This PDF file contains the front matter associated with SPIE Proceedings Volume 11252, including the title page, copyright information, table of contents, and author and conference committee lists.

Conventional CARS microscopy requires scanning a point focus through the specimen limits imaging speed. We present a spatial frequency projection imaging (SPIFI) method for CARS microscopy to spatially multiplex CARS microscopy. A spinning disk modulator is used to rapidly modulate the Stokes field with a rapidly swept spatially periodic transmission grating. SPIFI-CARS images are obtained by Fourier transforming the single pixel signal. Images of CARS and second harmonic generation from histological slices will be presented. The physics of image formation and the impact of multiplexing on SNR will be discussed. Prospects for scaling to high speed CARS imaging will be discussed.

Stimulated Raman scattering (SRS) offers a drastic speed advantage over conventional vibrational spectroscopic imaging techniques – making it ideal for studying fast biochemical dynamics. We developed an experimental paradigm that applies spectral stimulated Raman scattering (SRS) imaging to study the mechanisms of infrared (IR) photostimulation of neuronal cells. Infrared neural stimulation (INS) is a label-free optical neuromodulation technique with high spatial and temporal precision. Using SRS, changes in lipid and water vibrational signatures in live cells during INS were observed, suggesting that lipid membrane deformation accompanies IR exposure. The speeds afforded by SRS enables unprecedented observation of fast cellular biophysical dynamics.

We systematically investigate the changes of lipid and protein contents at all developmental stages of Caenorhabditis elegans, including L1 to L4 larvae, and adults, using the recently developed hyperspectral stimulated Raman scattering (hsSRS) microscopy. The gut granules in larvae, which are known as lysosome-like organelles, are specifically identified and the temporal change of these gut granules are tracked at different stages. By acquiring the spectral images of C. elegans at different ages and analyzing the lipid spectra change statistically with hsSRS, we will foster a better understanding of the physiological and pathophysiological processes in living organisms.

Candidemia remains the fourth most common cause of nosocomial bloodstream infections. For more than a half-century, amphotericin B (Amp B) has been the last line of defense in the treatment of life-threatening fungal infections. However, during the past several years, severe infections due to Amp B-resistant Candida spp. isolates have been increasingly reported. Here, through polarization stimulated Raman scattering microscopy, Amp B was found to accumulate largely in the cell membrane of Candida spp. in a highly orientated approach, and the interaction between Amp B and ergosterol was investigated as well. Moreover, we found that the correlation between Amp B and ergosterol in Amp B-susceptible Candida spp. is different from that of Amp B-resistant Candida spp., which provides us important information to understand the working mechanism of Amp B, and to achieve fast determination of the Amp B susceptibility of Candida spp.

Hematocrit, the volume fraction of red blood cells in whole blood, is a crucial metric of animal health in pharmacokinetic and disease model experiments. While robust, current methods for measuring hematocrit can be prohibitory for longitudinal animal models where animal well-being restricts serial blood collection volumes. Here we present in vivo hemoglobin concentration quantification in a mouse ear model using bimodal ratiometric imaging of transient absorption of hemoglobin and stimulated Raman scattering of water. Additionally, we leverage the intrinsic high resolution of nonlinear imaging to demonstrate a low volume method for ex vivo size and volume quantification.

We present a simulation of stimulated Raman scattering that could make deep super resolution imaging possible with chemical selectivity and relatively low power levels. For this, we directly use speckles as a structured illumination pattern in a coherent Raman scattering processes, in particular Stimulated Raman Scattering. Using off-the-shelf conjugate gradient-based algorithm, we demonstrate that the method enables super-resolution better than conventional raster scanning techniques.

Silicon nitride waveguides offer a high nonlinear refractive index and tight mode confinement, ideal for efficient four-wave mixing (FWM) processes. We present a light source for coherent anti-Stokes Raman scattering (CARS), with the potential to be set up as an all-integrated device, based on spontaneous FWM in silicon nitride waveguides with only a single ultrafast fiber-based pump source at 1030 nm wavelength. Broadband signal and idler pulses are generated with only 5 nJ input pulse energy, such that the idler and residual pump pulses can be used for CARS measurements, enabling chemically-selective and label-free imaging over the entire fingerprint region.

