3D image scanning microscopy with engineered excitation and detection

Scanning microscopes are important research tools for investigating 3D specimens. Modern beam shaping techniques can be combined with suitably designed data processing algorithms to improve instrument versatility and imaging performance. Here we introduce image scanning microscopy with freely programmable excitation and detection pupils and investigate point spread function (PSF) designs for parallel 3D information acquisition. The volumetric data is collected in a single 2D scan without the requirement of physical refocus. By sculpturing the excitation and detection PSFs into helical shapes of opposing handedness, we are able to capture sample information in a volume whose axial extension measures more than four times the z resolution. In a more generalized approach, jointly optimized phase masks are used in both pupils to shape the PSFs. As an exemplary case, we study the use of beam-splitting phase masks for the parallel scanning in multiple planes. The image reconstruction algorithm optimally integrates this information according to the various signal-to-noise ratios. Generalized PSF engineering scanning systems provide resolution improvement relative to confocal microscopy while accelerating data collection. We analyze the opportunities, trade-offs, and limitations of the approach.