Posted by
George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Two-photon-microscope-questions-tp7583010p7583029.html
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Hi Aro,
Intensity 2200 of feature of interest vs 2000 of background. Camera
maximum 65535. Not enough information. Also, N=1, so more cells, and
proper controls will help. If you increase exposure time 10x you might
get values of ~22,000 vs 20,000, which would help. Personally, I would:
1. acquire more data, including no primary antibody controls.
2. acquire Z-series and deconvolve (whether widefield or confocal ).
3. acquire other fluorescence channel(s) to understand how
autofluorescent your cells and/or tissue are. I routinely acquire at
least one channel that is not supposed to have any fluorophores in it
(440nm or 458 nm excitation and "Cyan FP" emission band works well for
many kinds of cellular autofluorescence).
4. If it is a protein antigen that is supposed to be synthesized by the
cells you are detecting the antigen in or on, corroborate with RNA FISH
(more on this later).
The imaging hardware we have is able to discriminate single molecules
even in brightfield - see
http://web.stanford.edu/group/blocklab/GutierrezAJP2010.pdf(a reference I am recycling from the recent single microtubules thread).
On a clean background, a few counts above the camera offset can be
enough. In or on cells that have autofluorescence, this is much harder.
For increasing signal over background -- when using 'clean' antibodies
-- I recommend tyramide signal amplification (TSA). This is commercially
available from both Molecular Probes/Invitrogen/LifeTech/ThermoFisher
and from PerkinElmer. Perkin Elmer is making a push with TSA for 3plex
and 6plex immunofluorescence,
http://www.perkinelmer.com/catalog/category/id/opal-multiplex-tissue-stainingespecially in conjunction with their Vectra spectral imager. Good ideas
though I recommend PeroxAbolish instead of microwaves to kill
horseradish peroxidase - my former UMiami colleagues, Dr. Takahashi and
Prof. Ichii, had outstanding CD4 and CD8 TSA immunofluorescence in their
work published in
http://www.ncbi.nlm.nih.gov/pubmed/21929847(the published images do not do justice to how bright and specific their
staining was).
There is a famous quote by Arthur Kornberg, "Thou shalt Not waste clean
thinking on dirty enzymes"
http://sandwalk.blogspot.com/2007/10/biochemist-arthur-kornberg-1918-2007.htmlTo which I'll add: "do not waste time or money on stupid antibodies", so
if you are using polyclonal antibodies (not reproducible, the rabbit is
probably dead already), reagents from companies like 'Santa Crap' that
don't do quality control, and especially companies that the USDA has
cited for violating the US animal welfare act,
https://awionline.org/sites/default/files/uploads/documents/SanatCruz-1-15-0023COMPLAINTRedacted.pdfThe first two papers in what will be a series were recently published in
Journal of Histochemistry and Cytochemistry, are open access, and
provide good advice:
http://jhc.sagepub.com/content/62/10.toc * Denis G. Baskin and
* Stephen M. Hewitt
Improving the State of the Science of Immunohistochemistry: The
Histochemical Society’s Standards of Practice
J Histochem Cytochem October 2014 62: 691-692 doi:10.1369/0022155414538453
* Free Full Text <
http://jhc.sagepub.com/content/62/10/691.full>
* Free Full (PDF)
<
http://jhc.sagepub.com/content/62/10/691.full.pdf+html>
* Stephen M. Hewitt,
* Denis G. Baskin,
* Charles W. Frevert,
* William L. Stahl,
* and Eduardo Rosa-Molinar
Controls for Immunohistochemistry: The Histochemical Society’s
Standards of Practice for Validation of Immunohistochemical Assays
J Histochem Cytochem October 2014 62: 693-697 doi:10.1369/0022155414545224
* Abstract <
http://jhc.sagepub.com/content/62/10/693.abstract>
* Free Full Text <
http://jhc.sagepub.com/content/62/10/693.full>
* Free Full (PDF)
<