The recent advent of Optical Photothermal IR (O-PTIR), has enabled for the first time, submicron infrared microscopy in far-field reflection mode with the combination of Raman for simultaneous and true correlative IR+Raman microscopy. These unique and exciting synergistic capabilities are now spawning interest in life science application [1]. In this presentation, examples of life science applications, ranging from live cell imaging of epithelial cheek cells in water, with submicron resolution of organelles, single bacteria IR imaging to ultra-high resolution images of breast tissue calcifications showing previously unknown levels of heterogeneity of calcifications that with traditional FTIR/QCL microscopes are not possible.

Photoacoustic tomography (PAT), in the forms of computed tomography or microscopy, provides in vivo functional, metabolic, molecular, and histologic imaging. PAT has the unique strength of high-resolution multiscale imaging across organelles, cells, tissues, organs, and small-animal organisms with consistent optical absorption contrast. The wavelength of the excitation laser can be broadly tuned to target various endogenous or exogenous molecules. PAT has the potential to empower omniscale biology and accelerate trans-scale clinical translation. Potential medical applications include imaging of the breast, brain, thyroid, muscle, joint, skin, vascular system, lymphatic system, prostate, esophagus, colon, cervix, bladder, and uterus.

The absorption of laser pulses by tissue leads not only to the generation of acoustic waves, but also to nanometer to sub-micrometer scale displacement. After the initial expansion, a quasi-steady state is achieved in a few microseconds. Previously we introduced the concept of thermo-elastic optical coherence tomography (TE-OCT) to “visualise" the rapid thermo-elastic expansion by measuring the Doppler phase shift rather than listening" to the acoustic wave as in photoacoustic imaging. In this study, we built a microscopic setup for high-speed 3D TE-OCT imaging, by means of thermo-elastic optical coherence microscopy (TE-OCM). The repetition rate of pulsed laser was set to 100 Hz and the line rate of the OCT system is 1.5 MHz. The OCT beam and the laser pulse were focused upon the same location on the sample FWHM spot sizes of 300 μm for the pulsed laser and 40 μm FWHM for the OCT beam. For each laser pulse, an M-mode OCT image consisting of 90 A-lines was acquired. The Doppler phase shift was extracted by comparing the phase signal before and after the pulse arrival. Within 6 minutes, a 3D TE-OCM image (10 × 10 × 4 mm³) can be acquired and processed. Imaging experiments were carried out in swine meat using 1210 nm excitation wavelength to highlight lipid in tissue. The results show that no significant displacement was detected in swine muscle while strong displacement was observed in lipid, owing to the optical absorption features. Furthermore, fatty tissue is easily identified in the 3D TE-OCM image while the conventional OCT images provides the structural information.

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.

Biological tissues have complex structures, dynamics and interactions between their constituents. When probing mechanical properties, differences are observed across spatial and temporal scales owing to the tissue viscoelastic response. Quasistatic mechanical testing, ultrasound and AFM-based techniques provide the traditional approach to measure stiffness based on the Young’s modulus. A novel technique in the fields of biophotonics and biomechanics is Brillouin spectroscopy, which is a contactless optical method to detect viscoelastic properties from the propagation of thermally-driven acoustic waves or phonons at high frequencies, GHz. A longitudinal elastic modulus is detected, whose significance in mechanobiology and clinical settings is currently emerging.

Blood oxygen saturation (sO2) plays an important role in maintaining energy homeostasis throughout the body. Clinical and research tools have been developed to monitor sO2 at a wide range of temporal and spatial scales. However, real-time quantification of sO2 at single red blood cell (RBC) resolution remains challenging. Such capability is critically important to study energy metabolism in heterogeneous tissues including brain and tumor tissue. In this work, we develop a label-free ratiometric transient absorption microscopy technique to image hemoglobin sO2. By exploiting differences in transient lifetime kinetics between oxyhemoglobin and deoxyhemoglobin, we directly quantified the sO2 of single RBCs.

