Two-photon microscope achieves in vivo calcium imaging with near-millisecond precision

Neural circuits in the brain fire on the millisecond time scale. Although two-photon calcium imaging can image this in vivo, its temporal resolution isn’t as high as electrical recordings. Now, researchers from the Brain Research Institute at the University of Zurich have designed a two-photon microscope that makes use of an acousto-optic deflector (AOD) to image at rates up to 500 Hz.

Standard two-photon setups use galvanometric mirrors for scanning and can typically sample at up to 10 to 15 Hz. Using an AOD instead allows movement of the laser focus between any two positions in a field of view in milliseconds and also lets the imaging be concentrated on the structures of interest. However, AODs require correction of spatial and temporal laser beam distortions, which the researchers accomplished by modifying a single-prism compensation method to improve transmission and dispersion compensation over a large field of view.

Using this setup they were able to measure fluorescence from neurons in the mouse neocortex at a 180 to 490 Hz sampling rate, detect single action potential evoked calcium transients with signal-to-noise ratios of 2 to 5, and calculate spike times with near-millisecond precision.

Research paper:
High-speed in vivo calcium imaging reveals neuronal network activity with near-millisecond precision
Benjamin F Grewe, Dominik Langer, Hansjörg Kasper, Björn M Kampa & Fritjof Helmchen,
Nature Methods 7, 399 - 405 (2010) 
doi:10.1038/nmeth.1453

Fluorescent proteins for mouse embryo imaging

Fluorescent proteins have led to numerous discoveries in biology and have become so important that the work on the original green fluorescent protein won a Nobel prize last year. In the last decade or so, researchers have added many new colors to the original green fluorescent protein, have fine tuned these proteins to make them brighter and easier to use, and have introduced new techniques using fluorescent protein characteristics that were modified or even discovered.  

With so many new developments I was glad to see that researchers from the Sloan-Kettering Institute in New York have published an excellent review paper in Biotechnology Trends that puts the most important information tied to fluorescent proteins in one place.  The review somewhat focuses on mouse embryo imaging, but doesn't skip over the history or the basics.

The writers include a helpful table detailing the excitation/emission, oliogmeric state, commercial availability and reference for almost 30 of most common ones. I have read the original publications on many of these proteins but did'nt realize just how many "common" ones there were until seeing this list. I found myself clicking on several of the references to remind me which research groups were involved in development or discover of the various fluorescent proteins. 

The authors believe that combining fluorescent proteins with mouse genetics is essential for unraveling mechanisms involved in regulating embryonic development. Thus, they detail studies several applications related to mouse imaging and point out that development of mouse strains for live imaging has been a limiting step in many applications.

The authors do include discussion of some alternative technologies such as quantum dots and small molecule probes, but seem to think that fluorescent proteins are the way of the future, at least for mouse embryo imaging. I think that quantum dots are quite promising for imaging and are often easier to use than fluorescent proteins. 

Do you think quantum dots will eventually replace fluorescent proteins?

Trends in Biotechnology paper: Live-imaging fluorescent proteins in mouse embryos: multi-dimensional, multi-spectral perspectives