http://jhc.sagepub.com/content/62/10/693.full.pdf+html>
For protein antigens that you expect are being made in the same cells
that you are measuring them in, the 'Central Dogma' of DNA codes for
mRNA codes for proteins, means that you could use:
1) gene knockout (more definitive) or RNAi (less definitive, but still
pretty good) to eliminate the gene or expression. ZFNs, TALENs and
CRISPR/Cas9 all work. Humin Zhao has published TALENs at $5 each
http://www.ncbi.nlm.nih.gov/pubmed/24237314 -- which is cost competitive
with the guide RNAs needed for Cas9. CRISPR Cas9 is now over 300
references for just 2014 (with one month left), for a tool only
published in August 2012 (
http://www.ncbi.nlm.nih.gov/pubmed/22745249)
-- this decade's answer to previous revolutions in PCR and RNAi. These
also intersect with light microscopy as in, for entry points,
http://www.ncbi.nlm.nih.gov/pubmed/25307933http://www.ncbi.nlm.nih.gov/pubmed/24556431http://works.bepress.com/gmcnamara/63http://works.bepress.com/gmcnamara/422. RNA detection
a. in live cells by molecular beacons, or SmartFlares (commercial name
from EMD Millipore) = NanoFlares (academic name,
http://www.ncbi.nlm.nih.gov/pubmed/25404304 and
http://www.ncbi.nlm.nih.gov/pubmed/18034495).
b. single molecule RNA FISH, for examples:
Stellaris FISH ...
http://stellarisfish.smugmug.com ... one of the
galleries is a bit of a tease with respect to new hardware,
http://stellarisfish.smugmug.com/StellarVision/RNAscope
QuantiGene
http://www.panomics.com/products/gene-expression/single-plex-assay/overviewRNAscope and QuantiGene are the same branched DNA technology, from two
different vendors.
I've been doing Stellaris FISH experiments for the past several months,
and posted some of the data online, for example
http://works.bepress.com/gmcnamara/64/George
p.s. All of Kornberg's commandments at/
http://sandwalk.blogspot.com/2007/10/biochemist-arthur-kornberg-1918-2007.html/Biochemists also know him for creating the Ten Commandments of
Enzymology. Unlike the author of the original ten commandments, Kornberg
was able to modify and amend his commandments as new developments came
along (Kornberg, 2003).
/Thou shalt…
* I. Rely on enzymology to resolve and reconstitute biologic events
* II. Trust the universality of biochemistry and the power of
microbiology
* III. Not believe something just because you can explain it
* IV. Not waste clean thinking on dirty enzymes
* V. Not waste clean enzymes on dirty substrates
* VI. Use genetics and genomics
* VII. Be aware that cells are molecularly crowded
* VIII. Depend on viruses to open windows
* IX. Remain mindful of the power of radioactive tracers
* X. Employ enzymes as unique reagents
/
My email signature line used to be:
"Old Soldier's never die, they just fade away" - Douglas Macarthur.
"Old antibodies die, please throw them away" - GM.
/
/
On 11/30/2014 11:55 AM, PengKe wrote:
> Dear lister:
> I'm recently puzzled by a question that I felt might have puzzled some of you as well and I would like to receive some suggestions and opinions from you.
> Question: In immunofluorescence analysis, sometimes we detected objects that showed very weak signals, so how can we decide whether the signal is really there or not?
> To make the question a little more specific, I will give some artificial numbers. Let's say the background has the signal intensity of 2000 and the potential object showed a signal intensity of 2200. The saturating intensity of the camera is 65535. In this case can one claim the 2200 signal represent a real object?
> A related question might be: is there a golden standard about how much higher a signal intensity needs to be above the background to be defined as an object?
> I'm looking forward and I'll be very grateful to your opinions.
> Best wishes,
> Aro
--
George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales
http://works.bepress.com/gmcnamara/42