Selecting fluoresence optics

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1

Hi everyone,

I’m building a delta stage for imaging fibers spun from lignin. They’re tricky to image using a normal optical microscope because of their transparency, so fluorescence imaging can be a decent alternative. When I use violet light (390nm), the emission is centered somewhere around 780nm.

I’m not extremely familiar with fluorescence microscopes, so I was hoping someone could give me their thoughts on a few questions I had.

  1. Since the emission is in the NIR range, would swapping the regular Raspberry Pi camera for a raspberry pi camera module 3 NoIR be a good call?
  2. If I’m only using the microscope for this application, could I use a violet LED instead of a white one and forgo using an excitation filter?
  3. Would a dichroic filter like this one from Edmund Optics be appropriate?
  4. Could I get away with using a type of colored glass filter like this one from Edmund Optics as my emission filter?

Thanks in advance for any help you can offer!

2

Hi @jbrownie, that sounds like a great project. There is another thread on fluorescence, Fluorescence optics for V7? , but what you are imaging is rather different. Particularly your excitation and emission wavelengths are very far apart, which makes separating the wavelengths a lot easier.

Going through your questions:

  1. Yes, you would want a NoIR version of the Pi camera. However it will need to be a Pi Camera 2 (not a Camera Module 3) in order to work with the OpenFlexure software.
  2. A violet LED will probably be necessary as a white LED will not have any light below about 440nm.
  3. Many of the filters from that range look good. The 550 and 600nm long pass have quite a lot of transmission below 400nm, so would not be suitable.
  4. You can probably get away without any filter, but a coloured glass filter may help. The camera will be very insensitive at 390nm, and you could use the red filter on the camera as the emisdion filter, just look at the red channel only in the images.

I have a question as well, what is your need for using the Delta Stage rather than the standard OpenFlexure Microscope?

3

I am currently working on a similar set of optics. I am working on a fluorescent bead with similar optics. The bead will fluoresce with UV and excites in red. I am trying to use a ring around the objective holding UV LEDs. I can turn on the LED when taking a fluorescent image or use the visible LED. In my case I’m using a green LED for visible imaging. My plan is to use the red channel to read the fluorescent signal, using the png image from the camera. So far I can see the fluorescent signal with just one UV LED, looking at the red channel. My goal is to find ways to improve the resolution of the image without using any filters (if possible). My plan is to compare the results with a DAPI filter cube using a CY5 emission filter on the cube on a standard inverted microscope. Just some ideas to consider.

4

Thanks, @WilliamW, for your thoughts and advice! This gives me more confidence about my approach.

Why I’m looking to use the Delta Stage rather than the standard configuration simply comes down to needing a reflection configuration. My fibers are spun onto an opaque substrate (e.g. aluminum foil) and can’t be transferred to a glass slide until there is a decent amount of fibers. Some of the intended uses for the microscope will be for verification that I am spinning fibers and quick optimization of some processing conditions.

5

All of the types of optics are available for the OpenFlexure Microscope as well as for the Delta Stage. The instructions are currently only in the Delta build, but the reflection/fluorescence optics modules are available for the Microscope, you can find them in the customisations section of the instructions.

The assembly and build are the same for either geometry, but the orientation of the camera and beamsplitter are different so you need the optics module from the right assembly.

The design of the Microscope is more developed than the Delta Stage, so if there is no particular reason that you need the Delta geometry I would recommend that you use the Microscope.