pgFocus alignment prblem

[Ken Jiiii]

Hi everybody,

Finally I have receiver the lenses and especially the translation stage to move the 50:50 beamsplitter in my setup.

To align the pgFocus with the laser I started as Kyle wrote:
- laser (focused to infinity since it has a small lense in front of it); clean the profile with an aperture
- beam alignment with two mirrors (before the BS) so that it leaves the objective straight
- put lense in beam path and focus on BFP (appropriate position found when light output is maximized)
Cross-check: I turn the screw of the translation stage while turning the beam leaves the objective in an steeper and steeper angle.

- add oil an coverslip (I use a 1mm capillary)
- turn screw of translation stage (approcimately half the size of the diameter of the entrance pupil of objective)

Now I can detect a spot which fades when I turn the translation stage in either direction.
The returning beam comes from the beamsplitter in a strange angle so that I get it on the pgFocus with two mirrors.

Unfortunately my laser beam profile is not as nice as Kato's and it changes the shape and intensity when I move the specimen in z-direction.

I attached two pictures which show this. By the waay if it matters: There is no filter in front of the pgFocus and the signal also does change when I move in xy. Something must be completely wrong.

Has anyone an idea what I can improve or what I did align poorly?

Thanks for your help!!

Regards

[Karl Bellve]

From the look of the peaks, it looks like you are off axis with respect to the sensor. You might be as much as 60-80° off.

The easiest thing to do is rotate the sensor 90° to see if it helps, and if it does, go back through your optical path and make sure everything is aligned properly to match the alignment of the sensor.

Cheers

Karl Bellvé

Biomedical Imaging Group

Molecular Medicine

University of Massachusetts Medical School

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[Ken Jiiii]

Thanks for your quick reply Karl.

I tried to find out in which direction the beam is going by having a look at it with a camera.
(Images attached, brighter spots are above the specimen, fewest intensity is in focus according to previous peaks. Spot moves along x-Axis.)
I aligned the spot on the camera so that I can move the camera along a rail without the point changing its position.

[Karl Bellve]

Well, you are seeing translation of the beam, which is good and it appears to be on axis (X). 

I am wondering if you are not coming in at a steep enough angle, especially since one of your images appears to be much brighter than the other two.

I see that you said you used oil. Try using water above the coverslip. 

Objective->oil->coverslip->water.

Cheers 

Karl Bellvé
Biomedical Imaging Group
Molecular Medicine
University of Massachusetts Medical School

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[Kyle Douglass]

Hi Ken,
I'm writing this from my phone, so please forgive my short response.

I noticed in your images that your z-axis stage undergoes 25 um of displacement. This is much larger than the noise magnitude that is typical of drift.

Do you need the full 25 um of displacement for your samples? I don't remember exactly, but I think my usable range in z is only 20 um or 30 um with a 100x objective.

Kyle

[Ken Jiiii]

Thanks for your responses.

Sorry Karl, I didn't explained my self well. I meant I added oil on the objective and put the coverslip with the water/bead-solution on it. So actually I have the following as you said: Objective-Oil-Capillary-Water

About the angle, of course I can move the translation stage a little further with decreases the intensity but I cannot reproduce such a nice peak is in the 190um (+10um above focus) for the specimen in focus.

Kyle, no I think I would not need the full 25 um I just displaced the z-stage to see an actual movement of the spot. So from the focus a +-10um movement of the stage shoudl b sufficient.

[Kyle Douglass]

Hi Ken,
I thought I would also point out that you can increase your sensitivity by increasing the distance that the pgFocus light sensor sits from the beam splitter. If you work out the geometry, you'll find that the same angular displacement of the beam will result in a larger transverse shift in its position by moving the sensor farther away. Essentially, the beam will shift by larger amounts in the light profile plots for the same amount of objective-coverslip displacements.

I've done this on my own setup such that the typical shift in the pgFocus offset readout that I can see by eye is less than the equivalent displacement for one DAU unit. What this means is that I can see the individual electronic "steps" that the pgFocus is applying to the offset.

Hope this helps,
Kyle