Re: Fluorophore(s) to make a "reference solution" **vendor response**

[Kilgore, Jason A.]

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**Vendor Response**

Hi Emmanuel,

For 488 nm excitation, fluorescein is fine (catalog F1300 from Thermo Fisher Scientific).  (as Michael suggests)

For 561 nm excitation, sulforhodamine 101 should be fine (catalog S359).  I haven't used Rose Bengal, myself.

For 400 nm excitation, I would suggest a Cascade Blue derivative, such as Cascade Blue hydrazide (catalog C687)

They should be able to be combined, though I don't know if there may be FRET effects.

Cheers,

Jason

Jason A. Kilgore
Technical Application Scientist
Molecular Probes / EVOS Tech Support
Life Sciences Solutions

Thermo Fisher Scientific
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Eugene, OR  97402-9132
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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of MODEL, MICHAEL
Sent: Saturday, January 28, 2017 5:31 AM
To: [hidden email]
Subject: Re: Fluorophore(s) to make a "reference solution"

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Hi Emmanuel,


We have done some work on it a while ago. If you use a very concentrated solution of pretty much any fluorophore, its fluorescence would not depend on the depth of the liquid (because light penetrates only into a very small depth) and they practically don't bleach for the same reason. We used Na fluorescein in water for green fluorescence, Rose Bengal for red (it is bright in water and still brighter in DMSO) and acid blue 9 for far red.


Model MA. 2014 (updated from 2002, 2006). Intensity calibration and shading correction for fluorescence microscopes. Curr Protocols in Cytometry. Unit 10-14.

Model MA, Blank JL. 2006. Intensity calibration of a laser scanning confocal microscope based on concentrated dyes. Analyt Quant Cytol Histol. 28:253-261.

Model MA, Burkhardt JK. 2001. A standard for shading correction and calibration in fluorescence microscopy.  Cytometry. 44:309-316.


Mike



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From: Confocal Microscopy List <[hidden email]> on behalf of Emmanuel Levy <[hidden email]>
Sent: Saturday, January 28, 2017 7:48 AM
To: [hidden email]
Subject: Fluorophore(s) to make a "reference solution"

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Hi,

We want to make a "reference solution" that will be imaged each time the microscope is being used. The purpose is two fold:
- make different experiments comparable in terms of absolute intensities
- monitor laser intensity stability over weeks

We work with multiwell glass bottom plates so a solution would be easy to use (e.g., we can spike it in an empty well)

We're thus looking for 3 fluorophores (for the 400/488/561 lasers) that can be mixed together and are stable in the same buffer.

Importantly, they should be stable at room temperature so we could leave the solution next to the microscope for everyday use. Also, water is preferable over organic solvents for compatibility with hardware autofocus.

If you have suggestions we'd very much like to hear them.

Thank you,

Emmanuel