Dr. Niles,

I would like to use your post in the "Microscopy 101" column of Microscopy
Today, may I?

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Thank you for your attention.

Phil
>
>I have measured fluorescence spectra of FM 1-43 in a variety of environments.
>In water (or .1 M salt, or .1 M salt + 1% BSA or .1 M salt + 0.5% C12E9),
>the emission spectrum is very broad with a ~symmetrical "peak" centered at
>648 nm.  Quantum yield is low.
>
>When the dye is allowed to partition to a polar-nonpolar interface, and the
>aqueous dye is either given very small room in which to maneuver (a
>hydrocarbon-water emulsion) or is removed (like washing externally labeled
>cells with dye-free buffer), the spectrum becomes polychromatic (indicating
>creation of the emission dipole by a change in rotation/vibration/excitation
>modes of the molecule, i.e., the 2 rings become coplanar with the styryl
>linker as the molecule lays at the interface) and the largest amplitude peak
>is at 593 nm (measured with 0.25 nm bandpass).  Quantum yields jump by a
>factor of 10-40 depending on the wavelength.
>
>So the upshot is that aqueous dye is redder than interfacial dye, and dye in
>vesicles is a mix of both.  However, I calculated based on the interfacial
>energy of FM 1-43 that ~98% of the dye in a 60 nm diameter endosome or
>synaptic vesicle is sitting at the membrane (it doesn't partition into the
>membrane).
>
>Walter Niles
>
>>Hello,
>>we are usinf the fluorescent dye FM1-43 to measure synaptic events. In
>>the catalog of Molecular Probes it is said that the emission maximum is
>>at 598 nm. Since I have the feeling that it is lower in my preperation,
>>I would like to know if anyone uses this fluorescent dye and has some
>>information for me.
>>
>>Thanks
>>[log in to unmask]
>>
>>
>Walter D. Niles, Ph.D.
>Department of Pharmacology (M/C 868)
>University of Illinois College of Medicine
>835 South Wolcott Avenue E-403 MSB
>Chicago, IL  60612
>
>telephone:  312-355-0236
>fax:            312-413-0222
>email         [log in to unmask]

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