Laser imaging set up

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quant_laser
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Joined: Mon Sep 05, 2022 12:45 pm

Laser imaging set up

Post by quant_laser »

Hi all,
This is a a bit of an oddball request. This question and post are NOT about macro/microphotography. However, the experience you folks have with macrophotography is the reason why I am posting it here. This is a FANTASTIC forum BTW!

So, I am a scientist in the process of building a laser spectroscopy set up. That portion of the set up is something I can easily do etc. However, what I also want to do is to simultaneously image the image plane where the laser will be focused down. Since all of the light is going to be coming from the back end of a Nikon Plan Fluor 20X, 0.45 NA objective, I am hoping it should work. This would be an infinity corrected objective BTW. I have attached a schematic for what I am trying to do. The advice I want about is mainly is in the red colored arrow path and the red oval in this picture. I have the ability to move the camera in along the Y axis (X and Z too, if need be). There would be back lighting and a 95/5 beam splitter which would let 95% of the light go straight on while diverting 5% of the light to the camera.

In a sense, this is like a microscope objective on the end of a DSLR for macrophotography. Except that,

1) The light path is in a "double L" shape
2) Part of the light path is not covered
3) I would like to use one of those smaller BASLER type cameras instead of a DSLR. Though, I could use a DSLR if need be.


Based on the extensive, PRACTICAL knowledge of the collective community here, I was wondering if you could advise me on the steps which may go wrong? For example, after the beamsplitter, what sort of optical elements would I need to place? A tube lens? if so, which ones? If I use a BASLER camera, they come with their own sets of lenses etc.

Also, I need to mention that I am NOT looking for imaging perfection here. That is, the goal is not to obtain a spectacular image. I need to be able to just visualize the interior and the FOV of the cuvette onto which the laser will be shined. Also, if the laser is turned on, I should be able to visualize the spot it is hitting within the FOV.

Thank you very much in advance! I know this is not a standard question. However, I have seen enough science to know that people with hands-on experience and DIY folks have the most creative thoughts and ideas!
Warm regards
Quant-Laser

PS - Hope y'all are having a wonderful long weekend!

quant_laser
Posts: 4
Joined: Mon Sep 05, 2022 12:45 pm

Re: Laser imaging set up

Post by quant_laser »

Here are the images. This is my first post and day on this forum...
Nikon_Objective.jpg
Laser_setup-2.jpg

blekenbleu
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Re: Laser imaging set up

Post by blekenbleu »

The schematic mostly describes an epi fluorescent microscope with additional mirrors.
Do you want a camera to sense the UV illuminated spot or just whatever visible fluorescence is excited?
So long as your camera lens can focus to infinity, no additional tube lens need be implicated.
FWIW, Basler seems not to offer any lens longer than 75mm;
the objective, rated 20x for 25mm field with 200mm reference focal length, would not be able to fill a very large sensor.
Metaphot, Optiphot 1, 66; AO 10, 120, and EPIStar 2571
https://blekenbleu.github.io/microscope

quant_laser
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Joined: Mon Sep 05, 2022 12:45 pm

Re: Laser imaging set up

Post by quant_laser »

Thank you Blekenbleu!

I do NOT need to capture fluorescence emission at all. I just need to image (using bright field light from the backlighting) the image plane which will have some live cells. Within this bigger field, I need to identify the location where the UV laser spot is focusing... So basically, this way I can move my image plane around so that I park the UV laser spot in a precise manner where I need to.

With the objective, I can buy the appropriate size sensor camera. So basically, the whole exercise to try to be able point the focused laser spot where I want Within the image field.

Thank you for any suggestions!
Regards

Lou Jost
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Re: Laser imaging set up

Post by Lou Jost »

There's an issue you may not have considered. Laser light makes speckles on a sensor due to interference, and this can make unusable photos. You may have to do something to break the coherence of the light. My favorite decoherence trick is to shine the beam through a dilute milk solution. An alternative is to shine the beam through a fast-rotating frosted glass disk. Such devices are commercially available though hard to find

RogelioMoreno
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Re: Laser imaging set up

Post by RogelioMoreno »

Actually I am trying to implement confocal on my microscope so I will try to help with the concepts that I have learned:

1. You will need to expand the laser beam to fill the back focus plane (BFP) of the objective, you can learn about beam expander on the following link:
https://www.newport.com/n/how-to-build ... -expander
You have to found the size of the back focus plane of your objective (for your reference the 20x/0.75 has 12mm BFP diameter, the 20x/0.6 has 9.6mm BFP diameter).

