This is not really about microscopy, but I end up building enough things that I thought this might be useful to other people as well: How to Build your Everything Really Really Fast is an instructable from an MIT instructor on fabrication for hobby-type projects, but a lot of it would be relevant to fabricating a custom mechanical assembly for a microscope.
A very cool new paper has just come out in Nature Methods: Misha Ahrens and Philipp Keller have built a light sheet microscope capable of imaging the whole zebrafish brain in about 1.3 seconds. Coupled with fish expressing neuronal GCaMP5, they’re able to image neural activity in the whole fish brain. Here’s one of their movies:
At Photonics West, back in January, I saw a booth from InfiniLED which was promoting their microLED arrays. These are LEDs that are around 25 μm in size and can be fabricated into arrays with spacings between LEDs only a bit larger than the size of the LEDs themselves. There is an article by and about them in the newest issue of Photonics Spectra. Another company pursuing the same technology is mLED. Both companies are still in pretty early stages of technology development, but demo systems are available from both.
An LED array from mLED
These could be a nice technology for photoactivation applications. The array sizes are still a bit small, but mLED, for example, has a 4096 pixel array of 16 μm pixels spaced 50 μm apart, and their LEDs can be manufactured to emit at 405 nm. Particularly if they could get to hundreds of thousands of LEDs, it’s easy to imagine placing one of these LED arrays at a conjugate imaging plane and using it to do spatially directed photoactivation.
We’re building a new microscope in the NIC. One of the goals of this microscope is to be fast. We’re getting a 100 frame per second Hamamatsu Flash 4.0 camera, and we’d like to be able to operate the rest of the hardware at that speed (or as close to it as we can achieve). For piezo Z-stages and LEDs, this isn’t that hard, but what about an XY stage?
One approach is to do what Austin has done – have the stage trigger the camera to expose when it’s reached position, and the camera trigger the stage’s next move when it’s done. This works pretty well, as you can see. Another approach, however, would be to have the stage move continuously and strobe the illumination when each field of view is centered over the camera. Can this be done?
In the past decade, some interesting fluorescent protein dye complexes have been published. These are antibodies which bind a non-fluorescent dye (a fluorogen) and stabilize it in a conformation that makes it fluorescent – for example . I’ve been curious about these for a while but the lack of commercial availability made them inconvenient to work with. It turns out that the scFvs from  – they called them fluorogen activating proteins or FAPs – are now commercially available from SpectraGenetics. There are two versions, a green (FITC-like) FAP (which is presumably the thiazole orange binding scFv) and a far red (Cy5-like) FAP (which is presumable the malachite green binding FAP). The far red FAP comes with two different fluorogens – one which is cell permeable and one which is cell impermeant (the only green fluorogen is impermeant). This means you can distinguish between protein on the cell surface and protein that is inside the cell .
There are probably other clever things you can do with these; if you try them out, let me know.
- A. Simeonov, M. Matsushita, E.A. Juban, E.H. Thompson, T.Z. Hoffman, A.E. Beuscher, M.J. Taylor, P. Wirsching, W. Rettig, J.K. McCusker, R.C. Stevens, D.P. Millar, P.G. Schultz, R.A. Lerner, and K.D. Janda, "Blue-fluorescent antibodies.", Science (New York, N.Y.), 2000. http://www.ncbi.nlm.nih.gov/pubmed/11030644
- C. Szent-Gyorgyi, B.F. Schmidt, B.A. Schmidt, Y. Creeger, G.W. Fisher, K.L. Zakel, S. Adler, J.A.J. Fitzpatrick, C.A. Woolford, Q. Yan, K.V. Vasilev, P.B. Berget, M.P. Bruchez, J.W. Jarvik, and A. Waggoner, "Fluorogen-activating single-chain antibodies for imaging cell surface proteins.", Nature biotechnology, 2007. http://www.ncbi.nlm.nih.gov/pubmed/18157118
- G.W. Fisher, S.A. Adler, M.H. Fuhrman, A.S. Waggoner, M.P. Bruchez, and J.W. Jarvik, "Detection and quantification of beta2AR internalization in living cells using FAP-based biosensor technology.", Journal of biomolecular screening, 2010. http://www.ncbi.nlm.nih.gov/pubmed/20488980
- J.P. Holleran, M.L. Glover, K.W. Peters, C.A. Bertrand, S.C. Watkins, J.W. Jarvik, and R.A. Frizzell, "Pharmacological rescue of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) detected by use of a novel fluorescence platform.", Molecular medicine (Cambridge, Mass.), 2012. http://www.ncbi.nlm.nih.gov/pubmed/22396015
The highlight of day two of ABRF, for me, was the talk by Robert Campbell on fluorescent reporters. He started off by saying that existing fluorescent proteins were quite good and unlikely to get dramatically better. He spent the rest of his talk discussing his labs work to improve fluorescent protein reporters, where there is a lot of work still to be done. One of his guiding principles is “redder is better”.
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.
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.
I had the pleasure last week of demoing a Yokogawa CSU-W1 spinning disk confocal – this is the new large field-of-view Yokogawa scanhead. Andor and Technical Instruments arranged a demo for us and paired the scanhead with an Andor Zyla camera. I’ll have more to say about the demo later – it’s a pretty cool confocal – but for now I want to focus on some field of view (FOV) issues it raised.