Imaging – Page 3 – The Live Cell Imaging facility

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!

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.

Imaging Africa workshop

Great initiative from the other side of the pond. Please forward to your imaging friends/colleagues working in African universities:

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We are pleased to announce Imaging Africa—a workshop initiative aimed at developing the microscopy knowledge and expertise of African life scientists.

Imaging Africa is an intensive, 4-day workshop + 1-day symposium focused on exposing students to a plethora of microscope technologies and impactful applications. Topics range from portable, cellphone-based microscopes to advanced super-resolution modalities. Furthermore, students will be introduced to experimental applications such as biosensors and optogenetic tools. These theoretical and practical classes will run in parallel with an in-depth quantitative image analysis course, which will provide the students with the skills necessary to reveal meaningful information from microscopy data.

With the generous support from the Gordon and Betty Moore Foundation, the Chan Zuckerberg Initiative, the Howard Hughes Medical Institute Janelia Research Campus and UNC-Chapel Hill, the Imaging Africa workshop is free of financial burden to all attending students. The expenses associated with air travel, accommodation, and food will be covered by Imaging Africa. Eligible applicants must currently be at an academic institution in the continent of Africa.

The workshop will be hosted at the University of Cape Town’s Institute of Infectious Disease and Molecular Medicine, South Africa from the 13th to the 16th of January 2020 and will be followed by a research symposium on the 17th of January 2020. Please visit www.imagingafrica.org for more information. Applications for the workshop close on the 15th of October 2019.

Please help us in making a meaningful impact on African researchers by forwarding this information to your friends and colleagues from any and all African institutions.

Sincerely,

Teng-Leong Chew, HHMI Janelia Research Campus, USA Dan Fletcher, Univ of California-Berkeley, USA Klaus Hahn, Univ of N. Carolina-Chapel Hill, USA Musa Mhlanga, Univ of Cape Town, S. Africa Kelly Rogers, Walter & Eliza Hall Institute, Australia Digby Warner, Univ of Cape Town, S. Africa