MicroscopyKarolinska – Page 13 – The Live Cell Imaging facility

Skip the secondary, Part 2! :)

In one of the LCI earlier posts, you can read about easy and powerful it is to skip using a secondary antibody while still having a bright signal.

We have not had any feedback on the Kromnigon technology but we got to hear great praise and see superb images of a similar product called Mix-n-Stain by Biotium.

You can now directly label your stock of primary or favorite tag ligand (Snap, Clip, Halo, TMP) in just 30 min! Each primary gets 3-5 fluorophores according to the Mix-n-Stain brochure so there is no problem with dim directly labelled antibodies as used to be the case in older labeling technologies.

Skipping the secondary means gaining time but also no more headache about matching antibody and tissue species so you can stain your tissue with 7 or 8 antibodies if you image with narrow filters or spectral unmixing.

Bye bye ‘No primary’ controls! It is high time to switch to an isotype control: buy an antibody with the same isotype as your favorite antibody and label it in the same way. This allows you to detect any aspecific binding of your primary antibody.

Last but not least, skipping the secondary means less animals used to produce them. That alone is a bit plus!

It costs 100€ or so to label 50 ug of antibody. Definitely worth a try! 🙂

Learn about Expansion microscopy

Next wednesday morning, David Unnersjö-Jess from KTH/Scilife will give a talk at the Live cell imaging facility (9:30, DNA seminar room), about a new and exciting aspect of microscopy: Expansion microscopy.

This is a technique where one ‘blows up’ the sample while keeping all proteins in place and at the same relative distance from each other. The sample is simply ‘inflated’. One can then take images of it with a normal microscope but the resulting image give a much higher resolution than normal microscopy.

After the seminar, David will have an open discussion with anyone who would like to try the technique.

Super resolution morning

On Thursday (15th of March), Teng-Leong Chew, director of the Advanced Imaging Center at the Janelia Research Center (Virginia, USA), will come to Stockholm and present what his facility can potentially do for you.

The AIC offers a crazy service where visiting scientists can use the super resolution systems they develop there with the help of their experts. This service is free of charge, including accommodation.

This can allow you to quickly run a project involving STED, PALM/STORM, SIM, adaptive optics or super resolution lattice light sheet microscopy.

Together with Leong’s presentation of his facility, there will be a few seminars by the Live Cell Imaging facility as well as several parts of the Advanced Light Microscopy facility at Scilife.

This is a great opportunity to catch up with what is available to you here and in the US. Hope to see you there! 🙂

Gluing samples

If your sample floats in its dish and you are desperate to image it, here is a good tip:

Vetbond is a good place to start! Glues super fast and is compatible with live cell imaging! 🙂

There are plenty of other similar products so please leave a comment to tell us if this works for you or not or which is your favorite tissue glue!

Skip the secondary! :)

Kromnigon makes fluorescent labels called FlexiStain that stick to biotin-labelled primary antibodies.

This means that you skip the species matching headache when using multiple antibodies and your labeling is faster! I have not tried myself but they offer free trial kits so give it a try! You are welcome to leave comments on this posts to tell us if it works or not! 🙂

Note: We have not had very good experience with these dyes. Check this post instead.

DAPI is so passé!! ;)

If you are tired of DAPI bleeding through your weak green channel but are stuck to using blue for the nucleus, you might want to give a try to Syto41.

Must better excitation efficiency at 405 nm and much narrower emission spectrum!

And Syto also comes in other colors. 🙂

The LCI facility has moved to Neo! :)

That’s it! We have done it! We have moved to Neo! 😀

The trip to Neo (just next door to Novum) started at the end of 2015! 2 years and lots of planning meetings later, all the companies involved (mostly Bergman Labora and Zeiss but also Alveole, La Vision Biotech, MMI and Andor) started dismantling, moving and reassembling our 7 microscopes on the 4th of December. Now, one and a half month later, everything is nearly done! Exciting! 🙂

Adaptive Optics

The next big thing in microscopy comes straight from the sky! Apparently astronomers have been using adaptive optics for years to improve their images and it is only getting into our microscopes now!!

Adaptive optics takes the nightmarish situation seen in b and puts it back straight as in a! This is done by measuring the wave front and deforming a mirror to reshape the wave front to perfection!

Sounds like a dream but I have actually seen it in action at the 2016 AQLM course and there was a definite WOW effect! 🙂

Check this review to learn more

Expansion microscopy

Just imaging being able to image very tiny details in your sample on your favorite microscope without using super resolution!!

Expansion microscopy is definitely worth trying if your sample is fixed and what you want to measure is smaller than the detection limit of the microscope (around 250 nm)! There are people at Scilife who have implemented it and he is happy to collaborate! Just ask us! 🙂

This is just one example among many where expansion microscopy did wonders!