Find below three interesting methods papers relevant for neuroscience. All three of them are, in my opinion, worth a quick read.

Acoustic cameras to localize ultrasound vocalization of mice

Sterling et al. (2023) from the lab of Bernhard Englitz addressed the problem how to localize ultrasound vocalizations (USVs) from rodents, in particular mice. The localization of USVs and their reliable attribution to a specific mouse can be challenging, especially when mice are interacting snout-to-snout. The authors managed to perform sound localization with a precision of less than 5 mm (91% of USVs correctly assigned). How do they achieve this?

The authors combined two complementary approaches. In a first approach, they used an “acoustic camera” formed by an 8×8 array of 64 ultrasound microphones (https://sorama.eu/products/cam64). This approach provided spatially precise localization but was less reliable for high frequencies. In a second approach, they used an array of only 4 but high-quality ultrasound microphones (https://www.avisoft.com/). Figure supplement 1-1 nicely compares the noise spectra of these two microphone types. The second approach was more important to detect USV events, while the “acoustic camera” approach was essential for localization of the detected events. A nice methods paper!

Glyoxal fixation for improved antibody staining

In this study, Konno et al. (2023) from the lab of Masahiko Watanabe refined and tested an alternative protocol for immunohistochemistry (IHC), following up on a study by Richter et al. (2018). The authors replaced the standard fixative (4% paraformaldehyde, PFA) by a different solution (glyoxal) and find that this modified fixation protocol enables improved diffusion of and labeling with antibodies, including antibodies against important proteins such as synaptic adhesion molecules, receptor proteins and ion channels.

The authors mention a few caveats in the Discussion: Glyoxal fixation improved signal for certain antibodies but not for all, without an indication why this could be the case. The sample seemed to be less than hard and fixed for glyoxal than for PFA fixation. Moreover, they report that “ultrastructural images of neurons and synapses” were more strongly compromised by glyoxal fixation compared to PFA fixation. This last comment seems interesting, since Richter et al. (2018) in their original publication on glyoxal fixation stated that ultrastructure was better conserved in glyoxal than in PFA. I’m curious whether glyoxal might become the new standard for IHC during the next decade!

Near-infrared co-illumination protects fluorophores

Fluorescence occurs when fluorophore molecules transition into an excited state and, during the relaxation process into the ground state, release a photon. This process can go awry when the molecule transitions from the excited state into a dark (triplet) state or other non-desired states where the molecule is stuck (photobleaching) or which result in chemical reactions that generate oxidative stress (phototoxicity). As an experimentalist, it is essential to find the balance between (a) as much power as necessary (to be able to observe the structures of interest) and (b) as little power as possible (to prevent bleaching and toxicity).

In a surprising study, Ludvikova et al. (2023) find that near-infrared (NIR) light prevents photobleaching and phototoxicity. The authors performed imaging of standard fluorescent proteins (EGFP) in cell culture using one-photon widefield microscopy with an excitation wavelength of around 470 nm. They find that co-illumination with NIR light (approx. 900 nm) reduced bleaching induced by 470 nm. It is not fully clear from the study why NIR co-illumination prevents transitioning into triplet states, but the evidence that this indeed happens seems very convincing.

It would be interesting to see how this can be generalized to different illumination intensities and patterns, e.g., to confocal or light-sheet microscopy. And it poses the intriguing question whether two-photon microscopy of green fluorophores, which typically uses a wavelength in the NIR (around 920 nm), also takes advantage of this protective effect. For two-photon microscopy, illumination already occurs at around 900 nm, so it could be that the same photons that induce fluorescence also protect from bleaching/toxicity. It seems however challenging to test this hypothesis experimentally. As a sidenote, I’m reminded of my own experiments (reported in this blog post) where I found that long-lived dark triplet states seemed to be less relevant at 920 nm than predicted from experiments performed at 800 nm. I’m hoping that follow-up studies will further dissect the NIR co-illumination effect and study its relevance for other imaging modalities, especially confocal and light-sheet imaging!

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References

Konno, K., Yamasaki, M., Miyazaki, T., Watanabe, M., 2023. Glyoxal fixation: An approach to solve immunohistochemical problem in neuroscience research. Sci. Adv. 9, eadf7084. https://doi.org/10.1126/sciadv.adf7084

Ludvikova, L., Simon, E., Deygas, M., Panier, T., Plamont, M.-A., Ollion, J., Tebo, A., Piel, M., Jullien, L., Robert, L., Le Saux, T., Espagne, A., 2023. Near-infrared co-illumination of fluorescent proteins reduces photobleaching and phototoxicity. Nat. Biotechnol. 1–5. https://doi.org/10.1038/s41587-023-01893-7

Richter, K.N., Revelo, N.H., Seitz, K.J., Helm, M.S., Sarkar, D., Saleeb, R.S., D’Este, E., Eberle, J., Wagner, E., Vogl, C., Lazaro, D.F., Richter, F., Coy-Vergara, J., Coceano, G., Boyden, E.S., Duncan, R.R., Hell, S.W., Lauterbach, M.A., Lehnart, S.E., Moser, T., Outeiro, T.F., Rehling, P., Schwappach, B., Testa, I., Zapiec, B., Rizzoli, S.O., 2018. Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy. EMBO J. 37, 139–159. https://doi.org/10.15252/embj.201695709

Sterling, M.L., Teunisse, R., Englitz, B., 2023. Rodent ultrasonic vocal interaction resolved with millimeter precision using hybrid beamforming. eLife 12, e86126. https://doi.org/10.7554/eLife.86126