ABRF Report, Day 1

I just got back from attending the meeting of the Association of Biomolecular Resource Facilities in Palm Springs. It was a nice little meeting, and not just because it was in sunny Palm Springs.

PSCC

The view from the convention center.

This is the first time that I had been to the ABRF meeting and my understanding is that this is the first time that microscopy had its own track at the meeting. The meeting is basically a meeting of core managers, and it was dominated by next-generation sequencing, along with some mass spectrometry and flow cytometry. Not surprisingly, I attended primarily the microscopy sessions.  What follows are a few things I thought were noteworthy.

The microscopy sessions opened Sunday morning, and were supposed to start with a talk by Jennifer Lippincott-Schwartz. However, she had to cancel at the last minute due to the sequester, and some hurried rearrangements gave Jonas Ries the opening talk. He gave a nice talk on his work on STORM microscopy, focusing mostly on his work with GFP nanobodies [1]. These nanobodies are commercially available from Chromotek, and were mentioned favorably by a few others. Among other things, they will penetrate the yeast cell wall and so can be used in fixed yeast without spheroplasting.

Jonas presented a lot of two-color dSTORM using Alexa647 and Alexa700.  He also discussed Binding Activated Localization Microscopy [2], which is analogous to PAINT [3]. In this technique, single molecule spots are generated by binding of a DNA-binding dye to DNA (they used YOYO-1); the dye is relatively non-fluorescent until DNA bound, giving the mechanism of contrast enhancement. Using the reducing / oxidizing ROXS buffer [4], they are able to get very high localization precision and very high labeling density, giving high super-resolution contrast. (As an aside, the acronyms in this field are out of control: BALM has now been used for two different superresolution approaches, the other being Bleaching Assisted Localization Microscopy [5]).

There were also talks introducing STED, SIM, and STORM from Leica, Applied Precision / GE, and Nikon. Nothing too new there, although Leica is now doing time-gated STED detection to improve the resolution.  In the afternoon, there was a discussion on super-resolution methods, in which a couple useful things came up. Good fixation is really critical for super-resolution, and in some ways we are relearning what the electron microscopists already knew. The general consensus is that formaldehyde isn’t enough and you need to add glutaraldehyde for most samples (at least at low concentration). However, this introduces autofluorescence (which can be quenched with sodium borohydride) and also may block certain antibody epitopes. Mounting can also be an issue – Alison North described artifacts arising from 3D-SIM imaging of yeast mounted in PBS: the yeast act like lenses. This goes away if you mount the yeast in glycerol so they are index matched. There was general consensus that high precision coverslips should be used for super-resolution imaging as well.

I’ll have more to say about day two tomorrow.

References