A connection between Mueller and Jones representations for polarization analysis in nonlinear optics for structures buried in turbid media is discussed and applied to second harmonic generation (SHG) imaging of thick collagenous tissue samples. Polarization analysis for buried interfaces and structures is complicated by heterogeneous phase retardance as incident light fields approach the focal plane. Further complexities arise in nonlinear optics, where multiple incident fields interact to form a unique polarization-dependent response. The Stokes-Mueller framework provides sufficient generality to describe fields composed of many superimposed polarizations, i.e., depolarized or partially polarized light. However, the Stokes-Mueller framework is intrinsically incompatible with Jones/Cartesian representations of nonlinear optical phenomena most commonly used to relate recovered tensor elements back to molecular-scale structure and orientation. To address this challenge, a mathematical framework bridging the more general Stokes-Mueller framework to the more utilitarian Jones/Cartesian framework was developed. This framework was previously applied to the limiting case of completely depolarized light, predicting SHG intensity emitted by z-cut quartz and enabling direct recovery of collagen orientation in thin tissue samples. In the presented work, the Stokes-Mueller framework was applied to imaging thick tissue sections, where native turbidity of the tissue induces significant depolarization of the incident fundamental beam. By modulating the incident polarization state rapidly with an electro-optic modulator, ten images of unique incident polarization were simultaneously acquired. The Stokes vectors for the incident fundamental light and second harmonic generation were measured, and the Stokes-Mueller framework was utilized to fit to the underlying Jones tensor elements for collagen, which are directly related to molecular-scale structure and orientation.

Chronic in vivo optical imaging of the spinal cord is an effective way to study the biological processes during and after spinal cord injury (SCI) in mouse models. It normally relies on an implanted spinal chamber to provide continuous optical access to the spinal cord. However, the chronic window consists of multiple layers of transparent materials with various optical properties and irregular thickness, which induce large optical aberration. Therefore, the image quality of multiphoton microscopy as well as the precision of femtosecond laser axotomy were dramatically degraded. In this work, we developed an adaptive optics (AO) microscope system integrating stimulated Raman scattering (SRS) and twophoton excited fluorescence (TPEF). Using our system, the aberrations induced by the spinal cord window were measured and compensated accordingly, enabling both high-resolution imaging and precise laser axotomy of the mouse spinal cord.

The rapidly increasing need for systems biology stimulates the development of super-multiplexed methods for simultaneous imaging multiple biomolecules with distinct colors. The consensus is that complex biology requires multiplex technology. Fluorescent microscopy has inherent limitation due to the fundamental color barrier. In this end, vibrational imaging coupled with novel probes has the potential to address this grand challenge. Here we will present our recent advance along this direction including novel probe development, new labeling and amplification protocols, and application to tissue imaging. Exciting results on super-resolution super-multiplex imaging and high-dimensional single cell analysis will be discussed.

We present a rugged all-fiber optical parametric oscillator (FOPO) for coherent Raman imaging (CRI). The FOPO shows ideal performance for hyperspectral video-rate coherent Raman imaging with pulse durations of 7 ps at 40 MHz and switching times of less than 5 ms from 700 to 3530 wavenumbers. Due to our patented all-fiber setup the FOPO runs on a regular laboratory cart. We present shock measurements that resemble typical situations in a clinical environment as well as long-term stability measurements. The presented robust fiber laser system is ideally suited to allow easy access to hyperspectral, high-speed CRI for translational research.

Stimulated Raman Scattering (SRS) microscopy is a powerful method for imaging molecular distributions based on their intrinsic vibrational contrast. However, SRS is hindered by a parasitic background signal which often overpowers the signal in low-signal applications. Frequency modulation (FM) has been used to suppress this parasitic background. However, many FM-SRS methods require the addition of multiple optomechanical components and extensive realignment. We report a new approach for alignment-free FM-SRS. In conjunction with a parabolically amplified Stokes pulse, we demonstrate near complete background suppression and also utilize the technique to increase the contrast of minority species in heterogeneous biological samples.

