Documentation for fluorescence microscopy

[rwb27]

There are bits of code around for adding a fluorescence filter cube for epifluorescence imaging. However, this is not documented in the assembly instructions (because it's probably too confusing). It would be good to document the fluorescence stuff.

[rwb27]

The filters I used were from Comar Optics, I used one long-pass, one short-pass, and one long-pass at 45 degrees in the standard epifluorescence configuration. I'll add order codes here when I've found them.

The stock filter sizes are too big for the microscope; I bought larger ones and cut them down to size to fit into my filter cube. If you buy the slightly larger filters you can get at least 4 filter cubes out of one set, if you're good at glass cutting (!).

[JunaidBinMasood]

Dear Richard,

Thank you for sharing the standard configurations document. I have a concern. After the Incoming Light Waves pass the excitation filter which allows the wavelengths of 450nm to 490nm (Figure 6 of standard configuration document) the Dichromatic Mirror completely reflects them onto the specimen. After reflection from specimen the wavelengths of 450nm to 490nm are not allowed to pass through the Dichromatic Mirror while rest are transmitted (as figure 6 suggests). So how is the visualization of the specimen taking place then? I mean which wavelengths will be transmitted back to the eye-piece as the 450nm to 490nm are being blocked by the Dichromatic already. Attached is the figure highlting the question in a more visual way.

Looking forward to your reply.

[JunaidBinMasood]

If you could please suggest where the Dichromatic Mirror was ordered from? The filters were ordered from Comar I saw.Waiting for your order codes. Thank you.

[rwb27]

Hello, in response to your question on the diagram above, the principle of fluorescence microscopy is that you absolutely don't want any of the excitation (illumination) light to come back to your camera port. The light that you are using to see the sample is the fluorescence emitted from the sample, which will be at longer wavelengths than the excitation light - in the diagram you pasted in, the spectrum of the sample's fluorescence is the white curve, which is predominantly green light. This should pass straight through the beamsplitter and the emission filter.

[rwb27]

The filters were all from Comar - the beamsplitter is a long pass filter at 45 degrees, the emission and excitation filters are I think both bandpass filters used at normal incidence. Exactly which ones you need will depend on the dye you are using and the wavelength of your excitation LED.

[JunaidBinMasood]

I re-consulted the document you uploaded (standard epifluorescence configuration) and found that it is the Strokes' shift which causes the reflected beam from specimen to go to a higher wavelength causing the excitation light to pass through the Dichromatic (new shifted wave-band now lying in it's permissible region). Thank you for replying.

[kevincvpp]

Nowadays, I am working on a project of a low cost lensfree imaging and the fluorescence is a big challenge. I would like to share this configuration using a prism such as it has been proposed by Ozcan (Lab Chip. 2010 April 7, Wide field-of-view lens-free fluorescent imaging on a chip) for build a lensfree fluorescence imaging. Usually, in lensfree imaging, it is not practical to use filter cubes. What do you think?

[kevincvpp]

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2863091/pdf/nihms194845.pdf

[rwb27]

That's a really elegant way to do it - I'd not come across that before. If photobleaching isn't a problem, it should get pretty good rejection of the excitation light without needing expensive filters, as indeed they say in the article. That said, if you've already got a field of view of 25x35mm, you probably don't need a mechanical stage like this one...

The filter cube in this microscope does the same job as the prism in the article you reference - essentially filtering out the excitation light. Using the cube, excitation light passes through the sample and out the back, so it doesn't hit the sensor (i.e. you excite and detect from the same side of the sample). This is roughly equivalent to the prism arrangement in the paper; it also stops excitation light from passing through and flooding the sensor. The difference is, of course, that the prism allows the sensor to be much closer to the sample, which you need for lens free imaging.

In a lot of applications, illuminating and detecting through the same objective is convenient and helpful, because it means the intensity in the sample is quite high (because the objective lens concentrates the excitation light very strongly). That's why the openflexure microscope uses a cube. If your application works nicely with a lens free imager, the prism method may well be better - I guess it should generate quite nice dark-field images too, if the filter is removed.

[davidmsachs]

Hi Richard,

I'm playing with this a little, and wouldn't mind a little documentation here. I see in the stl folder you have a filter cube design, and in the OpenSCAD you have an option for fluorescence that creates a square hole that the cube fits into. That part seems straightforward (although I think the filter cube is usually between the objective and the tube lens). How do you handle the illumination? If you haven't finished the documentation, a picture of the final assembly would be helpful.

[rwb27]

Hello,
great - the illumination design needs some serious work! That said, my current "solution" is to push-fit a 5mm LED into a holder that then slides on to the front of the filter cube. The whole thing can then be pushed into the optics module. The "tube lens" in this microscope isn't strictly the same as the tube lens in an infinity-corrected microscope; the beamsplitter is deliberately between the lens and the LED/sensor so that the front of the LED is imaged onto the sample. I often try to rough up the LED with a bit of fine sandpaper to diffuse the light a bit. Proper Koehler illumination would definitely be preferable, but I've not done the optical design for that!

I hope that's helpful - the filters should push-fit in a fairly sensible way; the excitation filter (if needed - LED spectra are fairly narrow but sometimes not quite narrow enough) I just glued on to the front of the LED holder. That could do with some improvement!

[davidmsachs]

Thanks, that's very helpful!

I also want to confirm some stuff about the specific LEDs and filters. I didn't see any specifics in this repository, but I found some documentation from that 2015 iGem project that I'm assuming you were somewhat involved in. They have this in their instructions:

1 Blue LED (GFP) Mfr: Kingbright; Part No. L-10934VBC/DS-D
1 550nm dichroic mirror, 25x16mm (GFP) Mfr: Comar; Part No. 550 1Y 116
1 490nm excitation filter, 25x16mm (GFP) Mfr: Comar; Part No. 495 1K 116
1 500nm emission filter, 25x16mm (GFP) Mfr: Comar; Part No. 515 1B 116

Is that what you'd recommend for GFP? For RFP, I noticed some attempts on their website, followed by: "Note: this did not work in practice". Have you had any success with RFP? And if so, do you have recommendations about specific filters and LEDs?

Incidentally, I've been printing stuff with black PLA/PHA from Meltink, which has a matte finish. I find that it helps a little for reducing any unwanted reflection.

Thanks again,
David

[rwb27]

No worries. Those look like the right filters - but they aren't the right size (I designed it to be quite small to fit with the rest of the optics). I usually ordered them deliberately too big, then used a cutting wheel/diamond scribe to scratch a line across the glass and snap it to size. 12x16mm is about right, but check by measuring the holder first!

I've only ever tried with GFP/Fluorescein wavelengths, so can't comment on RFP, other than saying that Comar have a pretty wide range of dichroics and you can find LEDs at most useful wavelengths.