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Hi George
I'm puzzled as to why you suggest PMTs detectors are non-linear. With proper dynode chain design linearity exists over ~5 orders of magnitude brightness. If the filtering into the A/D converter is too fast for the sampling you could get aliasing effects but that's another story. Photon counting mode is more likely to be non-linear due to pulse pile up at high rates. The intrinsic linearity of PMTs was carefully explored a while ago as they formed the core of all photometry.
Regards Mark
On 10/11/19, 2:17 AM, "Confocal Microscopy List on behalf of George McNamara" <[log in to unmask] on behalf of [log in to unmask]> wrote:
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Hi Jeff,
Confocal Sweet Spot! I love your phrase!!!
I posted a visual at linkedin,
https://www.linkedin.com/posts/georgemcnamara_confocal-sweet-spot-jeff-reese-nih-activity-6588214058011410432-IBV7/
I suggest a slight simplification to one, easy to remember value: 0.666
Airy units.
GPU deconvolve all the data, please!!!
Best confocal file formats: OIR and LIF (Olympus FV3000RS and Leica SP8)
... of course I've retired two Zeiss LSM510's. Good to know that CZI
supports 60,000x60,000 pixels (another reply in thread).
PMT gain (HV = High Voltage): my Olympus rep, Jason Brenner, simplified
this for our FV3000RS: the optimal HV setting for our four GaAsP and two
GaAs PMTs is 500 (that is, 500 mV). Yes there are times I suggest
UNoptimal values (usually in steps of 50, but maybe I'll try 666 mV next
time I am at 0.666 Airy Units), but if these PMTs have an optimum,
likely everyone's has an optimum. Since the user has control over the
HV, and the HV controls the amplification of electrons in the PMT (and
ignoring a lot of PMT details), I'll just claim that very few readers
(or my) PMT based confocal microscope are really reporting a 2x change
in fluorophore for intensity level 1000 vs 2000 (after subtracting the
PMT offset floor from the raw data output). More on quantitation later.
PMT offset: depends on the detector, mode (FV3000RS galvo mode is 12-bit
range, resonant scan mode is 10-bit range), I usually teach with Hi-Lo
LUT enabled, "no blue, no red", while live, no averaging.
PMT multiplier: 1 ... and a request to confocal companies with this:
please kill this GUI element!
Better than PMT: Leica HyD in 'counting mode' counts photons. Yes, there
is some counting saturation limit (we have 2nd gen, ask your Leica rep
how much faster counting SMD HyDs are), and other companies have similar
Hybrid detectors, with their own tweaks (and benefits and price points),
I believe first with Hybrid was Becker&Hickl (FLIM, now fast FLIM), now
also ISS, PicoQuant, probably others.
Disclosure: I am registered for the free (fast) TCSPC workshop hosted by
Becker&Hickl and Boston Electronics, Oct 31-Nov 1, 2019, Bethesda MD.
I've also hosted a 2018 Leica FALCON demo, and various microscope demo's
and talks.
https://www.becker-hickl.com/events/13th-annual-workshop-on-advanced-tcspc-techniques-in-the-biomedical-science/
I agree with Jeff: fastest scan speed possible. In particular, Leica SP8
starts up with default of 400 Hz, I tell all users to use 600 Hz (or
faster if their objective lens choice and zoom <--> field of view is
appropriate).
Best way to make live cells brighter: drop EGFP ---- soooooo 1996 ---
Nathan Shaner's AausFP1 is 5x brighter and ultra narrow (for FPs)
emission peak (10x with tandem dimer)
https://www.biorxiv.org/content/10.1101/677344v2.full Plasmids not in
addgene yet (its a preprint), sequence in paper and supplemental info
(and user's might want to use their own codon and/or other expression
optimizations).
***
Confocal quantitation - some thoughts:
-- the lasers on confocal microscopes are not perfectly stable, and
often very unstable (could be the AOTF intensity controller more than
the laser modules - my recollection is Bob Zucker, US EPA, explained
this to me ... and no reason to think any of the confocal companies have
made any serious effort to prevent these fluctiations). So no reason to
think AM vs PM, or even a few minutes apart, stable (to say nothing of
corner to corner at low zoom).
-- I pointed out above there is no reason to think that anyone's PMT
based confocal microscope value 1000 vs 2000 is really a 2x change in
fluorophore concentration (per voxel etc). Or that similar values would
appear if the specimen was scanned AM vs PM (see previous paragraph).
So: most -- that is PMT point scanner - confocal data is UNquantitative
with respect to whatever intensity measurement the user thinks they are
measuring of (a major use of confocal) single cells.