Raman spectroscopy is an essential optical tool for molecular fingerprints. The vibrational modes of biologically important molecules including proteins, nucleic acids and lipids have been studied to provide insight into their structure as well as insight into the metabolic processes and biomarker expression of cells. To explore hyper-Raman scattering as a complementary technique to Raman scattering, we build a laser system that can perform Raman and hyper-Raman scattering studies using a single setup. Using three amplification stages we are able to generate 8 ps, 1064 nm pulses at repetition rates up to 30 MHz. Converting the 1064 nm source laser to 532 nm, we achieve fast hyper-Raman detection and collect our spectrum with a commercial spectrometer and CCD. Using a single optical setup, we collect and compare Raman spectra at 532 nm to hyper-Raman spectra at 266 nm for water, ethanol and L-tartaric acid. Furthermore, we observe changes in the hyper-Raman peak intensities of an aqueous L-tartaric acid solution when selecting different laser repetition rates highlighting the need to control laser power and repetition rate to identify and mitigate thermal effects in biomolecules.

Worldwide, there has been an increase in the number of cases of non-Hodgkin lymphoma (NHL). Burkitt lymphoma comprises of 30-40% of pediatric NHL cases and is a rapidly growing tumor. Access to efficient diagnostic paradigms are therefore crucial for quick therapeutic intervention. Currently, the identification of Burkitt lymphoma and other NHL involves histologic and genetic testing which can be costly and slow. Also, the process of fixing tissue and staining biopsy samples can lead to inconsistent results. Recently, Raman spectroscopy has exposed potential biomarkers in B-cells that could be indicative of cancer. However, slow acquisition speed limits the viability of adapting Raman spectroscopy in a clinical setting. Here we demonstrate a high-speed method to visualize Burkitt lymphoma cells and non-malignant B-cells which does not involve chemical alteration or destruction of cells. Preliminary results indicate higher collection of lipid droplets in malignant B-cells compared to normal B-cells. Using a support-vector machine learning algorithm, we were able to exploit these chemical differences and classify malignant cells from non-malignant cells with a sensitivity of 80% and specificity of 81.2%. Further work into refining this process can lead towards faster identification of cells and could potentially provide deeper insights into the chemical processes that occur within malignant blood cells.

As an alternative imaging technique to conventional IR microscopy, a mid-infrared photothermal microscopy has been suggested to achieve spatial resolution at the submicrometer level and the inherent chemical contrast upon vibrational excitation. It also has substantial potential for real-time imaging of live organisms to observe the cellular dynamics without photodamage or photobleaching of fluorescent labels. We performed real-time imaging of oligodendrocytes to investigate cellular dynamics and observed a photothermal contrast associated with traveling protein complexes on an axon of live neutrons. We anticipate that mid-infrared photothermal imaging will be of great use for gaining insights into the field of biophysical science, especially with regard to cellular dynamics and functions.

Infrared spectroscopic imaging has emerged as a powerful label-free diagnostic tool to study the molecular composition and organization in biological tissues and cells. We report infrared spectroscopic imaging using polarized light to study differential absorption of plane-polarized light by an oriented sample to detect valuable information, such as, birefringence and dichroism. For instance, the organization of collagen, specifically fiber orientation and alignment, is crucial in understanding the progression and metastasis of cancer. Recent advancements in the development of Quantum Cascade Lasers (QCL) sources have opened new avenues for high SNR measurements in the field of IR spectroscopy. In addition, QCL sources are intrinsically polarized and orientation information can be obtained at discrete frequencies with different polarization orientations, allowing much faster acquisition than a corresponding FT-IR approach. We demonstrate improved performance in terms of fast and comprehensive polarimetric image acquisition and analysis using custom-built QCL microscope and evaluate its impact on applications by analyzing the important spectral bands of surgical tissue sections.

