# Optics alignment of a confocal scanning microscope

I am facing a challenge in my project regarding optical alignment.

See the figure:

The challenge is with the vertical optical system alignment. I considered placing a mirror and check back if the image and source coincide. But since the light is too low on power (less bright).

How can I align it so well that everything is absolutely vertical and good?

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Your source seems to be a $\approx 30\,mW$ emitter which is attenuated by the pinhole and following optical components. Which components are well aligned? Is the question for vertical alignment the M1-M2 optical path in your picture? –  Stefan Bischof May 16 '13 at 21:48
Everything until the Microscope system is aligned. The vertical components are M1 M2 and M3 and O. M1 and M2 are basically periscope assembly. I just want to make sure, the beam trajectory is vertical and parallel to the optical breadboard. –  abhilash sukumari May 16 '13 at 22:04
Please flip your beamsplitter in your image. Then your detection path (green) is able to end on your CCD camera. –  Stefan Bischof May 17 '13 at 16:31
yes that was just a representation of Beamsplitter. However, do you suggest me any other target that I can use that can fluorescence a visible PSF ... Anything that can be found in common ? –  abhilash sukumari May 17 '13 at 16:37
Also I forgot to add that ( for people who are into optics domain ) , this is a confocal scanning set up. The alignment and choice of the pin hole decides the Depth of Focus on the target. Just a info for people who are interested. –  abhilash sukumari May 17 '13 at 16:40

The basic concept of optical alignment is to perfectly align the beam step-by-step on all optical elements. Enshure your beam has the right height and angle before alignment. Your aim to align the beam on microscope optical axis. Using point sources like (e.g. Flourescent beads, microspheres, Quantum dots, quantum wells, NV centers in diamond) allows an easy alignment to a point spread function on your CCD sensor.

On-axis with an iris aperture

A quick method uses an iris aperture to make a parallel beam to the optical axis. Insert Iris $A$ between M3 and M2. The open iris aperture should allow the beam to transmit in center. Place a mirror perpendicular to opical axis at the translation stage with remove microscope objective and ocular. The beam is reflected on the plane mirror and possibly hits the back side of iris aperture.

Iterate angle and position alignment

Using M3 to center the beam on back of iris apeture, while oving aperture up and down. Possible position errors shoule be aligned with mirror M1 an M2. These have the largest impact on position. Start iterating with angle alignment. This coarse alignment should allow to insert microscope elements and fine align the beam to microscope objective.

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Very good information. Using an Iris should help. Is there any other daily seen object that can replace the fluorescent beads? I dont own any beads as present. –  abhilash sukumari May 16 '13 at 23:26
I do not know an everyday-life light source to be used as a point source in this case. Flourescent beads (googling will give microspheres for under 300 dollars), Quantum dots, quantum wells, NV centers in diamond... whichever goes with your excitation wavelength. –  Stefan Bischof May 17 '13 at 16:54
Excellent summary. A company I do a lot of work for used to make a commercial laboratory setup very like Abhilash's and Stefan's advice is spot on. I would add: when choosing fluorescent spheres, don't be too tempted to go for really small ones (which would give you an accurate PSF). Choose something about 5 micron diameter - they are small enough to align, but bright enough to see well for alignment. After you've got your setup working, THEN go for something smaller if you need to measure performance. These guys give 5um spheres for \$125: spherotech.com/2012%20Price%20List.pdf –  WetSavannaAnimal aka Rod Vance Jul 23 '13 at 13:12

A low tech version of a point-like source at the stage plane is to use a pinhole (very small, depending on magnification of your objective, say 1um - 10um). Then illuminate the back side of the pinhole using a laser. You will get a quasi-point source, of course not good for determining PSFs but maybe enough for alignement...

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