Re: How sensitive are the spectra of common probes in a cellular environment? - Vendor Reply

[Kilgore, Jason A.]

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**Vendor Reply**

For something like this, I would think you would need a spectral deconvolution imaging system, such as the Zeiss META or Leica SP confocal.

For Alexa Fluor dyes, at least, you likely wouldn't see any significant difference.  Nucleic acid stains have some precedent, though it is due to binding to different nucleic acids (Acridine orange is a common example), not due to simply being in a cytoplasmic environment.  Some dyes will have pH-dependent shifts (such as Oregon Green, Fluorescein derivatives, SNARF dyes or LysoSensor dyes), such as in acidic organelles like early or late endosomes, lysosomes, or autophagosomes.

Jason

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

Thermo Fisher Scientific
29851 Willow Creek Rd.
Eugene, OR  97402-9132
1-800-955-6288 then option 4, then option 6, then option 2.
Or dial direct at [phone]
[hidden email]
www.lifetechnologies.com

This communication is intended solely for the individual/entity to whom it is addressed. It may contain confidential or legally privileged information.  Any unauthorized disclosure or copying is prohibited and may be unlawful. If you have received this communication in error, please notify the sender immediately and delete it from your system.



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of [hidden email]
Sent: Monday, October 17, 2016 12:40 PM
To: [hidden email]
Subject: How sensitive are the spectra of common probes in a cellular environment?

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Dear confocal listserver,

I'm wondering if anyone out there can provide me with some information on what might be a simple question. I'd like to know how sensitive the spectra of common probes like Alexa dyes, fluorescent proteins, nuclear stains (DAPI), cyanine dyes, etc. are to the cellular environment. Ie: How different will the spectra of these probes be when attached to various biomolecules and present in different local cellular environments?

When I say "common probes" I'm excluding those fluorescent probes that are designed/used to detect/infer chemical/environmental changes via spectral shifts like calcium dyes, membrane potential dyes, or probes undergoing FRET (Fura, Di-8-ANEPPS, Laurdan, etc.).

I've tried searching through the literature databases, but I can't come up with any studies that have looked at this in any systematic way. My guess is that if there are spectral shifts associated with these common probes, they must be fairly small, otherwise it would be impossible to reliably look up the spectra of any of these probes online. If you know the answer to my question, can you also point me to any relevant literature/books that discuss this topic in detail?

Thank you as always,

John Oreopoulos

[John Oreopoulos]

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Yes Jason, you would need a spectral imaging system or a  
micro-spectroscopy instrument to determine this, neither of which I  
have access to, at least right now. I'm hoping someone on this  
listserver does have an instrument like that and has maybe  
investigated it.

Do you have any data for the probes you mentioned, or do you know of  
any publications that show some examples?

John

Quoting "Kilgore, Jason A." <[hidden email]>:

> *****
> 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.
> *****
>
> **Vendor Reply**
>
> For something like this, I would think you would need a spectral  
> deconvolution imaging system, such as the Zeiss META or Leica SP  
> confocal.
>
> For Alexa Fluor dyes, at least, you likely wouldn't see any  
> significant difference.  Nucleic acid stains have some precedent,  
> though it is due to binding to different nucleic acids (Acridine  
> orange is a common example), not due to simply being in a  
> cytoplasmic environment.  Some dyes will have pH-dependent shifts  
> (such as Oregon Green, Fluorescein derivatives, SNARF dyes or  
> LysoSensor dyes), such as in acidic organelles like early or late  
> endosomes, lysosomes, or autophagosomes.
>
> Jason
>
> Jason A. Kilgore
> Technical Application Scientist
> Molecular Probes / EVOS Tech Support
> Life Sciences Solutions
>
> Thermo Fisher Scientific
> 29851 Willow Creek Rd.
> Eugene, OR  97402-9132
> 1-800-955-6288 then option 4, then option 6, then option 2.
> Or dial direct at [phone]
> [hidden email]
> www.lifetechnologies.com
>
> This communication is intended solely for the individual/entity to  
> whom it is addressed. It may contain confidential or legally  
> privileged information.  Any unauthorized disclosure or copying is  
> prohibited and may be unlawful. If you have received this  
> communication in error, please notify the sender immediately and  
> delete it from your system.
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List  
> [mailto:[hidden email]] On Behalf Of  
> [hidden email]
> Sent: Monday, October 17, 2016 12:40 PM
> To: [hidden email]
> Subject: How sensitive are the spectra of common probes in a  
> cellular environment?
>
> *****
> 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 confocal listserver,
>
> I'm wondering if anyone out there can provide me with some  
> information on what might be a simple question. I'd like to know how  
>  sensitive the spectra of common probes like Alexa dyes, fluorescent  
>  proteins, nuclear stains (DAPI), cyanine dyes, etc. are to the  
> cellular environment. Ie: How different will the spectra of these  
> probes be when attached to various biomolecules and present in  
> different local cellular environments?
>
> When I say "common probes" I'm excluding those fluorescent probes  
> that are designed/used to detect/infer chemical/environmental  
> changes via spectral shifts like calcium dyes, membrane potential  
> dyes, or probes undergoing FRET (Fura, Di-8-ANEPPS, Laurdan, etc.).
>
> I've tried searching through the literature databases, but I can't  
> come up with any studies that have looked at this in any systematic  
> way. My guess is that if there are spectral shifts associated with  
> these common probes, they must be fairly small, otherwise it would  
> be impossible to reliably look up the spectra of any of these probes  
>  online. If you know the answer to my question, can you also point  
> me  to any relevant literature/books that discuss this topic in  
> detail?
>
> Thank you as always,
>
> John Oreopoulos
>