Coherent Raman and infrared microscopy have a huge potential in life science applications since both technologies probe the specific vibrational properties of samples and thus can identify and differentiate molecules. Easy to use, rugged and computer controlled light sources are essential for the translation into life science and medicine. We will present our latest developments on light sources dedicated to these applications.

Rapid diagnosis on endoscopic biopsies is crucial for decision makings during gastrointestinal endoscopy. We applied stimulated Raman scattering (SRS) microscopy on fresh biopsy specimens without fixation, sectioning or staining. We further combined SRS with deep convolutional neural network for automated diagnosis of early gastric cancer. Our preliminary results indicated that SRS histology integrated with deep learning algorithm provides potential for delivering rapid diagnosis that could aid the surgical management of gastric cancer.

Fingerprint stimulated Raman scattering (SRS) produces label-free chemical maps of molecules in living systems with higher specificity compared to CH vibration region. However, due to the weak signal levels in the fingerprint window, it remains challenging for fingerprint SRS to study highly dynamic or large-scale samples. Here, we push the design space of SRS using deep learning, which can recover the signal-to-noise ratio to the levels comparable to measurements with 100 times longer integration time. Combined with an ultrafast 50-kHz delay-line tuner, we can generate real-time images of cholesterol, fatty acid, and proteins of living cells and large-area tissues including the whole brain.

Stimulated Raman scattering (SRS) images often suffer from low signal to noise ratio (SNR) due to absorption and scattering of light as well as limited optical power. We use deep learning to significantly improve the SNR of SRS images. Our algorithm, based on a U-Net convolutional neural network, significantly outperforms existing denoising algorithms. The trained denoising algorithm is applicable to images acquired at different imaging powers, depths, and experimental geometries not explicitly included in the training. Our results identify potential towards in vivo applications, where ground-truth images are not always available to create a paired training set for supervised learning.

Raman spectroscopy permits label-free molecular quantitation of biological samples in situ in a non-destructive manner. Combining machine learning with Raman spectroscopy has increased its potential for use in molecular imaging and discrimination of living cells and tissues in biological research fields. In this work, Raman spectroscopy was paired with machine learning techniques to classify specimen of similar tissues. Raman spectra of rat long bone, rabbit long bone, and rabbit crania were collected and classified into their respective categories. The spectra were truncated to the range of 400 to 1800 wavenumbers. To train and validate the machine learning algorithms, the data were randomly split such that 80% (n = 499) of the data were used for training, and 20% (n = 125) were used for validation. Three approaches were taken to prepare the data for classification. The first approach utilized all Raman intensities between 400 and 1800 wavenumbers to perform the classification. The second approach reduced the dimensions of the dataset using Principal Component Analysis (PCA) prior to performing classification. The third approach also reduced the dimensions of the dataset by extracting intensities of peaks that are of interest for bone analysis and using these peaks for classification. The peaks chosen were Amide I, Amide III, Proline, CH2 wag, and Carbonate. Raman spectra were classified using supervised learning techniques for each data preparation approach. The supervised methods include Support Vector Machine (SVM), Decision Tree, Random Forrest, and Naïve Bayes. The three groups were successfully sorted into their respective classes by the applied classification algorithms. The most successful classification models were achieved by reducing the dataset to peaks of interest, and performing classification utilizing Support Vector Machine achieving a validation accuracy up to 98.40%. This proof of concept has potential to be applied to numerous research applications that require sensitive discrimination between similar tissues.

We present several approaches to broadband stimulated Raman scattering (SRS): Fourier-Transform SRS (FT-SRS), Photonic Time Stretch SRS (PTS-SRS) and a multi-channel lock-in amplifier (M-LIA). In FT-SRS the broadband Stokes pulse is sent to a birefringent delay line that creates two collinear replicas, whose interferogram is measured by a single detector. The pump-induced change of the interferogram, measured by a lock-in amplifier, gives, after Fourier transform, the SRS spectrum. In PTS-SRS the broadband Stokes pulse is temporally stretched by an optical fiber to a duration of a few nanoseconds and sampled by a high-frequency analog-to-digital converter, allowing measurement of SRS spectra at MHz repetition rates. Finally, a M-LIA allows sensitive measurement of the SRS spectrum at a number of frequencies from 4 to 32.

