In the last few months, I built a special kind of 2P-microsope. In the meantime, I encountered some papers on microscope techniques which I found interesting and worth a side-note.
- Using AODs instead of galvoscanners for point-scanning: High-speed in vivo calcium imaging reveals neuronal network activity with near-millisecond precision. In contrast to resonant galvoscanners, you can still define arbitrary scanning paths. In a more recent paper which I don’t find right now, it is shown that by defining a scanning path for the AODs not by the path but only the endpoints of the path, you can go effectively as fast as you want. (This is not shown for population calcium imaging, but imaging of dendrites/spines.) The perspective of not being limited by scanning in principle is quite promising.
- Using several beams for scanning several z-layers: Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing. The idea is quite simple, it’s based on the fact that the typical fluorophor lifetime (1-3 ns) is shorter than the time window between two laser pulses (typically 12.5 ns), so that pulses with different z-focus are delayed by some nanoseconds; the fluorescence can be gated.
- Instead of using a moving objective, they used a moving mirror to do the z-scan: Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates. I liked very much the idea of putting the mirror on two two galvanometers instead of a fast piezo as I would have done it in the first place. Piezos at the objective holder as the standard method to change the z-focus are quite fast right now (settling time 2-4 ms at maximum), but they induce vibrations in the setup and are only that fast if they carry a light load and have a limited traveling range (ca. 100 µm).
- Temporal Focusing is a method to provide z-sectioning in a widefield setup. This was the reason why I came to Vienna in 2013: Brain-wide 3D imaging of neuronal activity in Caenorhabditis elegans with sculpted light. I spent five months on improving this setup.