As usual the lectures at the LCI microscopy course will broadcasted live online, free of charge and there is no need to register.
Title: Microscopy: improve your imaging skills – from sample preparation to image analysis
The aim for this course is to improve the microscopy skills of students and researchers who have already used a microscope to acquire digital images of fluorescent samples but feel that more knowledge could help them.
Applications are closed but all lectures will be broadcasted live and open to anyone without registration.
The course covers the following topics:
- Optics, image formation, fluorescence, fluorophores, microscope and microscopy types
- Objectives and refraction index, Cameras and detectors
- Noise and background, Cameras and detectors, Bit depth and saturation, Multicolour imaging
- Resolution and contrast, Sample preparation, Immunostaining
- Nyquist sampling, Confocal and wide field settings, Scaling up and speeding up, High throughput/content
- Volume imaging, deconvolution, multiphoton, Clearing and expansion
- Live cell imaging, Fourier, AI, Super Resolution microscopy
- Data handling, OMERO.figure, Requirements for image analysis, Colocalization
- Image processing and analysis
Check the course schedule and details of how to join the Zoom webinars. Scroll down to read the kind testimonies of our dear students! 😊
Here is the course syllabus.
Hope you enjoy the LCI facility microscopy course 2021!
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!
Fluorophores are evolving fast! Here is a paper about a bunch of new fluorophores isolated from the jelly fish Aequoria. This includes the brightest fluorophore ever isolated: a new green fluorescent protein called AausFP1 that is almost 5 times brigther than GFP! Respect! 🙂
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.
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.
Imagine starting a study about some cool protein.
You find some useful articles on Google Scholar. In one paper, an antibody is mentioned. The name of the company that sold it to the authors is mentioned in the Material and Method but unfortunately that company has closed down or has been swallowed by one of the Pharma giants so you cannot order. Then you realize that the company was not producing any antibodies anyway, they were buying it from another company so there is no way to trace and buy the same antibody. Nightmare…
Then imagine that instead, the paper mentions the RRid number for that antibody. You do not know about what that is but you check and find this paper that explains it all.
Now suddenly, not only you can find on the Scicrunch website which company produces this antibody and which resells it so you can buy it, but you can also search pubmed for the RRid and find all the articles that mention it, opening your eyes to lots of results about your protein that have been published specifically with using that antibody. Now you can also check if the antibody gives consistent results!
And imagine being to do this for your favorite mouse model as well. See all publications that have mentioned your mouse RRId!
But it relies on you writing the RRid of your antibody or mouse in your next publication so think about it! 😀
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! 🙂
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!
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!