Raman Spectroscopy enables fast, sensitive and label-free chemical analysis of a large range of materials and has become a routine analytical tool in a wide range of material science and process-control applications. As the Raman signal is weak it is critical that the illumination laser has a very high level of spectral purity, for efficient detection of the Raman signal. Most materials can be characterized by studying Raman shifts down to 100 cm-1, but in some cases, for instance for determining the crystallinity of pharmaceutical compounds, it is required to study Raman shifts in the low-frequency regime; <100 cm^-1 . 785 nm is the most common illumination wavelength for Raman spectroscopy as it offers the best compromise between Raman signal strength and fluorescence background suppression. In this paper, we present a novel design for a frequencystabilized 785 nm diode laser using a highly reflective volume Bragg grating (VBG) element that offers not only a narrow spectral linewidth and low wavelength drift, but also a very high level of spectral purity. Using the VBG reflected light as output from the laser suppresses Amplified Spontaneous Emission (ASE) from the diode so that a very high level of sidemode suppression ratio (SMSR) in the laser output is reached within just a few cm^-1 away from the main peak without any external spectral filtering. This enhanced spectral purity directly from the laser enables simpler, more compact and more cost-efficient detection of Raman shifts in the very low frequency range.

Among various optical methods, fluorescence imaging has been the most widely exploited thanks to its superior sensitivity and specificity, but the resolvable colors are restricted to 2-5 colors because of the intrinsically broad and featureless spectra. Recently, this fluorescent “color barrier” was broken and super-multiplex optical imaging became possible taking advantage of well-designed Raman probes. However, the acquisition of the super-multiplex images is still relatively slow which impedes wider applications. Here, we demonstrate fast super-multiplex organelle imaging with high-speed color switching and acquisition, which accelerates the imaging speed by 2 orders of magnitude. We applied it in imaging cytometry, tracing mitosis and fast organelle motions in live cells. We anticipate that high-speed supermultiplex optical imaging can expand to a much wider field of biological researches.

Traditional drug detection technique is highly accurate but time consuming and labor intensive. Raman spectroscopy (RS) is a fast and non-destructive detection technique that provides detailed information on chemical composition, phase and morphology, crystallinity and molecular interaction of the sample. The current Raman spectrometer is mainly based on the use of Gaussian light, providing with good signal to noise ratio for a thin or transparent sample. However, owing to the scattering effect, the Gaussian beam will become diffuse in the scattering medium. This makes it not conducive to in vivo or deep imaging. Utilizing the long focusing characteristics and self-reconstructing properties of Bessel beam, we here presented a new scheme for RS, which used a Bessel beam as the excitation light. The Bessel beam-based RS was first verified with the standard samples, and then comparatively tested on several drugs. Taking the acetaminophen as the test sample, we compared the Bessel beam-based RS with the traditional Gaussian beam based one with or without a scattering medium. With the addition of a scattering medium, the signal-to-noise ratio of Raman spectra based on Bessel beam decreases less than that based on the Gaussian light, which demonstrated the great potential of the use of Bessel beam in in vivo or deep RS. This study provides great value for in vivo applications of Raman spectroscopy.

Gastrointestinal (GI) cancer is the second most commonly diagnosed cancer. Early cancer identification is critical to reducing mortality rates of GI patients. Conventional white-light endoscope, however, suffers from poor diagnostic accuracy of GI cancer during endoscopic examination. Raman spectroscopy represents a unique optical vibrational technique capable of harvesting biomolecular information in tissue with potential for tissue histopathological assessments. In this work, we report the development of a novel simultaneous fingerprint (FP) and high wavenumber (HW) fiber-optic Raman spectroscopy technique as well as its clinical applications for improving real-time in vivo diagnosis of GI cancer at endoscopy. We have demonstrated the diagnostic accuracy of >90% for GI cancer and precancer detection with FP/HW Raman spectroscopy. The rapid fiber-optic Raman spectroscopy also allows the

We present high-speed multicolor coherent Raman imaging enabled by a novel all-fiber light source. A high tuning speed of < 5 ms per arbitrary wavelength step (700 - 3530 wavenumbers) allows to switch the wavelength in a frame-by-frame manner when imaging with up to 100 frames/s. The pump (up to 200 mW) and Stokes pulses (> 400 mW) exhibit equal durations of 7 ps. The compact and environmentally stable system is predestined for fast multicolor assessments of medical or rapidly evolving samples with high chemical specificity.

