For typical confocal or two-photon microscopes that maintain (sub)cellular resolution, a high-magnification objective is needed (typically 16x, 20x or 25x). This in turn limits the field of view (FOV) to ⌀ 1.0-1.5 mm.

For imaging in the mouse brain cortex, which is basically a big unwrinkled surface of a size of the order of 10 mm, a bigger FOV would be nice to have for some applications. Recently, a couple of papers came out that tried to increase the FOV, while using optical engineering to maintain the resolution. (Please don’t hesitate to tell me if I missed a relevant publication.)

• Stirman et al. and Smith (Nature Biotechnology, 2016) designed the full optical path including the objective and get a ⌀ 3.5 mm FOV with an axial FWHM of ~12 um and a working distance of 8.5 mm. (Versions of the paper have been around on bioRxiv since 2014.)
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• Tsai et al. and Kleinfeld (Optics Express, 2015) used an off-the-shelf objective with a working distance of 29 mm and achieve a resolution of ca. 15 um FWHM in z in an 8 x 10 mm FOV.
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• Sofroniew, Flickinger et al. and Svoboda (eLife, 2016) basically did the same thing as Stirman et al. and achieved a ⌀ 5 mm FOV with an axial resolution of 4-7 um, depending on the lateral distance from the center position, and a working distance of 2.7-3 mm.

Few years ago, I would have expected such papers to be published in Nature Methods, but apparently the time has come where optical engineering and improvement of existing techniques is not considered enough for passing the novelty bar. However, the three papers offer some very interesting lessons on engineering a two-photon microscope, of which I want to pick a few:

• The use of large-aperture (15 mm) galvo scanners by Tsai et al. in order to avoid large scanning angles that would create large aberrations. (Thus the design cannot be used with resonant scanners which have much smaller apertures.) The large beam diameter at the galvos allows to use a low-magnification scan lens-tube lens pair, which demagnifies the scan angle to a lesser extent. It is important to understand that the scan lens-tube lens telescope magnifies the beam diameter, but at the same time decreases the scan angle by the same factor.
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• Due to the extremely large and heavy costum-designed objectives (click here for a picture of the Stirman et al. objective [update 2018: the picture has since been removed]), remote focusing is necessary for fast switching of the axial focus. Stirman et al. use optotune lenses; Sofroniew, Flickinger et al. take advantage of a remote mirror technique that has been developed 2007-2012, but use a voice coil motor for mirror displacement – interestingly, I had converged onto the same solution when I constructed my z-scanning module (see this previous blog post or the dedicated paper).
  The optotune solution by Stirman et al. is in my opinion less well-suited for remote scanning, since resolution cannot be maintained over large z-ranges due to optical issues (although this is not mentioned in the paper). It’s probably good enough for small z-ranges, but it has to be considered in the optical design from the beginning.
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• Sofroniew, Flickinger et al. use something they call a virtually conjugated galvo pair (VCGP) to avoid annoying relay optics. I do not understand why they came up with this strange name or whether this design has been used before, but the principle is quite nice.
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• Stirman et al. use temporal multiplexing to image two independently chosen locations using separated, delayed light paths, similar to this 2011 paper (also check out this thesis for less polished pictures).
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• A chapter that I found interesting to read is “Tolerancing and sensitivity analysis” in the Stirman et al. paper.
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• Sofroniew, Flickinger et al. employed oil interfaces close to the PMT surface to increase the photon collection NA.
  On a related note, it is interesting to read their speculations about inhomogeneities of the PMT photocathode.

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The problem with these microscope designs is – how to adapt them to one’s own lab? The goal should be to generate something which does not work for a paper only, but as a reliable and robust tool. I can see two (non mutually exclusive) possibilities how this could be achieved. The first is transparency, with the free and open distribution of Zemax files and software to anybody who wants it. In this spirit, I liked a lot figure 2 in the paper by Sofroniew, Flickinger et al., because it clearly shows the optical design a) as a scheme, b) as a CAD drawing, and c) as a real life picture.

A second way would be to license the design to companies like Scientifica, Thorlabs or maybe even smaller spin-offs/start-ups like Neurolabware or Vidrio Technologies. Similar to turn-key femtosecond lasers that revolutionized the field of 2P microscopy when they became available, one can hope for a company that puts together modular units that are stable, robust and working out of the box to enable complex microscopes (with z-scanning, with multi-region scanning, with simultaneous spatially patterned optogenetics, with multiple detection channels, with >1100 nm coatings for excitation, with adaptive optics for deep tissue imaging, etc.) in normal neuroscience labs. I’m probably not the only one who is fascinated by these technologies, but if someone has a neuroscientific question, he does not want to spend his whole life on the development of a high-end microscope. (Moreover, it would not be very rewarding, because this type of engineering and optimization process will not be rewarded by any kind of top-level publication.) Similarly, nobody wants to build his own femtosecond-pulsed laser for 2P imaging these days (although there are always exceptions, for example this one).