Conference 11656

Advanced Chemical Microscopy for Life Science and Translational Medicine 2021

Digital Forum: On-demand starting 6 March
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  • Welcome and Introduction
  • BiOS Hot Topics
  • Conference 11656 Paper Presentations - Session I
  • Conference 11656 Paper Presentations - Session II
  • Conference 11656 Paper Presentations - Session III
  • Conference 11656 Paper Presentations - Session IV
  • 1: Raman/CARS/SRS I
  • 2: New Methods for Chemical Imaging I
  • 3: Data Science in Chemical Microscopy I
  • 4: Raman/CARS/SRS II
  • 5: Translation to Clinic
  • 6: IR and Photothermal Chemical Imaging
  • 7: Raman/CARS/SRS III
  • 8: IR and Photothermal Chemical Imaging & New Methods for Chemical Imaging
  • Front Matter: Volume 11656
2021-03-05T08:34:48-08:00
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UPCOMING LIVE EVENTS:
Session R: Welcome and Introduction
11656-800
Author(s): Ji-Xin Cheng, Boston Univ. (United States); Wei Min, Columbia Univ. (United States); Garth J. Simpson, Purdue Univ. (United States)
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Introduction to SPIE Photonics West BiOS conference 11656: Advanced Chemical Microscopy for Life Science and Translational Medicine 2021
Session LIVE: BiOS Hot Topics
11618-700
6 March 2021 • 12:00 PM - 2:00 PM PST
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Every year, attendees look forward to Saturday Night Hot Topics, an evening spent hearing highly engaged, world renowned speakers reveal the latest innovations in their areas of expertise. Don't miss this year's outstanding list of speakers. View details: https://spie.org/photonics-west/event/bios-hot-topics/2589728
Session LIVE: Conference 11656 Paper Presentations - Session I
11656-700
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Join us for live presentations of papers on Raman/Cars/SRS + New Methods for Chemical Imaging
Session LIVE: Conference 11656 Paper Presentations - Session II
11656-701
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Join us for live presentations on Raman/Cars/SRS II + Data Science in Chemical Microscopy
Session LIVE: Conference 11656 Paper Presentations - Session III
11656-702
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Join us for live presentations of papers on Infrared and Photothermal Chemical Imaging + Translation to Clinic
Session LIVE: Conference 11656 Paper Presentations - Session IV
11656-703
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Join us for live presentations of papers on Raman/CARS/SRS III + New Methods for Chemical Imaging II
Session 1: Raman/CARS/SRS I
11656-29
Author(s): Jürgen Popp, Leibniz-Institut für Photonische Technologien e.V. (Germany)
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Here we will highlight our recent efforts in translating Raman spectroscopy towards routine clinical applications by developing compact clinically usable automated Raman spectroscopic instrumentation and their combination with other spectroscopic / optical modalities, to provide a multimodal approach with high TRL levels. We will start with novel multimodal spectroscopic instrumentation for precise surgical guidance and intraoperative histopathological examination of tissue and their combination with laser tissue ablation for tissue specific laser surgery and for therapy monitoring. Finally, we report on the application of non-resonant Raman spectroscopy for an early diagnosis of neurodegenerative diseases directly in the fundus of the eye.
11656-30
Author(s): Conor L. Evans, Wellman Ctr. for Photomedicine (United States)
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Coherent Raman scattering (CRS) imaging is capable of directly imaging the diffusion and uptake of drugs into tissue, enabling the direct quantification of pharmacokinetics and the extraction of key pharmacokinetic parameters such as Tmax. We will present our work translating this toolkit to the study of percutaneous pharmacokinetics in human skin and clinical studies. Our ongoing work incorporates new machine learning models to map drug PK to human skin morphorology, as well as portable coherent Raman imaging tools for pharmacokinetic studies directly in diseased skin.
11656-31
Author(s): Hyeon Jeong Lee, Zhejiang Univ. (China); Ji-Xin Cheng, Boston University (United States)
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Voltage imaging has become an emerging technique to record membrane potential change in living cells. Yet, compared to electrophysiology, microscopy approaches are still limited to relative membrane voltage changes, lacking important information conveyed by absolute membrane voltage. This talk will cover a spectroscopy approach to tackle this challenge. A spectroscopic signature of membrane potential was identified through stimulated Raman scattering (SRS) imaging, which enabled label-free, sub-cellular voltage imaging of mammalian neurons. We also employed pre-resonance electronic absorption to enhance SRS imaging sensitivity and specificity. microbial rhodopsin voltage sensors, providing a quantitative approach to measure membrane potential. Quantitative voltage imaging by SRS has enabled mapping of absolute voltage and has great potential in neurology and brain sciences.
11656-32
Author(s): Maximilian Brinkmann, Refined Laser Systems GmbH (Germany), Westfälische Wilhelms-Univ. Münster (Germany); Thomas Würthwein, Westfälische Wilhelms-Univ. Münster (Germany); Tim Hellwig, Refined Laser Systems GmbH (Germany), Westfälische Wilhelms-Univ. Münster (Germany); Nick Lemberger, Carsten Fallnich, Westfälische Wilhelms-Univ. Münster (Germany)
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We present high-speed multicolor stimulated Raman scattering imaging (SRS) enabled by an all-fiber light source. With a relative intensity noise level of -157 dBc above 10 MHz the light source is shot-noise limited up to a detector current of 0.75 mA. Compared to other fiber-based light sources optimized for SRS, the presented system is tunable in under 5 ms per arbitrary step between 700 and 3530 wavenumbers. The compact and environmentally stable system is predestined for fast multicolor assessments of medical or rapidly evolving samples with high chemical specificity.