Stimulated Raman Scattering (SRS) microscopy is a powerful nonlinear optical microscopy technique that images biological structures by exploiting the characteristic, vibrational contrast of the sample molecules. SRS provides a rich, chemically specific and biophysical image contrast that is in many ways complementary to the molecular contrast of fluorescence microscopy. Here, we present a range of applications of SRS, including label-free morphochemical imaging in model organisms, the characterization of organoids and spheroids, and investigations of brain tissues for neurodegenerative disease research. We show specifically that SRS can provide novel insights into the biophysical properties and biochemical composition of Amyloid-β plaques in a mouse model of Alzheimer’s disease. Our results highlight the potential of SRS to contribute to a deeper understanding of cell and tissue biology, and to serve as a powerful tool for preclinical and translational research.

Chemical characterization of materials at the nanoscale provides insights into their compositions and organizations. Infrared spectroscopy has been a powerful tool that directly indicates the identity and amount of functional groups of molecules by measuring the absorption of infrared light. However, nanoscale spatial resolution is hard to achieve for conventional Fourier-transform infrared (FTIR) spectroscopy because of the optical diffraction limit. Herein we reported a recently developed infrared microscopy and spectroscopy technique also based on infrared absorption of molecules – peak force infrared microscopy – that combines atomic force microscopy and infrared laser illumination. Sub 10 nanometer spatial resolution has been demonstrated on various samples, including block copolymers, hexagonal boron nitride flakes, and amyloid fibrils. Simultaneous chemical and mechanical mapping can be obtained with peak force infrared microscopy in that both information is encoded in the cantilever deflection curves during peak force tapping cycles. The high spatial resolution and multimodal measurement capability render peak force infrared microscopy a label-free chemical imaging technique for explorations of nanoscale across broad disciplines.

Label-free optical imaging is valuable for studying fragile biological phenomena where chemical and/or optical damages associated with exogenous labelling of biomolecules are not wanted. Molecular vibrational (MVI) and quantitative phase imaging (QPI) are the two most-established label-free imaging methods that provide biochemical and morphological information of the sample, respectively. While these methods have pioneered numerous important biological analyses along their intensive technological development over the past twenty years, their inherent limitations are still left unresolved. In this contribution, we present a unified imaging scheme that bridges the technological gap between MVI and QPI, achieving simultaneous and in-situ integration of the two complementary label-free contrasts using the midinfrared (MIR) photothermal effect. Our method is a super-resolution MIR imaging where vibrational resonances induced by wide-field MIR excitation and the resulting photothermal RI changes are detected and localized with the spatial resolution determined by a visible-light-based QPI system. We demonstrate applicability of this method, termed MV-sensitive QPI (MV-QPI), to live-cell imaging. Our MV-QPI method could allow for quantitative mapping of subcellular biomolecular distributions within the global cellular morphology in a label-free and damage-less manner, providing more comprehensive pictures of complex and fragile biological activities.

Chemical characterization of biological specimens in the mid-infrared (IR) window plays a central role in the analysis of their functionalities. Although recent advances in mid-IR microscopy have demonstrated detection of the sample’s chemical contrast at a sub-micron resolution using a visible probe beam, they have limited sensitivity at high-throughput. To overcome this limit, we employ wide-field interferometric microscopy to detect the minute change in the optical path induced by mid-IR absorption. Our technique enables high-speed fingerprinting of more than thousands of sub-200 nm nanoparticles at once. This method paves the way for high-throughput, ultrasensitive, and label-free chemical imaging of individual bio-nanoparticles at sub-micron resolution.

