Widening the View of Two-Photon Microscopy
Material below is adapted from the SfN Short Course Mesoscale Two-Photon Microscopy Engineering a Wide Field of View with Cellular Resolution, by Jeffrey N. Stirman, PhD, and Spencer L. Smith, PhD. Short Courses are daylong scientific trainings on emerging neuroscience topics and research techniques held the day before SfN’s annual meeting.
Mapping activity in the brain requires both a wide lens — to see connections extending across brain regions — and a fine focus — to see the cells forming those connections. Two-photon microscopy coupled with calcium imaging meets the criteria for a fine focus; it can detect activity within individual neurons or within a local population of neurons. But beyond a small window, two-photon excitation lessens and the resolution suffers. This narrows the field of view, making mapping long-range connections impossible. Fortunately, newly described tweaks to both the microscopes and imaging techniques are making “mesoscale” two-photon microscopy possible, allowing wider views than can span brain regions a millimeter or more apart. Recently, this type of imaging has been performed in vivo to map activity in the visual system of mice.
In a laser scanning microscope, the laser beam bounces off two mirrors that rotate back and forth to move the laser’s focus across the specimen. One of the main determinants of the field of view is the size of the angles these mirrors take as the laser scans. Higher scanning angles are required for viewing a larger field, so the first step in mesoscale two-photon microscopy is considering how to accommodate such high scanning angles. Accommodations include using objectives with high numerical apertures (contributing to better resolution) and short focal lengths (allowing wider views).
High scanning angles can also accentuate aberrations, distortions created by the imaging system. This is a problem in two-photon microscopy because a clear focal spot is needed to achieve two-photon excitation, and aberrations can prevent the formation of such a focal point, limiting the usable field. Again, accommodations can be made, such as using a compound lens, creating a custom lens, or correcting distortions with adaptive optics.
Finally, large fields of views can mean scanning takes a long time, a problem for some applications. Different scanning techniques can increase imaging speed. Resonant scanners, which scan at a rate four to twelve times faster than conventional scanners, provide high enough scanning angles and a wide field of view. Acousto-optical deflectors use sound waves to bounce the beam back and forth, improving speed a hundredfold but not necessarily widening the field of view. Arbitrary line scanning does a quick pass to find the cells of interest, and then directs the laser to those cells in particular, cutting down on time wasted scanning regions without active neurons. Additionally, these methods can be combined to use multiple beams to scan separate areas. The more beams, the faster the imaging speed.
Of course, other approaches (such as using two microscopes or quickly repositioning the microscope or sample) could also enable imaging of multiple brain regions. But by making certain accommodations to the microscope and technique, mesoscale two-photon microscopy can be achieved in a novel way. Mesoscale imaging will allow detection of both the cells making connections and the range of the connections, creating an activity map of unprecedented detail and scope.