Cool technique – Page 5 – The Live Cell Imaging facility

Free genetic and image analysis tools

Here are a few image analysis tools (constructs for FRET, software…) that might be useful to you.

Construct for fluorescence biosensors and optogenetic tools
Free Image analysis software

http://www.hahnlab.com/tools/index.html

Free Image analysis software

https://www.utsouthwestern.edu/labs/danuser/software/

Free Image analysis software

http://cismm.web.unc.edu/

Crest V3 spinning disk confocal demo

Tomorrow (17 sept) we will enjoy a seminar and a live demo about the Crest V3 spinning disk confocal which is being set up at our facility as I write! 😀

Very cool confocal!

enormous field of view (32 mm diameter)
fully confocal
can image at 100 frames per sec
spits out Nyquist resolution with the 60x objective!

You can come to the seminar (at 10 in the Gene seminar room at the LCI facility) or listen to it remotely (see here how to follow the LCI webinars).

You can even book a private demo to image your own samples.

Matching the refraction index of live samples

To image a thick sample, it is crucial to match the refraction index of the sample with that of the immersion medium between the sample and the objective. Typically, life samples are in an aqueous solution like culture medium which has a refraction index of 1.33. Unfortunately organoids often have a higher refraction index closer to 1.44 therefore as one images deeper into the organoids, light scatters due to the refraction index mismatch and the images become blurry.

This paper presents a product that has a high RI and is compatible with cell culture. Good to keep in mind for those who image organoids over time.

How to identify cells and nuclei in an image?

NucleAlzer is a great new deep learning tool to identify roundish objects like nuclei and cells in fluorescent or bright field images.

To test if the tool works for you before you download it, you can simply upload one of your images and check the result. Easy! 😀

Super-Resolution spinning disk demo at the LCI!

Dear microscope freaks

How would you like to run some gentle live sample imaging with a 60x objective with:

an xy resolution of 120 nm without software tricks (or even better after deconvolution),
the great contrast of a true confocal,
82 frames per second,
or decide to bypass everything, go widefield and image at 100 frames per second with a super large field of view (220×220 um)?

Sounds good to me! 🙂

For the next 2 weeks you can do that with the new toy on demo at the LCI facility!

The beast is a new sort of spinning disk confocal and is called SoRa (Super-Resolution Optical Reassignment). It is a collaboration between Nikon and Yokogawa.

We even have 2 cameras to compare (Prime95B and BSI from Photometrics).

Oliver Garner from Bergman Labora will give a short online presentation of how SoRa works on Monday (29^th) at 13:00. The presentation is done remotely and broadcasted live. You can join the audience from the comfort of your office chair by following the instructions here (please try beforehand to make sure all works).

Interested in trying it? Please contact us.

A dream!!

LCI product seminar: How to label organelles in live cells?

On Wednesday (13^th) at 9:30 in Lipid seminar room in Neo (KI Flemingsberg), please come and enjoy a short seminar presenting a new way to label organelles in live cells.

LabLife will present their product called Viromer Cytostain.

We will stream the seminar live so you can follow it even from your desk! 🙂

CUBIC seminar tomorrow at Scilife

Do not miss the seminar tomorrow morning at Scilife: the inventor of the CUBIC technique, Etsuo Susaki, will present his technique which can clear fatty and dense tissues.

Click here to know where and when.

And here is the link to the updated Cubic protocol.

Antibody-based technologies at SciLife

Apply to this course at SciLife and learn about Antibody-based technologies on the 25th of March.

Glyoxal instead of PFA

Poor PFA fixation often causes trouble in antibody staining. Folded cells, poorly preserved cytoskeleton… These artifacts appear when the stock of PFA gets older and degrades. Buying ready made PFA solutions, most of which contain 10-15% of methanol, can also lead to low labelling with some antibodies.

Glyoxal seems to be a good alternative. It had the added advantage that it is less toxic.

Check this article to know more. 🙂

How to stick a tissue section to a coverslip

As you all (nearlyish) know, one should never place a sample on a thick glass slide and add a coverslip. Instead, the sample should be placed on the coverslip then covered with a thick glass slide. And the coverslip should be 170 um thick (also labelled thickness #1.5).

Why is that? Because the coverslip is part of the design of the objective and all objectives from all manufacturers are designed to image through 170 um glass and assuming that the sample is directly in contact with the coverslip.

What about superfrost slides that one uses to make sure tissue sections don’t float away during antigen retrieval? No worry! You can make your own superfrost coverslips. It is cheap and you can prepare tons at the same time. Here is the protocol (and pasted below).

Not convinced? You will only see the difference when you compare side by side! The images of your tissue will be much sharper if the sample is on the coverslip because when you put your sample on the slide, either the thick glass or the mounting medium end up between the objective and the sample. The objective is not designed for this. ?

Here is the protocol:

Reagents Required
Gelatin-coating solution: 1 L deionized H2O, 5 g gelatin, 0.5 g chromium potassium sulfate dodecahydrate CrK(SO4)2 · 12H2O

Materials
Filter units
Histological slides
Hot plate with magnetic stirrer
Slide racks
Staining dish
Thermometer

Procedure

Prepare the gelatin-coating solution by dissolving 5 g of gelatin in 1 L of heated, deionized H₂O (temperature should not exceed 45 °C).
After the gelatin has dissolved, add 0.5 g of chromium potassium sulfate dodecahydrate. Chromium potassium sulfate dodecahydrate will positively charge the slides allowing them to attract negatively charged tissue sections.
Filter this solution and store at 2-8 °C until use. It is recommended that this solution be filtered again immediately before use (adjust to room temperature before filtration).
Place the histological slides into metal racks.
Note: The slides should be cleaned by washing them in soapy water and rinsing them thoroughly, first in tap water and finally in deionized water.
Dip the racks containing the slides 3 to 5 times (~5 seconds each) into the gelatin-coating solution.
Remove the racks containing the slides and let them drain. Blot excess solution from the racks onto filter paper (gently tap the racks against the filter paper for better drainage).
Place the racks containing the slides on the lab bench and cover them with paper towels to protect them from dust.
Dry at room temperature for 48 hours.
Dried slides can be put back into the boxes that they arrived in and stored at room temperature until use. Slides intended for cryostat sections can be stored at -20 °C.