). If you would like to help, please consider donating money (philanthropy) and/or equipment (vendors) to "make FPC happen" here.
- light microscopy,
- confocal microscopy,
- tyramide signal amplification,
- super-resolution microscopy
Publication DateWinter December 19, 2018
Citation InformationMcNamara G 2018 McTips 2018. Johns Hopkins University - Ross Fluorescence Imaging Center
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