Structured illumination on a Zeiss Universal

[Nidhogg]

Hello!

Sorry but I can't speak French!

I thought to share my SIM (structured illumination microscope) project on my Zeiss Universal on this interesting forum.

Structured illumination allows to record confocal-like images of fluorescent samples. Due to the slow acquisition rate of my system (10-15 seconds for each slice), only still, fixed samples can be recorded.

P1030564.jpg (120.85 Kio) Vu 50398 fois

Every part, like mechanical device for grid rotation, stage, camera, focusing knob, is controlled by two Arduino UNO rev3 and an Adafruit TFT 2,8" touch screen.

Here you can find a ppt of the project: https://lookaside.fbsbx.com/file/Zeiss% ... P7hYfZGAOA

Here the code for the SIM controller:

Arduino_Stack_SIM.txt

code for the SIM controller

(40.54 Kio) Téléchargé 155 fois

And here the code for the mechanical stage control:

Mechanical_Stage.txt

motorized stage controller

(3.8 Kio) Téléchargé 107 fois

Testing and fine tuning the system is still in progress, but the first results seem to go to the right direction. The contribute of out of focus parts is eliminated:

Widefield image, red autofluorescence in Sphagnum leaf

Basler_scA1400-17gm__21360943__20171218_184527670_0001rid.jpg (66.2 Kio) Vu 50407 fois

Reconstructed image from 15 frames (3 angles, 5 phases each of the grid)

Stack16_maxminrid.jpg (49.3 Kio) Vu 50397 fois

[Vardar]

Thank you Nidhogg !

No problem about writing in english here I guess, especially when illustrating such a fascinating system !
I will have a closer look at it for a better understanding.

Phil

[Bernard-J]

Wooaahh !! You made already a lot of work for an appealing project that seems in a very good way!
If this system will be able to deliver confocal-like images, this will be a great achievement.
May be another object would be a better choice for testing - what is the thickness of the slices in your example ?

For sphagnum leaf autofluorescence, I got this image from a single shot with a classical MO:

sphagnum-autofluo.jpg (34.31 Kio) Vu 50278 fois

[Nidhogg]

Thank you!
I don't have the means to measure the actual thickness of the slices. in theory it should be close to the objective depth of focus.
I know Sphagnum isn't the best choice, but I didn't have anything better to test! I should look for some cyanobacteria or maybe I will just use some higher magnification on plant cells. I also have acridine orange and calcofluor white staining to test.

Work is still in progress!

[Laurent]

Wow, nice work! Thank you for sharing it here !!! Do you maintain a blog where we could follow your progress?
I am waiting forward to get updates of this project...

[Nidhogg]

I don't have a blog yet because I don't have much time to write every project down. I'm also working on laser microdissection and on my new SEM and plasma sputter. Maybe in the future I will put everything on a blog!

[Bernard-J]

I know Sphagnum isn't the best choice, but I didn't have anything better to test! I should look for some cyanobacteria or maybe I will just use some higher magnification on plant cells. I also have acridine orange and calcofluor white staining to test.

Hello,
Concerning a better object than Sphagnum and if I understand correctly the needs for testing such structured illumination, you should have a nicely space limited and fluorescent part embedded in a bigger organism. A potential issue with calcofluor and AO is that these fluorochromes show a quite broad specificity. Nevertheless, I would suggest two possibilities:

1. Nuclei of buccal cells stained with AO. See below the AO-stained nucleus.. with a few bacteria.
A probable issue might be the quick fading of the AO staining under UV.

buccal_cell_AO.jpg (31.48 Kio) Vu 50074 fois

2. Rotifers stained with calcofluor. Calcofluor stains the trophi of the rotifers because trophi are partly made of chitin.
In my opinion, this would be a nice choice since rotifers are ubiquitous (e.g., in birdbaths) and trophi have superb and well limited structures.
I have recorded such stainings months ago and your question urged me to publish this short video:

https://youtu.be/65xKFafL1so

Hope this helps and keep us tuned!

[Nidhogg]

Thank you Bernard! Your suggested targets seem really good for the purpose. Since I made the stock solution for AO staining I didn't try to stain anything yet. So maybe it's time to start!
Both subjects seem cool and I will make some tests on them and update this post with the results!