Designing a large field-of-view two-photon microscope using optical invariant analysis

Jonathan R. Bumstead, Jasmine J. Park, Isaac A. Rosen, Andrew W. Kraft, Patrick W. Wright, Matthew D. Reisman, Daniel C. Côté, Joseph P. Culver

Research output: Contribution to journalArticlepeer-review

51 Scopus citations


Conventional two-photon microscopy (TPM) is capable of imaging neural dynamics with subcellular resolution, but it is limited to a field-of-view (FOV) diameter <1mm. Although there has been recent progress in extending the FOV in TPM, a principled design approach for developing large FOV TPM (LF-TPM) with off-the-shelf components has yet to be established. Therefore, we present a design strategy that depends on analyzing the optical invariant of commercially available objectives, relay lenses, mirror scanners, and emission collection systems in isolation. Components are then selected to maximize the space-bandwidth product of the integrated microscope. In comparison with other LF-TPM systems, our strategy simplifies the sequence of design decisions and is applicable to extending the FOV in any microscope with an optical relay. The microscope we constructed with this design approach can image <1.7-μm lateral and <28-μm axial resolution over a 7-mm diameter FOV, which is a 100-fold increase in FOV compared with conventional TPM. As a demonstration of the potential that LF-TPM has on understanding the microarchitecture of the mouse brain across interhemispheric regions, we performed in vivo imaging of both the cerebral vasculature and microglia cell bodies over the mouse cortex.

Original languageEnglish
Article number025001
Issue number2
StatePublished - Apr 1 2018


  • etendue
  • neurophysiology
  • optical design
  • optical invariant
  • scanning microscopy
  • two-photon microscopy


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