Confocal calibration & performance assesment

[F Javier Diez Guerra]

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Dear confocalists,

I would like to know about the preferred methods used by facility
managers to regularly asses the performance of their confocal (and
wide-field) microscopes. In other words, which are the most used and
reliable methods to check different features of confocal and wide-field
microscope peformance?.

Particularly, we'd like to check:

- illumination uniformity across FOV, now we use a slide coated with
fluorescent secondary antibody.

- XYZ Chromatic aberration (exciting 405, 488, 561 and 640 nm), now we
use 1um beads that can be excited with all laser lines.

- XYZ Resolution, we use smaller beads (0,17um) to build and analyze
PSFs (for each wavelength)

- Laser power and stability: for power, we use a power meter from
Newport placed at the objective exit, for stability, we capture long
time-series in reflexion mode with an empty preparation.

- We do not know how to check detector (PMT, GAsP, Hybrid, etc)
sensitivity and SNR.

Thanks for your input.


--
Fco. Javier Diez-Guerra, PhD

Profesor Titular UAM
Servicio de Microscopía Confocal
Centro de Biologia Molecular Severo Ochoa
C/ Nicolás Cabrera, 1
Campus de Cantoblanco
28049 Madrid
SPAIN

Tel     [phone]
e-mail: [hidden email]

[mcammer]

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The single biggest challenge to quality control is unassisted instrument use.  Yes, you need to check for all the issues in your list, but the single biggest problem is non-experts screwing around with settings and fundamentally misusing the instruments.  Most of our users have no idea whether the instrument is performing well or badly.  Fortunately, we use the instruments ourselves in assisted and training sessions so we catch problems, and there are expert users or at least observant ones who speak up when there are big problems (many users don’t recognize big problems or if they recognize them, don’t tell us).  But no matter how many times we explain the pinhole and optical path or say, "Even if you don’t understand what we're saying, follow the checklist and click on 1 Airy unit," someone disregards the note on the wall, website checklist, and spoken directions and opens the pinhole to max because they need more light.  

Brief answer:  preventative maintenance visits from vendors have consistently met targets for high quality imaging.  For more critical alignments, you need to step in.  Fluorescent Plexiglas and dishes of fluorescent dyes for uniform illumination; power meter with consistent method of measurement for laser power; beads in varying media for chromatic aberration; etc.  

Typically, after service calls instruments are within manufacturer's specifications but not up to yours.  One of our confocals has images from the 405 laser that at high magnification never precisely pixel aligned with the "visable" lasers, but within instrument spec.  I'm not happy with the images, but not a single users has ever noticed this.  An another scope, the PSF has always been slightly asymmetrically tilted, but, again, within manufacturers spec, and not a single users has noticed (more than seven years).  For reflection imaging, the 488 nm laser is clearly not stable, there are horizontal lines always, but it is within manufacturer spec and no user has ever noticed this as an issue with fluorescence.  Or chromatic aberration:  for some lenses I recommend offsets in Z for the 405 and 633 nm lasers based on testing with 0.1 um Tetraspeck beads, but to my knowledge no user has ever implemented them (although on the TIRF scope we have to or images are noticeably out of focus).  

What is users' biggest complaint?  The autosave feature doesn't work.  In training I tell them not to use autosave because it is buggy.  The written instructions say to avoid autosave.  But the window is there, they set it up, and then they cannot find their files.  Any time there is a software upgrade, it needs a thorough workout.  Add this to your list.

Enough screed for one morning...



Michael Cammer, Research Scientist, DART Microscopy Laboratory
NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016
C: 914-309-3270  [hidden email]    http://microscopynotes.com/ 
https://med.nyu.edu/research/research-resources/scientific-cores-shared-resources/microscopy-laboratory



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Fco. Javier Díez Guerra
Sent: Wednesday, August 30, 2017 8:10 AM
To: [hidden email]
Subject: Confocal calibration & performance assesment

*****
*****

Dear confocalists,

I would like to know about the preferred methods used by facility managers to regularly asses the performance of their confocal (and
wide-field) microscopes. In other words, which are the most used and reliable methods to check different features of confocal and wide-field microscope peformance?.

Particularly, we'd like to check:

- illumination uniformity across FOV, now we use a slide coated with fluorescent secondary antibody.

- XYZ Chromatic aberration (exciting 405, 488, 561 and 640 nm), now we use 1um beads that can be excited with all laser lines.

- XYZ Resolution, we use smaller beads (0,17um) to build and analyze PSFs (for each wavelength)

- Laser power and stability: for power, we use a power meter from Newport placed at the objective exit, for stability, we capture long time-series in reflexion mode with an empty preparation.

- We do not know how to check detector (PMT, GAsP, Hybrid, etc) sensitivity and SNR.

Thanks for your input.