2. May be you will not be able to see the fluorescence emitted by your subject when you have the back lighting on, maybe the best option is to use the 405nm to know where the ultraviolet laser will hit, the problem is that your DCM will not transmit 405nm so may be you will have to modify the system to replace the DCM for a 30/70 or 50/50 beam splitter (all depend how much light coming from the objective you want to reach the detectors) and put a long pass filter (something like 420 LP) after the 5/95 beam splitter (on the 95 side to block the exciter light to reach the detectors (I assume that they are PMT?)

3. To be able to see the laser spot on the camera you will need to magnify the image formed by the objective, for that you can use a long focal lens (on the side 5 of the 5/95 beams splitter) as tube lens, if you use a 200mm lens then the image on the camera sensor is magnify 20x. The side of the spot created by a 0.6 NA (your is 0.45, I do not have the number of the spot for that NA) objective is around 0.33um, so the size on the camera sensor is around 20 x 0.33um=6.6um may be too small to be seen on the image. If you use a 400mm lens then the image on the camera will be magnify 40x and so on.

Hope this help you.

Rogelio

rjlittlefield
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Re: Laser imaging set up

Post by rjlittlefield »

Regarding the red path, the biggest concern I have is vignetting caused by too much distance between the rear of the objective and the lens on the camera.

That problem will be exacerbated by small diameter of the lens on the camera, which goes along with the short focal lengths that would normally be used on a camera with a small sensor.

What are the dimensions of the equipment, and how large a field do you need to see at once?

--Rik

Macro_Cosmos
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Re: Laser imaging set up

Post by Macro_Cosmos »

Where are you placing the tube lens?
The optical path looks long and sophisticated, you will definitely need some kind of relay lens, or else the projection might be very small.
Is that a second beamsplitter going to 2 additional cameras?
Lou is correct, you will need to decoherent the lasers, a spinning diffuser works well.

No guarantees, but I did some loose reverse engineering on a confocal well plate WSI system which has a very similar optical setup. It could help you or might be of no use, I will see if I have time to draw it out tomorrow.

quant_laser
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Re: Laser imaging set up

Post by quant_laser »

Thank you to Lou Jost, Rogelio, RJ Littlefield and Macro_Cosmos!

All of the above replies have been really helpful without a doubt. Apologies for the delayed responses.

@Lou, decoherence is not really an issue since I need to only image the "reflection" and the laser spot will not be shined onto the camera sensor directly

@Rogelio, THANK YOU for the detailed reply! Yes, you make very salient and useful points...expanding the laser beam to back fill the objective is indeed on the agenda. I do not need to see the fluorescence light. But instead, merely where the laser beam is being focused. However, you bring a great point about the DCM being a barrier to "seeing" the laser. Indeed, you are right. I am now planning to move the camera INSIDE the box to overcome this. I will need to think a bit more about point #3. Yes, a tube lens will be needed...how much and where are the next questions I need to start thinking about. But all in all, your replies have been invaluable!

@RJL, This goes to Rogelio's point as well. I agree with you that it is too far. By moving it into the box, I hope mitigate this. A tube lens WILL be mandatory, but the question is where to place and how long does it need to be...

@Macro_Cosmos, placing the tube lens IS the key question! Need to give this some more thought...

J_Rogers
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Re: Laser imaging set up

Post by J_Rogers »

Are you planning to combine both wavelengths of laser? Or are they being used as single source emissions at different times? I cant give any detailed help without knowing exactly what type of spectroscopy you are planning on doing. It almost looks like Raman scattering, at which point id say skip the back light and throw a visible diode in the path the same way you have the UV lasers. Align with visible and then turn on UV lasers. If it is Raman scattering / laser absorption spectroscopy you want to limit the amount of additional optics in the path of the beams and whatever you are using to measure them. I assume the boxes on the right side of your diagram are your detectors.

Also, what do you have labeled ND? Neutral density filter?

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