Fluorescent dye with known diffusion coefficient, excited by 594nm (FCS)

[Krzysztof Berniak]

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Dear List,


I am looking for a fluorescent dye with a known diffusion coefficient,
which can be excited by laser line 594nm. I need it to calibrate and
determine a confocal volume in Fluorescence Correlation Spectroscopy (FCS)
measurement where I use mCherry fluorescent protein (unfortunately, I do
not have access to 561nm line, which is also good for mCherry).

Fluorescent dyes such as Atto 590, Atto 594 or Atto Rho 13 look nice, but I
have not found their diffusion coefficients in literature.

Any help or suggestions would be greatly appreciated!


Regards,

Krzysztof Berniak

Cell Biophysics

Jagiellonian University, Krakow

[mmodel]

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See https://outlook.office.com/owa/?realm=kent.edu&exsvurl=1


Diffusion coefficient weakly depends on molecular mass


Mike




________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Krzysztof Berniak <[hidden email]>
Sent: Tuesday, July 4, 2017 4:18 AM
To: [hidden email]
Subject: Fluorescent dye with known diffusion coefficient, excited by 594nm (FCS)

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Dear List,


I am looking for a fluorescent dye with a known diffusion coefficient,
which can be excited by laser line 594nm. I need it to calibrate and
determine a confocal volume in Fluorescence Correlation Spectroscopy (FCS)
measurement where I use mCherry fluorescent protein (unfortunately, I do
not have access to 561nm line, which is also good for mCherry).

Fluorescent dyes such as Atto 590, Atto 594 or Atto Rho 13 look nice, but I
have not found their diffusion coefficients in literature.

Any help or suggestions would be greatly appreciated!


Regards,

Krzysztof Berniak

Cell Biophysics

Jagiellonian University, Krakow

[Suvrajit Saha]

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Hi Krzysztof,

This list might be helpful.

https://www.picoquant.com/images/uploads/page/files/7353/appnote_diffusioncoefficients.pdf

Best,
Suvrajit


On 4 July 2017 at 01:18, Krzysztof Berniak <[hidden email]>
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.
> *****
>
> Dear List,
>
>
> I am looking for a fluorescent dye with a known diffusion coefficient,
> which can be excited by laser line 594nm. I need it to calibrate and
> determine a confocal volume in Fluorescence Correlation Spectroscopy (FCS)
> measurement where I use mCherry fluorescent protein (unfortunately, I do
> not have access to 561nm line, which is also good for mCherry).
>
> Fluorescent dyes such as Atto 590, Atto 594 or Atto Rho 13 look nice, but I
> have not found their diffusion coefficients in literature.
>
> Any help or suggestions would be greatly appreciated!
>
>
> Regards,
>
> Krzysztof Berniak
>
> Cell Biophysics
>
> Jagiellonian University, Krakow
>

--
Suvrajit Saha
Postdoctoral Scholar
Prof. Orion Weiner Lab
University of California, San Francisco ( UCSF)
Cardiovascular Research Institute (CVRI)
Smith Cardiovascular Research Building, Room 384, MC 3120
555 Mission Bay Blvd. South
San Francisco, CA 94158
<E-mail%[hidden email]>

[Romin, Yevgeniy/Sloan Kettering Institute]

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Hi Krzysztof

We use TRITC, which you should be able to get decent enough signal out with 594. From what I know, its diffusion coefficient should be similar to rhodamine, which is published

> On Jul 4, 2017, at 4:20 AM, Krzysztof Berniak <[hidden email]> 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.
> *****
>
> Dear List,
>
>
> I am looking for a fluorescent dye with a known diffusion coefficient,
> which can be excited by laser line 594nm. I need it to calibrate and
> determine a confocal volume in Fluorescence Correlation Spectroscopy (FCS)
> measurement where I use mCherry fluorescent protein (unfortunately, I do
> not have access to 561nm line, which is also good for mCherry).
>
> Fluorescent dyes such as Atto 590, Atto 594 or Atto Rho 13 look nice, but I
> have not found their diffusion coefficients in literature.
>
> Any help or suggestions would be greatly appreciated!
>
>
> Regards,
>
> Krzysztof Berniak
>
> Cell Biophysics
>
> Jagiellonian University, Krakow

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[Neil Anthony]

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Hi Jagiellonian,

One thing to think about is that whatever values you get, they might be 'off' the hardcore exact value, but as long as they are consistently 'off' that's the main point, and of course it's important that it's not too far off.  Molecular weight is one of the main factors, but it's not always that straight forward.  See one of the slides in a lab meeting prezi here from a while back: https://prezi.com/yt5gpx7jewbf/diffusion-and-taufcs/  Rhodamine 6G and Rhodamine B have exactly the same MW but different charged groups, and it can change the diffusion by a significant factor.

If you have access to control the scan patterns, I love this self-calibrating technique that scans the volume in a circle:  http://www.sciencedirect.com/science/article/pii/S000634950870660X  You do need to calibrate the radius, but that only needs to be done once.  You can sufficiently calibrate the radii with a dye solution and camera if you can't get a grid.

Neil



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Krzysztof Berniak
Sent: Tuesday, July 04, 2017 4:18 AM
To: [hidden email]
Subject: Fluorescent dye with known diffusion coefficient, excited by 594nm (FCS)

*****
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 List,


I am looking for a fluorescent dye with a known diffusion coefficient, which can be excited by laser line 594nm. I need it to calibrate and determine a confocal volume in Fluorescence Correlation Spectroscopy (FCS) measurement where I use mCherry fluorescent protein (unfortunately, I do not have access to 561nm line, which is also good for mCherry).

Fluorescent dyes such as Atto 590, Atto 594 or Atto Rho 13 look nice, but I have not found their diffusion coefficients in literature.

Any help or suggestions would be greatly appreciated!


Regards,

Krzysztof Berniak

Cell Biophysics

Jagiellonian University, Krakow

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