Photon counting is better (some PMTs can be operated in photon counting,
APDs are, but often slow, various Hybrid detectors nice behavior,
trusting Hamamatsu and whoever does the electronics to 'get it right',
or at least not mess it up too badly), but still subject to laser/AOTF
intensity fluctuation issues.
enjoy,
George
p.s. one of the other posts in this thread mentioned Leica SP8 pinhole
value control is hard to find and by default is set to 580 nm: In Leica
LAS X the drop down is easy to find, the "Airy 1.0" is easy to find, the
button uses the midpoint of the emission range when a single detector is
enabled (ex. 510-550 nm range --> 530 nm mid-point). If two detectors
(we have two HyDs) are on, it uses the midpoitn of those. Easy to turn
off one detector, click "1" using the other, then turn on both (user
choice).
On 10/10/2019 2:46 PM, Reece, Jeff (NIH/NIDDK) [E] wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> For the pinhole, I tell people to start with 1AU, but to feel free to optimize for the question being answered: adjust smaller to get more resolution (when signal allows); or larger if SNR, speed, and photostability requirements can't be met with other adjustments...
>
> When there is plenty of signal (most fixed specimens), I recommend reducing pinhole for better resolution, with a practical limit of roughly 0.5AU. The sweet spot is usually 0.6-0.7AU.
>
> I stress to the users, not to report methods merely as "confocal", or mentioning the AU only, but rather report the theoretical optical section thickness achieved with the pinhole, which has more meaning for the users' experiments and the readers. It bothers me when I read a paper and all they say is "confocal".
>
> Zeiss formats: In years past, I found that Zeiss lsm imports better than czi into Fiji, and even better at reporting the settings in ZEN (Black), although both formats appear to 'ReUse' correctly. Based on other more recent reports, my feeling is that these problems with czi are less true of newer versions of ZEN and Fiji, with Zeiss focusing now much more on czi. We have mostly older Zeiss scopes, so lsm is still my default, but I recommend people try both formats and see what works better for them.
>
> Other things you didn't ask about, but I'll quickly mention:
> ...Find practical upper limit of PMT Gain by scanning with laser off and inspecting detector noise in the background, with contrast increased if it's important to preserve quantitation in dim pixels.
> ... Decide on a scan zoom and stick with it, so you don't have to deal with changing zoom later for figures.
> ... For nominally correct sampling in xy, use the 'Optimize' button to choose the # of pixels. Same for z-stack delta which is for sampling in z. These values can be changed somewhat, always exceptions, various reasons. Generally multiply the answer by 1.5-2x if performing decon.
> ...Use the fastest scan speed possible.
> ... Don't hit any of the fancier 'automatic' buttons like auto-exposure, settings wizard, or auto-focus, which are limited as to the conditions in which they work correctly. But thank you for trying, software engineers.
> ...If you lose your focus, remember where your pinhole setting is, then open it all the way to see and focus your sample again (then return pinhole setting).
>
> Cheers,
> Jeff
>
> -----Original Message-----
> From: Cammer, Michael <[log in to unmask]>
> Sent: Thursday, October 10, 2019 12:16 PM
> To: [log in to unmask]
> Subject: training and bets practices for confocal
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> I have been providing the following guideline in introductory confocal training which I thought were critical for data integrity. But other people have been disagreeing with all three points. Interested whether there is a consensus. Does anyone disagree with the guidelines below? Any comments welcome.
> Cheers-
> Michael
>
>
> General Confocal Best practices:
>
> * The pinhole is what makes the confocal a confocal. Set at 1AU (which means 1 Airy unit) and click the 1AU button each time you change lenses.
> If you are opening it for imaging fixed samples, you should go use a widefield fluorescence scope instead.
> Except in special case of live cell imaging where you understand that images are not confocal, this is NOT AN ACCEPTABLE WAY TO MAKE IMAGES BRIGHTER. You won't hurt the instrument, but when you write your methods, you won't be accurately describing your microscopy as "confocal".
> * Offset. Always use at 0 or 1.
> Other numbers are wrong.
> * Digital gain. The preset is 1. Leave it there.
> * Use the Range Indicator button to make sure you have no saturated pixels<http://microscopynotes.com/imagej/saturation/index.html>. If you see red pixels, you need to turn down the Gain or Laser.
> Saving Files
> All files should be stored in Drive D:.
> Files left on the desktop, drive C, Pictures folder, etc will be deleted.
> .lsm or .czi always.
> CZI is best. These files can be opened directly into image analysis software. These files retain instrument settings, channel integrity, bit depth, and spatial scale that may be necessary for image analysis.
> If you save files as TIFor other formats, the integrity of color channels may be lost and you will have no metadata regarding instrument settings and spatial scale.
> Move data to your lab's shared server space.
>
>
>
>
> Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY 10016
> Office: 646-501-0567 Cell: 914-309-3270 [log in to unmask]<mailto:[log in to unmask]>
> http://nyulmc.org/micros http://microscopynotes.com/
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