McTips 2018 PDF (complete "20181217") go to: McTips 2018 download at https://works.bepress.com/gmcnamara/84
My McTips 2017 is available online at https://works.bepress.com/gmcnamara/81/
McTips 2017 Direct download is https://works.bepress.com/gmcnamara/81/download
McTips 2018 PDF (as of 201810002) go to: McTips 2018
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).
ThermoFisher Prolong Glass (without DAPI) is now the best choice, if imaging fixed specimens with oil immersion objective lens.
Prolong Glass info states needs to cure for 30+ hours.
* grow cells in imaging dishes (mattek or WPI-Inc ... or ibidi imaging quality coverglass chambers)
** at no time should cells be allowed to "air dry" = keep submerged.
* fix (i.e. formaldehyde), permeabilize if needed.
* immunofluorescence (i.e. http://www.nano-tag.com 2ndary nanobodies with each mouse mAb) ... can include DAPI and/or other counterstains here (example: fluorescent phalloidin).
* wash extensively (but quickly).
* "drip on" some Prolong Glass with imaging dish tilted, so that it forces aqueous media away ... pipet out the "run off", drop more (but not too much $) Prolong Glass ... goal is ~100% Prolong Glass, ~0% aqueous.
* allow to "cure" 30+ hours, in the dark, at room temperature, no lid, large volume of air (i.e. not small sealed box) to let volatiles escape. ... Probably simplest to go closer to 48 hours (and would be nice to be consistent in experiments).
20180803Fri ... connecting to our file server from Windows (win 7).
* ask George for the name of our file server - and please do not give out the name or IP address of our server.
* you are welcome to set up your own 'share drive to transfer your files (and can we please have 42 Terabytes of space on yours?).
Some Windows PC's are able to see our file server. Some are not. Today we -- "we" being 99% Jim Potter and 1% GM -- were able to trouble shoot the network acess issue.
(i) windows PC (win7 or ideally win10)
(ii) plugged into the JHU SOM network (Ethernet cable).
(iii) you have administrator privileges on the PC (does not need to be 'Administrator' login name).
1. Use JHARS to connect to JHU network (if you have not already done so) https://jhars.nts.jhu.edu/
1a. after setting up (or confirming) JHARS, probably useful to power off the PC, wait a few seconds, then power up and log in.
2. enable all the items in the Local Area Connection Properties dialog box (it is probably ok to enable more, but at minimum you need IPv6 and IPv4 and probably more).
3. using CMD prompt -> IPconfig / all (2nd screen shot below) ... see that DHCP Server 10p15.76.226
Windows Start menu ... cmd ... ipconfig / all
==> DHCP Server 10.15.176.226
==> Subnet mask 255.255.255.0 (if this is not correct, you may not be able to see JHU network at all!).
January 15, 2019 (20190115U) new book and eBook:
Basic Confocal Microscopy second edition
Chapter 1 includes:
#1. reminds me of my high school's National Honor Society slogan (which I'm paraphrasing here): "Those of you who think you're perfect, amuse those of us who are". (our NHS T-shirt had the original slogan and Mr. Wampole's face ... Mr. Wampolewas the advanced math teacher in addition to neing NHS chapter advisor).
#5. fluorescence intensity is 'at best only semiquantitative' ... this is sad & true
* (I blame it on 30+ years of researchers not requiring 'good intensity quantitation' of manufacturers AND manufacturers not making quantitation easy and reasonably priced).
* I suggest ACCM's Leica SP8 confocal microscope HyD detectors in photon counting mode enables users to come close to quantitation. There are still issues of laser performance (most lasers fluctuate in power), Z-drift and XY-drift (not a big deal for sngle focus plane, single field measurements), and specimen refractive index induced issues (if any mismatch in R.I., then Z affects intensity -- see Staudt and Hell "TDE" paper and Olympus silicone oil graph).
* Fast FLIM and - simpler and less expensive to get going and less data deluge - "fast photon counting" (FPC) can be implemented on any PMT or Hybrid or APD based point scanning confocal microscope. Re: Becker&Hickl fast FLIM or ISS FastFLIM" (and either would be less expensive to add to our FV3000RS confocal microscope than buying a new fully loaded Leica SP8 Falcon Fast FLIM ... bonus: Wolfgang Becker correctly disses Leica's featuring 'fast lifetime contrast' (FALCON) over fast TCSPC data).
Grey and Price 2018 Table 1.1:
Pawley J (2000) The 39 steps: A cautionary tale of quantitative 3-D fluorescence microscopy. BioTechniques 28:884–888