How sensitive are the spectra of common probes in a cellular environment?

[John Oreopoulos]

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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

[mmodel]

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Hi John,

I suppose your question is not about quenching/dequenching (those change a lot) but about the shapes of spectral curves?

Mike Model

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of [hidden email]
Sent: Monday, October 17, 2016 3:40 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

[Craig Brideau]

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Relevant:

https://www.ncbi.nlm.nih.gov/pubmed/26175127

http://peripheralnerve.org/meeting/abstracts/2016/10.cgi

Not very common probes, but they do have significant (i.e. useful) degrees of chromatic shift. I have not seen any real study of this for common probes. I suspect antibody bound probes would be less sensitive due to the isolating nature of the antibody preventing direct interaction with the sample. Local pH could still have a very significant effect, however.

Craig

On Mon, Oct 17, 2016 at 1:46 PM, MODEL, MICHAEL <[hidden email]> wrote:

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Hi John,

I suppose your question is not about quenching/dequenching (those change a lot) but about the shapes of spectral curves?

Mike Model

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of [hidden email]
Sent: Monday, October 17, 2016 3:40 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 Michael,

I'm mainly concerned with spectral (wavelength) shifts or spectral  
shape changes as opposed to quenching (intensity). Thanks for asking  
for clarification.

John



Quoting "MODEL, MICHAEL" <[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.
> *****
>
> Hi John,
>
> I suppose your question is not about quenching/dequenching (those  
> change a lot) but about the shapes of spectral curves?
>
> Mike Model
>
> -----Original Message-----
> From: Confocal Microscopy List  
> [mailto:[hidden email]] On Behalf Of  
> [hidden email]
> Sent: Monday, October 17, 2016 3:40 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
>

[Michael Giacomelli]

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Hi John,

It is important to double check under which conditions online databases report spectra.  For instance, the Chroma and Invitrogen spectral viewer tools report DAPI bound to DNA with an emission maxima at ~460 nm, while the omlc spectraviewer reports it in water without DNA (maximum around 490 nm).  I've been fooled before by quickly googling an obscure fluorophore, picking the wrong filter, and then finding that my images don't look good.  

If in doubt, you can (usually) find something on Google Scholar with enough searching.  Failing that, a lot of imaging cores have spectrally resolved confocal detectors.  For a few obscure fluorophores, I've just gone and measured the spectrum under the conditions I'll be using.  This is time consuming but it is the safest option.  

Mike

 

On Mon, Oct 17, 2016 at 3:39 PM, <[hidden email]> wrote:

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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

[George McNamara]

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Hi John,

To celebrate molecular motors win in Chemistry (not fluorescent proteins or optical tricks for a change), I suggest you check out the Suhling paper below. Yes, if you can afford spectral FLIM, that would be a good way to go -- maybe SAR/Andor/Oxford Instruments next instrument? To celebrate Roger Tsien, may the product slogan could be "Time flies like an arrow; fruit flies like a banana". Bonus: slogan already has its own wikipedia page, https://en.wikipedia.org/wiki/Time_flies_like_an_arrow;_fruit_flies_like_a_banana

As Jason mentioned, Alexa Fluor dyes -- especially Alexa Fluor 488 vs fluorescein, were designed to be pH resistant and otherwise well behaved. While putting together the fluorophore table at

http://www.geomcnamara.com/fluorophore-table

I happened across the theoretical performance of fluorescein at different pH's (blanks for FH2 and FH3+ mean not fluorescent). Basically, below pH 8, fluorescein is not maximal. BCECF is purchased to measure pH deliberately.

For fixed cells, I encourage testing out expansion microscopy (http://expansionmicroscopy.org/). Once the fluorophore(s) and whatever it/they are attached to, are crosslinked to "the matrix" (https://en.wikipedia.org/wiki/The_Matrix) expand at will and image in whatever mounting medium is best. Ed Boyden explains how diapers led to ExM,

https://www.ted.com/talks/ed_boyden_baby_diapers_inspired_this_new_way_to_study_the_brain

J Vis Exp. 2012 Feb 9;(60). pii: 2925. doi: 10.3791/2925.

Fluorescence lifetime imaging of molecular rotors in living cells.

Suhling K¹, Levitt JA, Chung PH, Kuimova MK, Yahioglu G.

Abstract

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level. While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues. Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment. Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment. A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the Förster Hoffman equation. This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity. Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm. Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosityeffects. In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

PMID:

 

22348887

 

PMCID:

 

PMC3415204

 

DOI:

 

10.3791/2925

[PubMed - indexed for MEDLINE] 

Free PMC Article

On 10/17/2016 2:39 PM, [hidden email] wrote:

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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