Re: Confocal calibration & performance assesment

Posted by lgelman on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Confocal-calibration-performance-assesment-tp7587224p7587234.html

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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
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-----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

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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

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