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.
Axially-offset differential interference contrast (ADIC) microscopy was developed for quantitative phase contrast imaging (QPI) by using polarization wavefront shaping approach with a matched pair of micro-retarder arrays. In ADIC microscopy, wavefront shaping with a micro-retarder array (μRA) produces a pattern of half-wave retardance varying spatially in the azimuthal orientation of the fast-axis. For a linearly polarized input beam, the polarization pattern induced from the linearly polarized plane wave through the μRA is identical to the interference between a slightly diverging right circularly polarized (RCP) and a slightly converging left circularly polarized (LCP) plane wave. Using a 10× objective, two axially offset foci separated by 70 μm are consequently generated from the patterned wavefront with orthogonal polarization states, serving as the sample and reference focal planes respectively for QPI. A paired μRA in transmission coherently recombines the two orthogonal components to recover the incident polarization state in the absence of sample. The large spatial offset (roughly 1/10 of the field of view) between the two foci provides a stable and uniform reference. Quantitative phase contrast images are directly recovered from sample-scan measurements with a single-channel detector and lock-in amplification with fast polarization modulation. This method has been successfully used for bio-sample imaging, nanoparticle detection and refractive index calculation of silica microbeads.
KEYWORDS: In vivo imaging, Raman scattering, Endoscopy, Endoscopes, Microscopy, Chemical analysis, Tissues, Hyperspectral imaging, Raman spectroscopy, Single mode fibers
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.
Spectroscopic stimulated Raman scattering (SRS) is a label-free chemical imaging modality enabling visualization of molecules in living systems with high specificity. Among various spectroscopic SRS imaging methods, a convenient way is through linearly chirping two femtosecond lasers and tuning their temporal delay, which in turn corresponds to different Raman shifts. Currently, the acquisition speed using a resonant mirror is 3 seconds (80 microseconds per spectrum), which is insufficient for imaging samples with high motility. In this work, we aim to push the imaging speed using a 50-kHz polygon scanner as a delay line tuner, achieving a speed of 20 microseconds per spectrum. At such high speeds, to overcome the signal level decrease due to reduced signal integration time, we apply a U-Net deep learning framework, which first takes pairs of spectroscopic SRS images at different speeds as training samples, with high-speed, low-signal images as input and low speed, high-signal ones as output. After training, the network is capable of rapidly transforming a low-signal spectroscopic image to a high-signal version. Consequently, our design can generate ultrafast spectroscopic SRS image while maintaining the signal level comparable to the output with longer signal integration time.
Rapid local hyperpolarizability tensor imaging of collagenous tissue was achieved with spatially encoded polarization dependent nonlinear optical measurements. Second harmonic generation (SHG) is sensitive to polarization-dependent measurements due to its unique symmetry requirement, providing rich information of local structures for protein crystals and biological tissues. Fast polarization-dependent measurements reduce 1/f noise and suppress motion blur for in vivo imaging. In this work, spatially encoded polarization dependent SHG was used for local hyperpolarizability tensor imaging of z-cut quartz and collagenous tissue by using a single patterned microretarder array (μRA). The μRA was designed with a pattern of half-wave retardance varying spatially in the azimuthal orientation of the fast-axis. When placed in the rear conjugate plane of a beam scanning microscope, the μRA enabled spatial modulation of incident light with polarization states varied at different positions in the field of view. The ‘snapshot’ approach was available for the polarization dependent measurements of a uniform sample so that one image included a complete set of polarization modulation from different pixels. Combining with sample translation, this method was able to recover local hyperpolarizability tensor of non-uniform samples. This strategy was successfully used to extract local nonlinear optical tensors for z-cut quartz and collagenous tissue with good agreements with traditional polarization dependent measurements, providing an alternate approach for fast polarization analysis of collagen tissue with minimal modifications on current beam scanning nonlinear optical systems.
As plane-polarized light propagates through a turbid medium, scattering alters the phase and polarization differently in different locations. The corresponding depolarization of the beam complicates recovery of the rich information content contained within the polarization-dependence of second harmonic generation (SHG) microscopy. A theoretical framework connecting Jones and Stokes formalisms for describing optical polarization allows prediction of the polarization-dependent SHG produced from “ballistic”, but depolarized incident light. Measurements with collagen-rich tissue sections support the predictions of the framework. Partially polarized SHG produced from a depolarized source enabled recovery of local orientation distribution for collagen and local tensor information. Bridging the gap between SHG instigated by fully depolarized light and partially polarized light more common to practical turbid systems, a method for predicting local nonlinear optical susceptibility tensor elements was developed and applied to collagen in thick sections. Recovered values for the tensor element ratio ρ are in good agreement with previous results for thin tissue and literature reports.
Fluorescence hyperspectral imaging is a powerful tool for in vivo biological studies. The ability to recover the full spectra of the fluorophores allows accurate classification of different structures and study of the dynamic behaviors during various biological processes. However, most existing methods require significant instrument modifications and/or suffer from image acquisition rates too low for compatibility with in vivo imaging. In the present work, a fast (up to 18 frames per second) hyperspectral two-photon fluorescence microscopy approach was demonstrated. Utilizing the beamscanning hardware inherent in conventional multi-photon microscopy, the angle dependence of the generated fluorescence signal as a function beam’s position allowed the system to probe of a different potion of the spectrum at every single scanning line. An iterative algorithm to classify the fluorophores recovered spectra with up to 2,400 channels using a custom high-speed 16-channel photon multiplier tube array. Several dynamic samples including live fluorescent labeled C. elegans were imaged at video rate. Fluorescence spectra recovered using no a priori spectral information agreed well with those obtained by fluorimetry. This system required minimal changes to most existing beam-scanning multi-photon fluorescence microscopes, already accessible in many research facilities.
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