Tweety turned into a power machine!

Our dear Tweety microscope, which was simplest and cutest of all the LCI systems, has muted into our most sophisticated power machine!

On the 24th at 10, we will run an online demo (link below) to show what our upgraded Tweety can deliver:

  • Much larger field of view (from 18 mm diagonal to 25 mm)
  • Upgraded single point confocal on the left side
  • Resonant scanner with 1024×1024 pixels (compared to 512×512 on our other resonant scanners), still the same speed (30 fps) and improved low noise
  • Spinning disk confocal on the right side with bypass to image wide field
  • 2 very sensitive cameras on the right side: one with the very large field of view and 11um pixels for best sensitivity and one with the normal field of view and 6.45 pixels for best resolution
  • Our great Primo is still on the back of the microscope to allow micropatterning of proteins at the bottom of a dish or micromanufacturing of wells in the shape/pattern of your choice
  • 2 wonderful silicon immersion objectives specialized for tissue imaging with automatic correction ring: 20x/1.05 and 40x/1.25

After the demo, the LCI users who have already been trained on our widefield systems can get access to Tweety for free after a mandatory short training.

Please add the demo in your calendar and make sure to test the link ahead of the meeting.

Link to the Zoom Meeting on the 24th at 10am: https://ki-se.zoom.us/j/7302561100

CLIJ2: Open Computing Language and ImageJ2, GPU power for everyone

CLIJ2 allows you to use ImageJ/Fiji on GPU instead if CPU processing, so much faster! 🙂

Here is a nice article about what CLIJ2 can do. By the way, this article is published on a new imaging forum called FocalPlane. Check it out! And here is the presentation of how to use CLIJ2 at one of the recent Neubias event in May 2020.

If you have an analysis pipeline built in Fiji, Icy or Matlab and processing takes a long time, CLIJ2 will help you a lot.

Freeware to turn widefield, TIRF or confocal images into super resolution images!

Check out this webinar tomorrow by Rickardo Henriques, the inventor of SRRF (surf), post processing magic for all types of images!

*Title:* Open and accessible cutting-edge technology for  super-resolution and machine-learning enabled microscopy

*When: *June 5th 4:00 pm CEST

*How to access/register*

And here’s a small teaser if you want to quickly see the topics we’ll cover.

Great webinars about sample preparation at Scilife

Hans Blom from the Advanced Light Microscopy facility at Scilife organises next week (10th and 1tth of June) 3 sessions of great seminars:

I especially recommend the CUBIC seminar to anyone who wants to image samples thicker than 100 um. This will teach you how to treat your sample to make it fully transparent so you can image through many cm of tissue!

🙂

One-step non-toxic clearing protocol

Do you know that clearing is not just about light sheet microscopy? Even if you have done your job well and your sample is directly on the coverslip (not on the slide), as soon as your sample is thicker than 10 um (1 cell diameter), you will see the effect of the refraction index mismatch.

What is that? Your sample and the mounting medium around it have a certain refraction index (or likely several). The objective you are using is designed for a certain refraction index (e.g. air, water or oil). If these refraction indices do not match what happens? as soon as you image a tiny bit away from the coverslip, the sample will look elongated, the intensity and contrast will drop very fast.

Sounds familiar? If yes, changing your mounting medium to match the objective will solve the problem. It works for light sheet but it also works for wide field or confocal imaging! Just change your mounting medium and you will see an enormous difference!

Here is an article describing a one-step clearing protocol. This basically is about using a different mounting medium. Easy, cheap and non-toxic! Give it a try!

Have a look at this post for more info.

The antibody validation nightmare

Ever wondered if that antibody you used throughout your whole PhD was actually also binding to something else than its supposed target protein?

Antibody validation in tissue staining is a very difficult task!

Here is a great step-by-step validation protocol published by EuroMabNet, a network of scientists who try to improve antibody validation.

And this paper gives a useful flow chart for antibody validation.

And here is the 5 pillars of antibody validation paper which explains what can be done to validate antibodies.

 

Transfecting hard-to-transfect cells

Here you can see a nice film of a beating cardiomyocyte.

It was transfected using Fuse-it vesicles full of the mRNA of LifeAct-tagGFP2. According to Ibidi, it also work well with primary cells which are typically difficult to transfect.

RNA-based transfection seems to be gaining speed compared to classical transfections using DNA.

If you try it, leave some comments here to tell us how it worked for you! 🙂

Fluorophores that blink for STORM without any special buffer

Sounds good! No more buffer that stops working after one hour of imaging!

This company sells SaraFluor650 secondary antibodies. This fluorophore seems to be a natural blinker which does not need to reducing environment. Apparently it doesn’t need very bright lasers either!

Apparently they have also developed a green variant. So you can run 2-colour direct STORM on your favorite TIRF system! 😀

They also sell pH sensors and more so check their website and if you try, write a comment to let us know how things worked!

Multiplexed immunostaining

It is not easy to find enough antibodies that work together to be able to label a sample with more than 4 antibodies at the same time. And even 4 is pushing it.

This paper describes a new immunostaining multiplexing method called 4i. The method is based on a special imaging buffer that prevents the antibody from being strongly bound to the sample due to the imaging process. This allows the authors to detach the antibody with gentle treatments, leaving the sample in a good shape and ready for another round of labeling and imaging.

Using this method they have successfully labelled the same sample with 40 different primary/secondary of the shelf antibody pairs!

Seminar on how to directly label your primary antibodies: Skip the secondary, part 3!

The LCI will host a seminar about GlyCLICK, a new way to directly label primary antibodies and stop using secondaries: 6th of November at 13:00 in the Lipid seminar room in Neo, KI Flemingsberg. The seminar will be broadcasted live. Here is how to find us and here how to follow the seminar online.

Everyone agrees that it would be great to be able to label our samples without using secondary antibodies.

  • less animals killed
  • shorter and cheaper protocols
  • no problem with isotype cross-reaction
  • no problem with secondary species when using many antibodies at once

There are many kits to label primary antibodies directly with fluorophores. The main disadvantage compared to primary/secondary stainings is that direct labels are often weaker because the final primary/fluorophore ratio is too low. Using an amplification method like TSA (Tyramide Signal Amplification) leads to a loss of resolution.

Over the years users at the LCI have tried this kit, this kit and this one. They gave mixed results depending on the antibody but we keep looking! Come to the LCI seminar about a new direct labelling technique that uses Click chemistry.