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.

See also:

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):

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/

Model WL-micro

Total Power (total spectrum) >200 mW

Visible power (400-750nM) >25 mW

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.