Which filter for pH fluorescence?

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Hss's picture
Which filter for pH fluorescence?


I would like to perform some fluorescent experiments with pH indicators for the first time and it will be nice if someone can tell me wich filter do I need for it. In the Lab there is one setup for calcium imaging and I want to use it for pH fluorescence too. I understand that osme differents filters are needed, so I´m trying to know wich filter I need (or wich filter combination).


The FFM's picture
 Look in the product details

 Look in the product details data sheet or on the web site for the company from which your are purchasing the pH fluorescent dye what are the excitation and emmission maxima for the dye.

You will then be able to determine which filter set that you will need to use

if you go to Chroma's web site http://www.chroma.com/products/fluorochrome

you can search by fluorophore or filter sets to determine the appropriate dye/filter combinations

Bluejay's picture
The traditional pH dye is

The traditional pH dye is BCECF-AM.  A dual excitation at 490 and 440nM with emission at 535 is suggested by the vendor, Invitrogen, formerly Molecular probes.   For single excitation use 490nM excitation. (See http://probes.invitrogen.com/media/pis/mp01150.pdf for the manual.)  Although I have used calcium dyes extensively, I've not had much luck with pH dyes.  The flurescent probes handbook from Invitrogen is also useful for general background and ideas.  The manual suggests that they BCECF is not very sensitive to small pH changes, plus the excitation and emission wavelengths are very close/overlap.  See also free, on the Invitrogen website, Molecular Probes Handbook chapter 20, entitled "pH indicators."  The SNARF dyes seem to be more recent.  Again, the filters used would depend on the dye characteristics.

Hss's picture
 ...umm, I´m not sure how it

 ...umm, I´m not sure how it works

It means that, when the dye is BCECF-AM, in the filter holder should be one filter for the 490nm excitation and one for the 440nm excitation, and a third one for the 535 emission?  

ps. I saw this web in wich the filters seem to be less expensive, or is just only ma impression; what do you think about it?: www.thorlabs.de/NewGroupPage9.cfm

Hss's picture

...so, you try already with pH dyes; do you know wich filters have the set up for your dyes?