Barrier filter for autofluorescence

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Andy Davies
Posts: 132
Joined: Tue Dec 09, 2014 3:25 pm

Barrier filter for autofluorescence

Post by Andy Davies »

I have been using a "conventional" Tiffen Yellow #12 as a barrier for autofluorescence but was reading on Bioquest's website of a "wide spectrum" barrier filter which allows reds to pass as well.

The #12 is designed to block everything below 500nm and above 560nm creating a bandpass that increases the contrast in B&W film and hence “blocks” red.

Any ideas as to what this wide spectrum barrier filter might be and where it can be sourced? I have messaged Starshade (Daniel Stoupin) who used to post on this forum but haven't had a response as of yet.

ChrisR
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Post by ChrisR »

What colour autofluorescence? Scorpions are blue, chlorophyll is red...?
Chris R

pbraub
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Joined: Fri Feb 02, 2018 1:23 pm

Post by pbraub »

I would rather ask: what color/wavelength for excitation do you want to use?
If you want UV excitation @365nm for example you could choose an appropriate longpass filter (400 nm LP for example) . Then you can detect all the higher wavelengths (all the colors).

Depending on the quality of your lightsource you might want to consider an excitation bandpass filter to clean your excitaiton light.

For UV autofluorescence I use a DAPI longpass filterset (like this one: https://www.chroma.com/products/sets/19 ... i-longpass)


The new interference filters can be bought from the usual suspects (chroma, thorlabs, semrock, edmund) and are quite expensive. But if you are patient some might come up on ebay.

https://www.chroma.com/products/single- ... %201080%5D

https://www.thorlabs.de/newgrouppage9.c ... oup_id=918

https://www.semrock.com/filtersRefined. ... =0&recs=10

Pau
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Location: Valencia, Spain

Post by Pau »

Are you referring to macro (or low magnification micro) with flashlights or to microscope with epifluorescence?

pbraub refers to microscope epifluorescence while Starshade breathtaking images are "macro"

If you use a UV LED flashlight approach for macro, an emission filter like UG1 or ZWB2 mounted in the flashlight is useful. For emission you need a longpass filter to cut UV and violet, a very light yellow like 420nm LP will be adequate.

A good source for surplus interference filters at low prices is Omega ebay store.

As usual, more info can lead to get better advice...
Pau

pbraub
Posts: 79
Joined: Fri Feb 02, 2018 1:23 pm

Post by pbraub »

@Pau
I was indeed referring to epifluorescence. Wouldnt you use a similar setup for "macro": Excitation filter on the UV Flashlight (for example) to cut any wavelengths higher than necessary and a longpass barrier filter. At least this would have been my approach.

Andy Davies
Posts: 132
Joined: Tue Dec 09, 2014 3:25 pm

Post by Andy Davies »

I'm using a Royal Blue 450nm LED source on the micoscope and Zeiss barrier filters at the moment.

For macro, I am also using a Royal Blue 450nm LED in the studio and a Tiffen #12 barrier filter.

In the field, I am using a white video light and dichroic excitation filter to generate Royal Blue 450nm.

Subjects studied so far have contained chlorophyll.

I am searching for what Bioquest studios refer to as a "wide spectrum" barrier filter. Look here under the section The Science of Coloours" http://bioqueststudios.com.au/gallery/

pbraub
Posts: 79
Joined: Fri Feb 02, 2018 1:23 pm

Post by pbraub »

Well, I'd guess a longpass with appropriate cut-on frequency (it is the widest spectrum you can get in terms of emission).

However they write on their hompage "... We employ a technique that we call “wide-spectrum fluorescence”..." and I am not 100% sure that they speak only about the barrier / emission filter.

This could also mean that they use several wavelengths for excitation with - for example a multibandpass exciter with UV, blue and green excitation and an appropriate emission barrier filter. This could also be compatible with the term "wide-spectrum fluorescence".

As long as they are not willing to share information this will be at best difficult to reproduce.

P

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