Adding laser to Zeiss Photomic III for Raman

[cmtalb01]

I have added a green laser to my Zeiss photomic III to create a Raman microscope. I am finding alignment super critical. I have seen the article on aligning the photomic (head, nosepiece, field diaphragm, etc.), but I don't think I have it as good as it needs to be. Does anyone have knowledge of how or who to contact about photomic alignment. Google search always brings up phase alignment only. I am getting Raman spectra embedded in fluorescence, but I am finding that alignment is super critical for good S/N. Thanks!

[Oktagon]

Wow! That's an interesting progect! I wish I though of something like this.
So, I think the forst thing is to simplify the light path by closing the side lever on the tube to prevent light from going through internal stand path. You might be better off with Universal in this case (and you can just buy universal tube which lacks the rear light path option).
The I think that the most important thing is to achieve coaxiality between the laser and central axis of the objective. As long as there is no significant horizontal shift within the beam splitting prizm inside the tube there should not be a significant Raman scatter. Also, dust anywhere would be absolutly and critically detrimental to achieving propper analytical signal. In general this is not an easy project you have undertaken!

[cmtalb01]

I admit, this is more challenging that I had originally thought. I have added a dual mirror system to bring the LASER into the microscope which allows me to adjust the beam for better control. That has helped significantly. I have also tried various long pass filters that are affordable on a home budget. At the end, even with the filters, I had to simply put a piece of cardboard in front of the spectrometer detector in a position that blocked the Raleigh scattering signal. I am now seeing Raman bands, but heavily masked by fluorescence. I have been working with a green 532nm LASER because of the ability to purchase cut-off filters. I have also experimented with a red 656 nm LASER as I had filters for this wavelength lying around. But fluorescence is still a problem. I have also tried cooling the CCD in the spectrophotometer with a Peltier to remove thermal noise. The last are that I need to check is the position of the fiber collection lens in the photo tube on the PhotoMic III. I have thought about trying an IR laser at 1056nm, but that will entail a significant expense for the laser and the filters (not to mention increase the safety concern. I do appreciate your feedback - this has been and continues to be a very fun endeavor - reminds me of my grad school days! Thank you for your help!!

[cmtalb01]

I've continued to work on this project and wanted to post an update. After much experimenting I came to the conclusion that the problem was alignment, although I could not rule out issues with the microscope optics. I took a side tangent and spent a number of months aligning the PMIII, testing various objective/eyepiece combinations, and evaluating the best camera setup to assure that the signal was getting to the fiber optic. After all of this I went back and assured myself that I could actually acquire Raman bands away from the microscope. I found three things that gave me a challenge and probably was a good part of the issue on the microscope. First, as suspected alignment was a big issue. I proved this be modifying a beam splitter assembly I found on the internet and 3D printing my version. Raman peaks immediately appeared. Next was finding a laser that was affordable with the smallest band width possible. I found one from China that appears to work, although it is by no means a Raman laser. Third was to change the grating int he spectrometer to get the highest resolution possible. I am not getting consistent Raman bands with good S/N and the best resolution possible. Next step is to move all of this back to the microscope. I will update when that is accomplished.

[Hobbyst46]

Congratulation on pursuing such a challenging project. Looks like you are half-way to a home-built confocal microscope;
BTW, there exist a technique - double-photon something if I recall - were the laser wavelength is effectively doubled... from 532 to 1064nm...
Are you using an Argon laser?