--
Fco. Javier Diez-Guerra, PhD

Profesor Titular UAM
Servicio de Microscopía Confocal
Centro de Biologia Molecular Severo Ochoa C/ Nicolás Cabrera, 1 Campus de Cantoblanco
28049 Madrid
SPAIN

Tel     [phone]
e-mail: [hidden email]

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[Volodymyr Nechyporuk-Zloy]

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Dear Javier Diez-Guerra,

You can use Argolight slide: http://argolight.com/argo-hm/

Best regards,
Volodymyr

[lgelman]

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Dear Javier,

We have been using for years beads and the Argolight slide mentioned by Volodymyr, in combination with a powermeter.
With the beads we can estimate the resolution power of our system, but also detect issues with vibrating fans in cameras or Z-motor drives. We check only one wavelength, I think that's enough, but measure every week 3 beads for each objective.
The powermeter is obviously to measure light source power (we measure at the objective, re-using always the same conditions). We observe very important fluctuations with Metal Halide ligh, as expected, but were very surprised to see that lasers on our LSM were also not always stable over years. Some decrease fast and die, others loose over a week 25% power and then stay stable for a year, then loose again 25%, etc... We could foresee many breakdowns and organize laser replacements in time thanks to these measurements.
The Argolight slide is used here to measure the response of the PMTs or camera, to check illumination homogeneity, to assess precisely microscope z-motor drive and Z-piezo, and to align cameras. That's a tool that also our users use a lot themselves in combination with our powermeter whenever they need to check the system before an important (quantitative) experiment.

Very best regards,

Laurent.


Laurent Gelman
Facility for Advanced Imaging and Microscopy
Head Light Microscopy
Friedrich Miescher Institute for Biomedical Research
Maulbeerstrasse 66
4058 basel
Switzerland
[phone]





-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Fco. Javier Díez Guerra
Sent: Wednesday, August 30, 2017 14:10
To: [hidden email]
Subject: Confocal calibration & performance assesment

*****
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.
*****

Dear confocalists,

I would like to know about the preferred methods used by facility managers to regularly asses the performance of their confocal (and
wide-field) microscopes. In other words, which are the most used and reliable methods to check different features of confocal and wide-field microscope peformance?.

Particularly, we'd like to check:

- illumination uniformity across FOV, now we use a slide coated with fluorescent secondary antibody.

- XYZ Chromatic aberration (exciting 405, 488, 561 and 640 nm), now we use 1um beads that can be excited with all laser lines.

- XYZ Resolution, we use smaller beads (0,17um) to build and analyze PSFs (for each wavelength)

- Laser power and stability: for power, we use a power meter from Newport placed at the objective exit, for stability, we capture long time-series in reflexion mode with an empty preparation.

- We do not know how to check detector (PMT, GAsP, Hybrid, etc) sensitivity and SNR.

Thanks for your input.


--
Fco. Javier Diez-Guerra, PhD

Profesor Titular UAM
Servicio de Microscopía Confocal
Centro de Biologia Molecular Severo Ochoa C/ Nicolás Cabrera, 1 Campus de Cantoblanco
28049 Madrid
SPAIN

Tel     [phone]
e-mail: [hidden email]

[Cole, Richard W (HEALTH)]

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Javier Diez-Guerra


The light microscopy research group ( https://abrf.org/research-group/light-microscopy-research-group-lmrg) with in the ABRF (ABRF.org) has been working to develop performance testing procedures and achievable results (in conjunction with NIST).  These performance tests have been performed in labs worldwide.

Here are some relevant publications: sorry for any perceived self-promotion

Stack, R., Bayles, C., Girard, A., Martin, K., Opansky, C., Schulz, K., and Cole, R. (2011) Quality Assurance Testing for Modern Optical Imaging Systems. Microscopy & Microanalysis 17(4):598-606. DOI: 10.1017/S1431927611000237

Cole, R.W., Jinadasa, T., and Brown, C.M. (2011) Resolution and Quality Control of Confocal Microscopy Optics. Nature Prot. 6 (12): 1929-1941. doi:10.1038/nprot.2011.407

Cole,R., Thibault,M., Bayles,C., Eason,B., Girard,A., Jinadasa,T., Opansky,C., Schulz,K., and Brown, C. (2013) International Test Results for Objective Lens Quality, Resolution, Spectral Accuracy and Spectral Separation for Confocal Laser Scanning Microscopes (CLSM). Microscopy & Microanalysis doi:10.1017/S1431927613013470

Jonkman, J., Brown, C.L., and Cole, R.W. (2014) Quantitative Confocal Microscopy: Beyond a Pretty Picture. Methods Cell Biol. 123:113-34  doi: 10.1016/B978-0-12-420138-5.00007-0

Brown, C., Reilly, A., and Cole, R, W.  (2015) A Quantitative Measure of field Illumination. Vol 26:2 J. Biomol. Tech. doi: 10.7171/jbt.15-2602-001

BTW Mike points are "spot-on." if we could just eliminate the user ...

Cheers

Rich

Richard Cole
Research Scientist V
Director: Advanced Light Microscopy & Image Analysis Core
Wadsworth Center
 
Research Assistant Professor
Dept. of Biomedical Sciences
School of Public Health State University of New York

120 New Scotland Avenue, Albany N.Y. 12208
518-474-7048 Phone
518-408-1730 Fax

Website http://www.wadsworth.org/research/cores/alm
 twitter.com/microscopejock