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Thinking Outside the Box: Microscopy for Immunologists

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Thinking Outside the Box: Microscopy for Immunologists

When you think of an immunologist, you will likely imagine someone who studies the immune system… or maybe a person who speaks in a completely different language (CD? IL? The list goes on.). You may also think of a slew of assays that almost exclusively “belong” to immunologists, including ELISA, ELISpot, Flow Cytometry, chromium release assay, and so on. While immunologists rely heavily on these assays, there is another commonality. Traditionally, all of these protocols require 96 well plates. Crazy, right? But here’s the thing: you don’t have to only use these assays to investigate immune questions. Break away and taste some of the other flavors!

Light Microscopy

Several analysis techniques already exist that don’t necessarily require a 96-well plate, like investigating DNA, RNA, and/or protein. So it’s a fairly easy jump to get into the world of light microscopy, even for immunologists. You can’t manage the same investigative panel that you would with flow cytometry, so it works best when you are interested in a few choice proteins or structures. And it results in beautiful images that can really bolster your research.

One tip: when you set up your cover slide, be sure to pre-coat your slides in L-lysine, especially when you are studying lymphocytes (i.e. T cells, B cells, NK cells). This group of immune cells do not adhere strongly to most surfaces, so without the L-lysine your cells will float away before you even get your antibody washed off!

Laser Capture Microscopy

If you’ve ever taken an image of a cell using a microscope and thought “Wow, that cell looks really interesting. I wish I could study it more!”, laser capture microscopy is for you. At it’s core, laser capture microscopy (LCM) is a method in which the researcher can actually cut out tissue subpopulations from larger pieces of tissue or even an individual cell while directly visualizing it simultaneously with a microscope.

In short, you first stain your tissue/cells of interest with antibodies just like you would for immunohistochemistry imaging. Then you dehydrate your sample and load it onto the laser capture microscope. Those cells that are labeled with antibody attach to an IR capture laser and are cut out of the surrounding tissue via the ultraviolet (UV) laser.

Now, LCM is in no way a new technique, but with immunology melding more and more with other fields (i.e. cancer, virology, etc.), using this kind of technique could be incredibly useful in the study of diseased tissue, where the researcher is interested in cells from a specific area. Moreover, since the entire morphology of the cell(s) remains intact, evaluation can be more precise.

Two-Photon Excitation Microscopy

The standard methodology to develop a 3D image is confocal microscopy. However, confocal laser scanning microscopes can only penetrate about 50 µm deep, which makes it difficult to use confocal microscopy when investigating thick, complex, and multi-cellular lymphoid tissue. An alternative to this is two-photon excitation microscopy, which emerged in 1990.1

What makes two-photon excitation microscopy so different is, you guessed it, two photons are sent out to excite the fluorophore! But why does this make a difference? Well, since the two excitation photons are almost simultaneously absorbed by the fluorophore, the emitted light “increases in proportion to the square of the excitation intensity”.2 So all of this specific light at the focal point allows for 1) accurate identification of the cell of interest, and 2) minimizes any unspecific capturing since the highest density of photons is at the focal point only.

All in all, two-photon excitation microscopy can actually penetrate nearly 400 µm, which is perfect for all those intact organs you’ve been itching to take a look at.

Analytical Histo-Cytometry

Now, what’s an immunologist to do when they would really like to not only see the composition of cells within tissue, but also WHERE those cells actually are? Have no fear! That’s where analytical histo-cytometry comes into play.

Similar to the other methods we’ve covered, histo-cytometry has been around for at least 5 years. But it doesn’t make it any less awesome! This protocol enables the best of both worlds: the phenotypical quantification of complex cell populations directly in the tissue. The methodology is complex, but amazing. It first builds on multiplex staining of cells using antibodies, much like you would perform in flow cytometry. Using conventional diffraction-limited confocal microscopy, researchers are able to accurately detect fluorophore signals on x, y, and z axes. Then, after several other computing steps, the machinery forms 3D volumetric surface objects to correspond to individual cells so that the read out is similar to flow cytometry phenotypic profiling and quantification.3 The merging of two fields is quite spectacular, no?

And that brings us to our wrap up of bringing immunologists into the light and away from the 96-well plate, at least for some of the time. What are your favorite cross-specialty assays? Comment below!


  1. Denk W et al. Two-photon laser scanning fluorescence microscopyScience 1990; 248:73–76.
  2. Cahalan, M.D. Two-Photon Tissue Imaging: Seeing the Immune System in a Fresh Light. Nat Rev Immunol. 2002; 2(11):872-880.
  3. Gerner, M.Y. et al. Histo-Cytometry: in situ multiplex cell phenotyping, quantification, and spatial analysis applied to dendritic cell subset micro-anatomy in lymph nodes. Immunity. 2012; 37(2):364-376.
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