Stimulated Raman scattering (SRS) microscopy enables the imaging of molecular events on a human subject in vivo, such as filtration of topical drugs through the skin and intraoperative cancer detection. A typical approach for volumetric SRS imaging is through piezo scanning of an objective lens, which often disturbs the sample and offers a low axial scan rate. To address these challenges, we have developed a deformable mirror-based remote-focusing SRS microscope, which not only enables high-quality volumetric chemical imaging without mechanical scanning of the objective but also corrects the system aberrations simultaneously. Using the remote-focusing SRS microscope, we performed volumetric chemical imaging of living cells and captured in real time the dynamic diffusion of topical chemicals into human sweat pores.
High resolution volumetric stimulated Raman scattering (V-SRS) imaging allows a precise measurement of chemical distribution in a three-dimensional (3-D) complex biological system. To compile a stack of multiplane images, current methods such as using piezo objective positioners or tunable lenses either yield low scanning speed, disturbance of specimen, or significant aberrations. Here, we develop a V-SRS microscope with a high-speed MEMS deformable mirror (DM) which has 140 actuators and a frame rate of 20 kHz using hardware-trigger. The DM conjugated with the objective pupil plane enables wavefront shaping at reflectance mode and remote focusing of both pump and Stokes beams on the sample. The depth scan range can reach tens of micrometers by using 40X and 25X objectives. Multiple 3-D cancerous cell images are obtained. We expect the V-SRS to have great potential to enable label-free studies of cell metabolism, brain function, and developmental biology.
An adaptive scanning optical microscope with extended depth of field (ASOM-EDOF) is described. The system is based on the ASOM developed previously*, and uses a custom-built Thorlabs 0.21NA objective with a 75mm pupil diameter that allows scanned imaging over a circular region of 40mm diameter using fast steering mirror. The microscope is configured for fluorescence imaging with epi-illumination. A 140 actuator, 5.5µm stroke DM is conjugated to the pupil of the objective, and is used in conjunction with a Shack Hartmann wavefront sensor in an adaptive optics loop to measure and compensate errors of the objective as a function of nominal scan angle. At a given scan angle, the microscope camera forms an image of a 200µm x 200µm region with resolution of about 1.4µm. Images recorded at different scan angles can be stitched together to form a larger image mosaic. At each scan angle, the DM has been calibrated not only to compensate astatic aberrations, but also to perform an axial focal sweep: changing shape from concave to convex at high speed during a single camera exposure. This type of extended depth of field (EDOF) imaging (without aberration compensation) has been reported previously**. By combining these two techniques (ASOM and EDOF), a single recorded camera frame includes in-focus light from objects at depths from the nominal objective focus to depths +/-250µm from that focus, corresponding to an extension of the depth of focus by a factor of 100x for this microscope. The image also includes out-of-focus light from all depths. After a simple deconvolution, one can recover the in-focus light from all swept layers, condensed into a 2D image. Calibration details and performance metrics are described, along with example images from large volumetric samples.
*Potsaid B, Wen JTY, “Adaptive scanning optical microscope: large field of view and high-resolution imaging using a MEMS deformable mirror,” Journal of Micro-Nanolithography Mems and Moems, , 10, (2008).
**Shain WJ, Vickers NA, Goldberg BB, Bifano T, Mertz J, “Extended depth-of-field microscopy with a high-speed deformable mirror,” Optics Letters, , 995-998, (2017).