Innovations in optical spectroscopy and microscopy have revolutionized our understanding in biological systems at sub-cellular levels. In this talk, I will discuss about our recent development by coupling stimulated Raman scattering (SRS) microscopy with chemical probes that could allow new subcellular bioanalysis in live cells. The introduced tags offer additional SRS contrast channel for quantification of biological contents that were previously difficult. Both physical and chemical principles underlying the optical microscopy will be presented, as well as our efforts in biomedical applications including cancer- and neuronal- metabolism.

Dimethyl sulfoxide (DMSO) is a biologically important solvent in part due to its dual miscibility with hydrophilic and hydrophobic molecules. Binary solutions of DMSO-water display non-ideal thermodynamics properties such as high viscosity and low freezing point due to hydrogen bonding. The unusual properties of DMSO-water solutions have been exploited to disrupt the formation of secondary structures of proteins during polymerase chain reaction assays and to act as a cryoprotectant for tissues. The exact coordination of the DMSO and water molecules remains unknown. Hyper- Raman scattering was employed for the first time to investigate binary systems of DMSO with water (H₂O). As a part of this study, hyper-Raman and Raman spectra of pure solutions were first acquired and compared against existing Raman and IR spectroscopic data. Then the corresponding measurements were taken with deuterated DMSO-d₆ and heavy water (D₂O) to validate the analysis and to isolate overlapping spectral features. The permissive selection rules of hyper- Raman scattering provide new insight into disruptions of the self-hydrogen bonded networks of DMSO and water and the establishment of hydrogen bonded networks.

Raman spectroscopy has become an essential characterization tool for chemical and biomedicine, capable of single-cell sensitivity with high spatial resolution. We applicate single-cell Raman microspectroscopy (SCRS) into cancer cells and aim to investigate the practical distribution of glycogen in the brain cancer cell and analyze the main pathway, moreover, try to give an insight of how glycogen metabolism affects Krebs circle, cancer metastasis and invasion. Here, we found the high glycogen points are more likely to concentrate at the cytoplasm randomly, and the glycogen concentration of cancer cell grown under stress condition is higher than that of normal condition.

Using coherent anti-Stokes Raman scattering (CARS) microscopy, we discovered heterogeneity of mitochondrial chemical compositions in cancer cells after hypothermia. Individual mitochondria associated with higher signals in CARS images are likely the result of fatty acid accumulation, which is caused by a reduced rate of fatty acid β-oxidation. Tracing individual mitochondria after reheating the cells to 37°C reveals degradation of these organelles through the mitophagy process. Further study will continue to unveil how stressed mitochondria would form in various conditions, and the fate of these organelles upon changes of their environment. Our results shed new light on mitochondrial function and cell metabolism.

Doppler Raman (DR) spectroscopy is a coherent Raman technique that combines impulsive Raman excitation with novel frequency shift detection to enable high-sensitivity Raman spectroscopy in the biological fingerprint region (500cm-1-1500cm-1) and the low frequency regime from 10cm-1 – 500cm-1. Using DR, we demonstrate nonresonant Raman spectroscopy on a suite of biologically significant targets involved in cell respiration including cytochrome c, adenosine triphosphate (ATP), flavin adenine dinucleotide (FAD), and nicotinamide adenine dinucleotide (NADH). High-sensitivity detection of low-to-medium frequency Raman vibrational modes may provide a tool to monitor states of cell respiration along with large molecular structural changes such as protein conformational dynamics.

For developing diffraction-unlimited Raman microscopy, a three-beam femtosecond stimulated Raman scattering (SRS) is deviced for simultaneously inducing two different SRS processes associated with Raman-active modes in the same molecule. Two SR gains involving a common pump pulse and two separate Stokes beams are coupled and compete: one SRS is selectively suppressed as the other Stokes beam intensities increases. Our theoretical description and experimental evidence support that the selective suppression behavior is due to the limited number of pump photos used for both of the two SRS processes. We anticipate a potential of this new switching-off concept in super-resolution label-free microscopy.