Paralens fluorescent attachment to detect DAPI and Hoechst?

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Paralens fluorescent attachment to detect DAPI and Hoechst?

Postby MicrobioMike » Jun 23 2017 4:13 pm

Hello all,

As a cheap alternative to a fluorescence microscope for my home lab, I am considering the Paralens Advance screw-on objective lens.

http://www.druckerdiagnostics.com/media/wysiwyg/paralens-advance/PLA_Manual_No_Spreads_.pdf:
(Please see page 9 of the PDF for an illustration of working spectrum)

The Paralens produces excitation at 385-480nm and emission at 480nm (as far as I can tell, please see PDF above!). Question: Will this work with Hoechst 33258 and DAPI stain (spectra as below)?

- Hoechst 33258: excitation: 352, emission: 461
- DAPI: excitation: 358, emission: 461

Thank you,

Mike
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Re: Paralens fluorescent attachment to detect DAPI and Hoech

Postby r.rosati » Jun 25 2017 9:41 am

Hi! From what I can see, I don't suppose it will work with DAPI considering the setup. That filter set is meant to be used with FITC (blue excitation, green emission) as opposed to DAPI (UV excitation, blue emission). It will also work with red-emitting fluorocromes, if their excitation range matches the setup.
It is true that the excitation filter would allow light in the 385-480nm range to pass, but the LED will only emit light at 410nm and above.
At page 1 (5th page of the PDF), the manual states, "The ParaLens Advance’s blue LED light source attaches to the filter set arm, emitting blue light with a 410-511 nm wavelength" (That would be "light output" on the figure at page 9).
Also, the dichroic mirror and emission filter setup are "in practice ok" (could be better) for emission wavelengths of 525-530nm and below, which again is OK for FITC, but it'll cut out any blue light the specimen might produce.

If you're looking for a DNA stain with excitation around 430-480nm and emission above 530nm, you could buy a DNA stain that works with blue light transilluminators. For example, you might be able to visualize SYBR Safe. Does the stain need to enter live cells?
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Re: Paralens fluorescent attachment to detect DAPI and Hoech

Postby r.rosati » Jun 25 2017 12:52 pm

You can try to reproduce "more or less" the filter setup on the Chroma website:
https://www.chroma.com/spectra-viewer?parts=14580,18221,17933
...noting that the setup is horrible, because some excitation light will bleed back. The objective's setup tries to circumvent this by using a LED light, that supposedly won't emit near the 500nm region.

Anyways, here is DAPI:
https://www.chroma.com/spectra-viewer?fluorochromes=10386&parts=14580,18221,17933#tabs-spectra_viewer_plot-left-2
you can see that DAPI's excitation spectra is far on the left of the blue LED light, and that its emission spectra is on the left too. So you would expect at most a "dim greenish glow" for DAPI.
And here is SYBR Green:
https://www.chroma.com/spectra-viewer?fluorochromes=10412&parts=14580,18221,17933
Not great either, but much better than DAPI.
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Re: Paralens fluorescent attachment to detect DAPI and Hoech

Postby MicrobioMike » Jun 25 2017 4:22 pm

Hi R. Rosati,

Thank you for your comprehensive reply and links! I learned quite a lot from your reply - and that's a neat tool at the Chroma website for visualising spectra. I understand now why the Paralens is not really suitable.

Ideally, I would like to be able to visualise live cells. Hence, I have purchased Hoechst 33258. I still don't have a microscope to use it with of course! I was considering buying a basic AmScope fluorescence microscope for around $2000, but that would use all the spare money I have and I am a little nervous about how good a quality the AmScope would be.

I posted the same question onto the DIYbio forum, and got the following response:
An alternative is to place your illuminating led and filter on one eyepiece of a binocular head. And a filter and camera on the other eyepiece. The led beam is semi focused on the specimen through the objective. Fluorescence light goes up the optical path and is divided equally into the eyepiece though only the camera sees the fluorescence image. Can send you an image of this setup if it helps.


I have a Zeiss Standard 25 binocular microscope. Do you know how I would go about implementing the above advice, and where / what components I could buy to make the modifications suggested?

Thanks again,

Mike
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Re: Paralens fluorescent attachment to detect DAPI and Hoech

Postby r.rosati » Jun 26 2017 3:21 pm

I don't know how the AmScope is, but I see it's very cheap. I don't know how well it would perform. Fluorescence microscopy is a bit of a sensitive technique. (On the other side, your Zeiss is a very nice piece of equipment).
I understand their idea, however I don't know if I can be very helpful in getting specific parts. They want you to use one objective as a light entry source for a UV LED, to illuminate the specimen; and you'd use the other objective to collect light back, which would include part of the UV light and any fluorescence. So you need a UV led light, at least a UV filter to block the UV coming back from the specimen, and a camera adapter.
The cheapest UV lamps I know of, are the handheld, battery-operated ones used to harden "UV-curing" glue. Not sure which wavelength they operate on, but they might be worth a try since they're about 10-15 dollars or so.
The cheapest UV filter is probably Plexyglas plastic. I know that there's a special place in hell for people who mention plastic in a thread about microscopes, but, oh well, I'm going there anyways. You could buy work glasses or a work shield that protects against UV and try it. Surely better using this setup with a camera though, instead of risking one's eyes.
The cheapest camera adaptor I know, is no camera adaptor and putting your phone's camera at the right distance from the objective so that the specimen is in focus. Not pretty, but it might work.

Best luck, protect your eyes, and don't hack too much your nice Zeiss microscope!
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