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Optical scattering poses a significant challenge to high-resolution microscopy within deep tissue. To accurately predict the performance of various microscopy techniques in thick samples, we present a computational model that efficiently solves Maxwell’s equation in highly scattering media. This toolkit simulates the deterioration of the laser beam point spread function (PSF) without making a paraxial approximation, enabling accurate modeling of high-numerical-aperture (NA) objective lenses commonly employed in experiments. Moreover, this framework is applicable to a broad range of scanning microscopy techniques including confocal microscopy, stimulated emission depletion (STED) microscopy, and ground-state depletion microscopy. Notably, the proposed method requires only readily obtainable macroscopic tissue parameters. As a practical demonstration, we investigate the performance of Laguerre–Gaussian (LG) versus Hermite–Gaussian (HG) depletion beams in STED microscopy. 

We numerically compare the null quality for STED microscopy generated by Laguerre-Gaussian beams with orbital angular momentum and donut beams generated by incoherent addition of orthogonal Hermite Gaussian beams when imaging deep biological tissue. 

Imaging sub-diffraction dynamics of neural nanostructures involved in behaviors such as learning and memory in a freely moving animal is not possible with existing techniques. Here, we present a solution in the form of a two-photon (2P), fiber-coupled, stimulated emission depletion microscope and demonstrate its capabilities by acquiring super-resolution imaging of mammalian cells. A polarization-maintaining fiber is used to transport both the 2P excitation light (915 nm) and the donut-shaped depletion beam (592 nm), which is constructed by adding two temporally incoherent and orthogonally polarized Hermite–Gaussian fiber modes. The fiber output is insensitive to bending or temperature changes and is the first demonstration toward deep tissue super-resolution imaging in awake behaving animals. 

We demonstrate a two photon (2P) fiber STED microscope in which the excitation and STED light are delivered to the sample in polarization maintaining (PM) fiber. 

The DyMIN method reduces photobleaching, a problem in STED microscopy. Labs implementing custom-built STED microscopes would greatly benefit from DyMIN capabilities. We present an inexpensive, open-source version utilizing an FPGA and multiplexer.