For high-throughput link of microbiome function and taxonomic identity at the single cell level, we established a stimulated Raman scattering (SRS)-fluorescence in situ hybridization (FISH) platform. SRS combined with the deuterium-based isotope probing enables chemical mapping and reveals metabolic activity of bacteria. Fluorescently tagged oligonucleotide probes identify different bacteria and are detected through two photon fluorescence (TPF) microscopy. As a proof-of-principle demonstration, we tested the platform in a mixture of two distinct gut microbiota taxa with different deuterium labeling levels. This established platform not only provides enormous potential to study microbiota in the complex environment, but also the simultaneous observation of phenotype and genotype in the general biological systems.
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.
Miniature stimulated Raman scattering (SRS) imaging systems such as an SRS handheld probe and endoscope will enable label-free in vivo optical-biopsy for human patient investigation. Towards the miniature system, the challenge remains at the design and fabrication of such an achromatic micro-objective with low optical aberrations. Recent advances in achromatic metalenses with diffraction-limited performance open the opportunity to tackle the challenge. Here, we demonstrate the first proof-of-concept of metalens-enabled SRS imaging. The metalenses hold great potentials for developing endoscopic SRS and nonlinear imaging system for future clinical applications.
Compared with conventional histology, Stimulated Raman scattering (SRS) microscopy provides high specificity, fast speed and label-free histopathological analysis of the lesions by mapping their chemical compositions. However, benchtop SRS microscopy is limited to its bulky size to access the tissues of interest in-vivo inside the human body. To enable SRS in-vivo label-free histology, here, we develop an implantable fiber-scanning SRS endoscope. The endoscope is capable of providing hyperspectral Raman images at C-H and C-D regions. We use a double-clad single-mode fiber to deliver the pump and Stokes femtosecond pulses through the core and collect back-scattering signals through the outer cladding. To remove the nonlinear background induced by the pulse interactions in the fiber, we temporally separate the two pulses by tuning a delay line. We custom-design a micro-objective made of high-dispersive ZnSe glass which enables a simultaneously focusing and recombining the two pulses at spatial and time domains on the sample for excitation. A piezo actuator is designed to resonantly scan the fiber cantilever with spiral patterns. By establishing this technology, we expect the SRS endoscope to have great potential in medical applications such as label-free image-based diagnosis and surgical guidance.
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.