Imaging – Page 2 – The Live Cell Imaging facility

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:

Sample preparation for super resolution. These seminars include the great technique of expansion where one can ‘image in super resolution’ without a super resolution microscope.
Clearing with CUBIC. Great clearing technique that works for all organs. The authors clear whole adult mice!!!
Light sheet microscopy by Bruker. Learn about the latest light sheet development.

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!

🙂

Super bright green fluorescent protein

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! 🙂

A microscopy-related job at EMBL Heidelberg with EuroBioImaging

Euro-BioImaging Bio-Hub at EMBL Heidelberg is looking for the Euro-BioImaging Industry Board (EBIB) Coordinator who will work in close collaboration with EBIB Chair and EBIB members located across Europe as well as with the Euro-BioImaging Hub Office.

The Euro-BioImaging Industry Board (EBIB) is currently comprised of thirteen different companies across the imaging field. In the EBIB, all companies work together as a single, professional entity, setting their own goals in order to proactively drive the interaction between the imaging industry on the one hand and European researchers, funders and the imaging facilities linked with the European research infrastructure, Euro-BioImaging on the other hand. In October 2019, the European Commission has officially established Euro-BioImaging – which provides life scientists with open access to a broad range of technologies and resources in biological and biomedical imaging – as a European Research Infrastructure Consortium (ERIC). The post holder will report to the Director Euro-BioImaging Bio-Hub at EMBL and the EBIB Chair. The responsibilities of the position include among others:

Proactively seeking opportunities for EBIB members to engage and collaborate with Euro-BioImaging Nodes, users and national networks;
Representing the EBIB at international events in a professional and politically astute manner;
Strategically prepare and operationally manage EBIB-driven meetings and workshops to understand the needs of the imaging community;
Coordinating “One Voice” activities to the political community at national and European level (European Commission, Brussels) in order to increase the imaging market;
Developing communication tools and activities to boost EBIB’s visibility in order to increase the number of industry members and strengthen EBIB’s presence in the scientific and political landscape;
Keep up-to-date with imaging-associated initiatives and provide EBIB with tailored insight into European research trends and culture changes;
Coordinating industry participation in Euro-BioImaging EU-wide training programs;

If you are interested please find here the complete job description and link for online submission of your application.

Please distribute also to your friends and colleagues, who might be interested in this job posting. Thank you!

Kind regards,

Antje Keppler, PhD

Interim Section Director

Euro-BioImaging Bio-Hub

Global BioImaging Coordinator

EMBL Meyerhofstr. 1, DE-69117 Heidelberg Tel. +49 6221 387 8847

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.

Broadcasted lectures from the LCI microscopy course and private demos of light sheet and cameras

Our course starts tomorrow! 😀

Target audience:

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 and want to improve their skills.

Registrations are closed but all lectures are open to everyone without registration.

The schedule and details of the venue are here.
All lectures are also available online live. The link and instructions to watch are here.
Make sure you check the schedule in case of last minutes changes.

If you are in Sweden, you are welcome to try some of the equipment on demo with your own sample.

To book at timeslot, please contact the responsible person directly.

Light sheet microscope from M2Lasers: Valentina Loschiavo Valentina.Loschiavo(at)m2lasers.com
- Fast imaging of large sample
- Overview function to navigate in the sample and find the region of interest
- 800x800um field of view with 1um min resolution
- Any immersion media
- Sample size up to centimetres
Wide Field microscope from Nikon with 3 different Andor cameras: Oliver Garner (oliver.garner(at)bergmanlabora.se)
- Nikon Ti2 microscope with 4 times larger field of view
- A front illuminated sCMOS camera: Good sensitivity and resolution, great speed, but a greyish background (Andor Zyla 4.2)
- A back-illuminated EM-CCD camera: highly sensitive camera with very dark background, but lower resolution and lower speed (Andor 897U)
- A back-illuminated sCMOS camera: same sensitivity and low background as an EM-CCD but better resolution and speed (Andor Sona)

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!

How to precisely measure the volume of a cell?

Measuring the volume of a cell is often done by labelling the cell membrane or its cytoplasm. Analysing large flat cells this way is easy but it is much harder for tiny cells like blood cells, yeast or bacteria.

Another way to measure volumes is to use a negative stain, i.e. where the medium is made fluorescent with a dye that does not go into the cell. The cell appears as a black hole in fluorescent images and unlike lipid-based membrane labelling, borders are even and easy to segment.

While many dyes can be used for live cells, one must choose large dyes when negatively imaging cells that have been fixed and permeabilized.

This paper and this one use high molecular weight (2000 KDa) Dextran to achieve these results and measure the size of bacteria.

This recent paper optimizes the technique.

Deep red fluorescent proteins

The microscopy field is moving away from blue dyes. This is because red light, used to excited far red and deep red fluorophores, is less damaging to live cells than near UV light which is used to excite blue fluorophores.

On top of that, red light penetrates deeper into thick samples.

So as the trend in microscopy is to move to thicker samples and use more live samples, far red and deep red fluorophores are becoming more attractive.

Here is an article describing 3 new fluorescent protein in the far red to deep red range. One can excite them with 640 nm or a 685 nm lasers or LEDs.

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!