Hi Timour,

Do you have a schematic of your setup/proposed setup? I've never used the tube lens in the microscope stand. Depending on the brand of your stand, the configuration of ports may be slightly different. I won't use a camera port. I'd use either the back port, or side port intended for mounting a confocal unit (that means free of tube lens) and either way use my own tube lens. Depending on your experience with optical design, you can either model the system in something like Z-max and find the position for the pinhole that way, or do just a rough estimate and leave the fine tuning to be done experimentally by moving the pinhole in all 3 dimensions by sufficiently precise positioners (manual or motorised for automation). To align it all, take a fluorophore solution (for the first alignment I'd take a bit more concentrated one and go by intensity only, for later fine tuning you can take FCS concentration and go by counts/particle). Yes, it might be a good idea to do the first alignment of the APD without the pinhole. The focused spot on the APD surface should be significantly smaller than the active area (if the APD surface is in the focus of the focusing lens), so the APD alignment is less sensitive than the pinhole alignment (there's a plateau outside of which the intensity drops steeply to dark counts). In general the alignment process is iterative, optimizing alternatingly all degrees of freedom until you converge to the optimum.

Good luck,

Radek

To think of it again, you should be able to use a camera port as well (and the tube lens that comes with it). The pinhole should then be in the plane where a camera chip would be when the camera is mounted using the appropriate C-mount adapter. Overall the pinhole position in Z (distance from tube lens) is far less sensitive than the X,Y position (the tube lens focal length is quite long), so you don't need to be too precise there and once you find the position, you won't need to touch it again any time soon (unlike the XY). So when I mentioned about motorised actuators, I meant them for XY, for Z it would be a waste.

Hi, thanks for the clarification. Yes, I'd start first with the APD without a pinhole to get this part right. Do you have two lenses between the pinhole and the APD or just one? As you mentioned, using an optical fibre is another option, it can replace the pinhole and the in-coupler replaces the tube lens. I am currently using a setup based on IX71 where i illuminate through the backport where i have a custom attachment with a dichroic and two fibre couplers, one for incoming laser, the other as tube lens and pinhole and a mirror in place of the filter turret. I haven't evaluated how much light is loss in the coupling compared to a standard pinhole, in general fibres have about 70 - 80% coupling efficiency, so there would be probably some losses there. There's a paper characterising multimode fibre in the roe of pinhole: Article A simple submicron confocal microscope with a fiberoptic output