McTips

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 

https://works.bepress.com/gmcnamara/84

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.

My advice:

* 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. 

0. assumptions:

      (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

local network

 

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!).

ipconfig all

 

 

 

 

January 15, 2019 (20190115U) new book and eBook:

Basic Confocal Microscopy second edition
https://link.springer.com/book/10.1007%2F978-3-319-97454-5
W. Gray (Jay) Jerome, Robert L. Price 2018

Chapter 1 includes:
Our Ten Commandments of confocal imaging are as follows.
1. The Perfect Microscope and the Perfect Microscopist Do Not Exist
2. Confocal Microscopy Is More Than a Confocal Microscope
3. During Specimen Processing the Integrity of the Specimen Must Be Maintained as Much as Possible
4. Photons Are Your Friends and Signal-to-Noise Ratio (SNR) Is King (GM note: and Queen and President and Premier ... and Dean, milliDean, microDean, nanoDean)
5. Quantification of Fluorescence in a Confocal Micrograph Is a Challenge and at Best Is Only Semiquantitative 
6. Scientific Digital Imaging and Normal Digital Imaging (Family Photography) Are Not the Same
7. Your Image Is Your Data: Garbage in Will Result in Garbage Out
8. The Resolution and Bit Depth Present in a Digital Image Are a One-Way Street
9. The JPEG (Joint Photographic Experts Group) Image File Format Is EVIL but Useful
10. Storage Media Is Essentially Free and Infinite
Notes:
* JHU staff can download eBook PDF for 'free' (that is, JHU has a subscription to publisher's content ... which comes out of NIH and other grants indirect overhead).
* Springer ofters MyCopy softcover edition $24.99 see "Buy" button at top right of https://link.springer.com/book/10.1007%2F978-3-319-97454-5

GM comments:

#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:

Reference: 

Pawley J (2000) The 39 steps: A cautionary tale of quantitative 3-D fluorescence microscopy. BioTechniques 28:884–888

https://www.future-science.com/doi/abs/10.2144/00285bt01 

 

Leica Microsystems - THUNDER Imager Tour at JHU SOM 3/3019

https://www.leica-microsystems.com/jhu-tour/ 

Leica - LIGHTNING and THUNDER

Leica THUNDER Tour - See Through the Haze with Computational Clearing
Johns Hopkins University, School of Medicine, Ross Fluorescence Imaging Center
* Summary: alternative to 'optical clearing' (which requires fixation and chemical clearing) for your fluorescent specimens.
* Demonstrations March 12-15, 2019. Two instruments:
   1. "Model organisms": Leica M205, 5x 0.5NA objective lens ... large field of view.
   2. "Live cells": Leica DMi8 inverted microscope, environmental controls (37 C, 5%CO2) available, full set of objective lenses.
* Seminar: THUNDER Imagers – Decode 3D Biology in Real-time.
RSVP please.
Computational Clearing – available exclusively on Leica Microsystems THUNDER Imagers – offers groundbreaking ease of use, throughput, speed and sensitivity for 3D tissue, live cell and model organism imaging. Unparalleled image quality from stereo, upright and inverted live cell microscopes, with no special sample preparation needed.

THUNDER Imager - how it works (pdf) ... two imaging systems platforms.

https://www.leica-microsystems.com/science-lab/thunder-technology-note/ 

LIGHTNING info (web link below - pdf download at bottom of that page) ... 'adaptive deconvolution' (GPU enabled).

https://www.leica-microsystems.com/science-lab/how-to-extract-image-information-by-adaptive-deconvolution

 
 
 
 
 

 

 

Equipment

MetaMorph Key 2 (#4646) (AxioImager Upright license)

July-August 2017: we are considering transitioning to keep this license to be kepy full time on the Zeiss AxioImager /...

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Li-Cor Odyssey CLx

Li-Cor Odyssey CLx fluorescence scanner. This instrument is hosted by Prof. Donowitz lab and use is intended for G.I. Center...

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Zeiss Axio Observer.A1 Inverted Microscope (S972)

Zeiss Axio Observer.A1 Inverted Microscope in Ross S972 has an Olympus DP80 RGB color + monochrome camera, capable of both RGB...

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MetaMorph Key 3 (#34334)

MetaMorph Key 3 is license #34334. Ross 9th floor users: If your preferred Windows PC does not have this license installed,...

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