Sample preparation – 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!

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.

Apply now to the LCI microscopy course 2020 :)

It is now time to register to the LCI intensive microscopy course (Jan/Feb 2020). Check out the course schedule.

Loads of fun workshops, informative lectures, intense discussions and our popular Student Imaging Challenge workshop where students get direct feedback on how to improve their own sample preparation/experimental design.

We always run two courses in parallel:

the full course (#2870, 6 points, apply here) where students attend all activities
the theory only course (#2871, 4.5 points, apply here) for students who only attend the lectures

As usual, all lectures are public and broadcasted live so you are welcome to just show up (how to find us) or watch remotely (how to connect) without registration. Check the program as it may be updated in case of (unlikely) last minute changes.

We welcome your feedback about the quality of the webinar and the content of the lectures.

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.

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.

LCI product seminar: How to label organelles in live cells?

On Wednesday (13^th) at 9:30 in Lipid seminar room in Neo (KI Flemingsberg), please come and enjoy a short seminar presenting a new way to label organelles in live cells.

LabLife will present their product called Viromer Cytostain.

We will stream the seminar live so you can follow it even from your desk! 🙂

CUBIC seminar tomorrow at Scilife

Do not miss the seminar tomorrow morning at Scilife: the inventor of the CUBIC technique, Etsuo Susaki, will present his technique which can clear fatty and dense tissues.

Click here to know where and when.

And here is the link to the updated Cubic protocol.

Matching the Refraction index of the sample with the objective

Together with the coverslip and the immersion medium (oil, water, glycerol or air), the sample mounting medium is part of the design of a microscopy objective. Matching the refraction index of the sample to the one recommended by the manufacturer of the objective will make the sample transparent for the objective, drastically improving fluorescence microscopy in samples thicker than a couple of um (i.e. anything except fluorescent beads!).

Not matching the refraction indices is equivalent to watching something through a wet window… Far from optimal! :-/

The refraction index recommended by the manufacturer is the same as the RI of the immersion medium: 1.52 for an oil immersion objective, 1.47 for a glycerol objective, 1.33 for a water objective, 1 for an air objective.

This article compares 7 mounting media and their effect on the refraction index of brain samples. CFM3 seems to be a cool mounting medium. The company that produces it has partially paid for the study but it sounds worth a try anyway!

In the same vein, this article presents a non-toxic way to change the refraction index of cell culture medium (not the sample) to improve imaging of live samples. Sounds pretty promising to grow live organoids which quickly become opaque. This will also be very useful when clearing samples as the sample chamber on a light sheet microscope is big so this is a cheap way to fill the chamber for imaging. 🙂

If you try any of these 2 chemicals, please leave a comment to let us know how it went! 🙂

Glyoxal instead of PFA

Poor PFA fixation often causes trouble in antibody staining. Folded cells, poorly preserved cytoskeleton… These artifacts appear when the stock of PFA gets older and degrades. Buying ready made PFA solutions, most of which contain 10-15% of methanol, can also lead to low labelling with some antibodies.

Glyoxal seems to be a good alternative. It had the added advantage that it is less toxic.

Check this article to know more. 🙂