pgFocus optics: BFP focusing lens

[Christian Niederauer]

Hi all, 

I am currently trying to implement a pgFocus-based focus stabilization system on a TIRF setup with a 60x 1.49 oil immersion objective.

I use a Thorlabs CPS808S collimated laser diode with an iris to clean up the beam profile. A 400mm focal length lens should focus the beam onto the BFP and a neutral density filter attenuates the beam. 

It travels through a beam splitter and is reflected using a shortpass dichro onto the TIRF dichro cube inside the microscope body, parallel to the excitation laser beam which is focused onto the BFP using a 200mm lens.

On the way back, it is reflected again by the shortpass dichro, and the portion which is reflected inside of the beamsplitter falls onto a linear CCD array.

I was wondering what kind of optics other people use to get a nice beam shape, and to focus the infrared laser onto the BFP and where they had put that lens. Placing it upstream of the translatable mirror forces me to use a very long focal length lens and to focus through the beamsplitter. Also, I think that closing the iris too far to improve the beam shape messes with the collimation of the beam.

Any input is welcome,

Chris

[Karl Bellve]

Hi Chris,

That is really cool. From looking at your picture, are you using an Arduino Mega clone? I really have to work on a pgFocus Shield to simplify it...

Is the 400mm lens in lieu of a tube lens? Since the focus beam is a laser, you shouldn't have to focus it. A

But, if you have a tube lens (or any lens) in place, you do have to change the angle of the laser beam to account for that lens. The laser beam should be straight and towards the edge of the BFP of the objective.

We also use an iris to clean up the beam but not a neutral density filter. Centering the iris on the beam is important.

I think you would have issues with using a ND with a laser. 

Also, the surface (coverslip) needs to be really clean to get a good return beam. 

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Kia ora,

Karl Bellvé

Kia ora, a common Kiwi salutation, means to wish somebody life and health in the Maori language

[Christian Niederauer]

Hi,

thanks for your input. I think I could improve the centering of the beam on the iris. The board is a ST microelectronics board (stm discovery kit) we had around.

About the tube lens: I am not sure if I get you correctly. I do have a tube lens to focus my fluorescence emission onto the chip of a sCMOS camera. The pgFocus pathway however is coupled into the excitation pathway and since it should be collimated in the sample plane, it has to be focused onto the back focal plane of the objective (just like the TIR beam used for fluorescence excitation) - which I try to do with the 400mm lens in the pgFocus pathway. 

I know that there are focus locking solutions using a dichroic mirror in the emission pathway, but I didn't want to risk loosing any fluorescence photons in the emission pathway. 

Chris

[Karl Bellve]

Hi Chris,

We don't use a lens in the laser path. We do have to account for the tube lens in our microscope, which does change the angle of the laser. You don't have to do this because you lack a tube lens in your excitation path. 

This means we tilt our laser (or use two mirrors) to make sure the laser is parallel to the optical axis and near the edge of the objective after it passes the internal tube lens. It should be simpler for you since you don't have an internal lens changing the angle of the laser. No focusing needed. It should just be a clean laser beam.

Neutral density filter shouldn't be needed either but I am not sure what sensor you are using for the return beam. pgFocus does auto-exposure, but you might be running your own code.

Cheers Karl

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[Christian Niederauer]

Hi Karl,

I agree that the alignment should be easier if I don't use a lens. However, if I don't use the lens, the objective will focus my collimated infrared laser onto the sample, which means there will be rays in different angles incident on the coverslip. (see Kyle Douglas' comment https://groups.google.com/d/msg/pgfocus/zIabaXbO5MY/RIVi_0cpBgAJ) 

Wouldn't that worsen the focus tracking? With the excitation tube lens in your setup, you shouldn't have this problem.

Indeed we are running our own code, since the institute´s approach is to have setup related code written by permanently employed software engineers which also maintain it over several generations of PhDs. 

If I don't put a neutral density filter in, the 4.5mW infrared laser is visible on the sCMOS camera against the single-fluorophores I am imaging, even through the dichro and a 525/50 bandpass filter. I will try with an additional shortpass filter instead of the neutral density and see if that changes the beamshape of the infrared laser to the better.

Thanks for your input,

Chris

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