Re: Confocal calibration & performance assesment **vendor response**

Posted by Kilgore, Jason A. on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Re-Confocal-calibration-performance-assesment-vendor-response-tp7587243.html

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** Vendor response **

Hi, Javier,

For a uniform fluorescence standard, I recommend the Fluor-Ref slides.  These are sold or re-sold by various places and are fairly inexpensive.  These are plastic slides that are autofluorescent in four different wavelengths and are incredibly uniform and photostable.  The drawbacks are that they are extremely bright, so much so that you often either have to throw in neutral density filters or image an off-wavelength slide.  Also, they are thicker than a standard slide.  Here's the webpage from Ted Pella:  http://www.tedpella.com/histo_html/fluor.htm.aspx or resold as a set of four from Fisher Scientific: https://www.fishersci.com/shop/products/NC0158348/nc0158348#?keyword=FLUOR-REF+SET+OF+4+SLIDES 

There are other published methods for uniformity slides (such as a simple solution of fluorescein under a coverslip, or putting dye in agar) or labeling coated slides, but nothing comes close to the Fluor-Ref slides for photostability, storage stability, and uniformity of intensity.

There is a good and fairly inexpensive option for some of the other needs you mention:  FocalCheck microspheres slides.  There are different slides, but probably Slide #1 is best for your needs, catalog F36909.  The top row has options where you align rings of different colors to check alignment, or rings and cores of the microspheres. There are also TetraSpeck beads of three different sizes (4, 1, and 0.5 um) that are broadly fluorescent, which you can use for PSF as well as alignment.  The bottom row has 6um beads of five different relative intensities, for doing intensity calibrations.  
Webpage from Thermo Fisher Scientific:  https://www.thermofisher.com/order/catalog/product/F36909
Manual: https://assets.thermofisher.com/TFS-Assets/LSG/manuals/mp36909.pdf 

We sell fluorescent beads of smaller sizes, too, if you need them (such as 20 nm) of different wavelengths, for PSF measures.

I hope this helps, but please feel free to contact me offline if you wish more info (such as a PowerPoint I have showing images from that last slide).

Jason


Jason A. Kilgore
Technical Application Scientist
Molecular Probes / EVOS Tech Support
Thermo Fisher Scientific
 
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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 5:10 AM
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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