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

Area of Science:

• Neuroscience
• Optical Engineering
• Microscopy

Background:

• Conventional two-photon microscopy (TPM) offers subcellular resolution but is limited by a small field-of-view (FOV).
• Existing methods for extending FOV in TPM lack a standardized design approach, hindering development of large FOV TPM (LF-TPM).

Purpose of the Study:

• To establish a principled design strategy for constructing LF-TPM systems using readily available optical components.
• To demonstrate a significant increase in FOV for TPM without compromising resolution.

Main Methods:

• A design strategy based on analyzing the optical invariant of individual components (objectives, relay lenses, scanners, collection systems).
• Component selection to maximize the space-bandwidth product of the integrated LF-TPM system.
• Construction and testing of an LF-TPM system using the proposed design approach.

Main Results:

• The developed LF-TPM system achieves a 7-mm diameter FOV, a 100-fold increase over conventional TPM.
• Maintained lateral and axial resolution of [Formula: see text] and [Formula: see text], respectively.
• Successfully performed in vivo imaging of mouse cerebral vasculature and microglia across cortical regions.

Conclusions:

• The presented design strategy simplifies LF-TPM development and is adaptable to various microscopes with optical relays.
• LF-TPM offers unprecedented FOV for studying large-scale neural structures and dynamics, such as interhemispheric brain regions.
• This advancement facilitates comprehensive analysis of brain microarchitecture and cellular activity in vivo.