11656-33
Author(s): Meng Zhang, Weili Hong, Boston Univ. (United States); Nader S. Abutaleb, Purdue Univ. (United States); Junjie Li, Pu-Ting Dong, Cheng Zong, Boston Univ. (United States); Pu Wang, VibroniX, Inc. (United States); Mohamed Seleem, Virginia Polytechnic Institute and State University (United States); Ji-Xin Cheng, Boston Univ. (United States)
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This work demonstrates a rapid platform that can determine the antimicrobial susceptibility testing (AST) in cation-adjusted Mueller-Hinton Broth medium, urine and blood by stimulated Raman scattering (SRS) imaging of deuterium oxide (D2O) incorporation at a single bacterium level. The total AST assay time with the value of the single-cell metabolism inactivation concentration (SC-MIC) obtained is less than 2.5 h from colony to results. The SC-MIC results of 37 sets of bacterial isolate samples were systematically validated by MIC determined by the Clinical and Laboratory Standards Institute criteria, with a category agreement of 94.6% and 5.4% minor error. Furthermore, SRS imaging of D2O metabolic incorporation can rapidly determine SC-MIC directly in clinical samples for urinary tract infection or septicemia blood infection.
11656-34
Author(s): Christopher B. Marble, Texas A&M Univ. (United States); Xingqi Xu, Zhejiang Univ. (China), Texas A&M Univ. (United States); Georgi I. Petrov, Texas A&M Univ. (United States); Dawei Wang, Zhejiang Univ. (China); Vladislav V. Yakovlev, Texas A&M Univ. (United States)
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Hydrogen bonding plays an essential role in biological processes. In this report we apply hyper-Raman scattering spectroscopy to probe the effects of the alkyl groups on hydrogen bonding in mixtures of DMSO-methanol. We characterize the dependence of hyper-Raman spectra on concentration and observe suppression of the hyper-Raman responses of the methanol alkyl group at intermediate concentrations. In addition, small frequency shifts in the vibrational frequencies of DMSO and methanol were detected. These results provide new insights into the nature of the hydrogen bonding in solution and into the details of the hydrogen bond’s interaction with the alkyl groups.
11656-35
Author(s): Luca Genchi, Andrea Bucci, Siarhei Laptenok, Carlo Liberale, King Abdullah Univ. of Science and Technology (Saudi Arabia)
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We present a high spectral resolution multiplexing acquisition mode for SRS microscopy based on a dual-beam femtosecond laser. A multi-channel Acousto Optical Tunable Filter (AOTF) generates spectral masks, given by the Hadamard matrix, by turning on and off different subsets of its 8 independent channels, corresponding to different wavelengths available within the broad bandwidth of the “pump” femtosecond laser. The SRS spectrum is retrieved by using the inverse Hadamard matrix. When additive noise is dominating, spectral measurements using the multiplexed method show the same signal to noise ratio of conventional single-wavenumber acquisitions performed with 4 times longer integration time.
Session 2: New Methods for Chemical Imaging I
11656-22
Author(s): Richard N. Zare, Stanford Univ. (United States)
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Several different experiments employing confocal microscopy for examining water microdroplets will be described, with the goal of understanding better why there are so many marked differences between the behavior of water microdroplets and that of bulk water.
11656-23
Author(s): Ashok Ajoy, Univ. of California, Berkeley (United States)
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The development of point-like quantum sensors based on wide bandgap materials, in particular the Nitrogen Vacancy (NV) center in diamond, has thrown up exciting new possibilities for the NMR of materials, molecules, and biological systems through optical means. I will describe how NV center endowed nanodiamonds could usher in a new form of a "deployable" NMR sensor with novel applications to signal enhanced NMR spectroscopy and imaging.
11656-24
Author(s): Terumasa Ito, Miyako Iritani, Fumiaki Matsuoka, Kazuhiko Misawa, Tokyo Univ. of Agriculture and Technology (Japan)
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Quantitative measurements of a small amount of chemicals in label-free tissue imaging by conventional SRS microscopy remain challenging because of background signals. We present a time-resolved, phase-modulated (PM) SRS microscopy method with increased signal contrast. In addition to removing background signals generated via amplitude modulation, PM-SRS can reduce intrinsic tissue background signals by temporally separating the excitation and detection processes. Furthermore, polarization artifacts in tissues can also be removed by rapidly modulating the relative delay between the pump and probe pulses. This new technology enables robust quantitative measurements in tissue and extends the potential of SRS imaging in biomedical applications.
11656-25
Author(s): Christian Oelsner, Eugeny Ermilov, Volker Buschmann, Frank Birke, Matthias Patting, Uwe Ortmann, Rainer Erdmann, PicoQuant GmbH (Germany)
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We will demonstrate the performance of a spectrometer-microscope assembly for characterization and analysis of samples in terms of lifetime, spectral and spatial resolution. This combined approach provides access to further information, which are not available when using only lifetime or steady-state experiments. The combination of both techniques in one setup can help to understand biochemical or physical processes by detecting changes in local environment such as pH, temperature, or ion concentration, and to identify molecular interactions or conformation changes via Förster Resonance Energy Transfer (FRET).
11656-26
Author(s): Dashan Dong, Xiaoshuai Huang, Liuju Li, Liangyi Chen, Kebin Shi, Peking Univ. (China)
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Optical diffraction tomography (ODT) is one of the most developing imaging technologies in recent years, it can perform long-term three-dimensional (3D) imaging on live cell noninvasively. However, a lack of molecular specificity in label-free ODT microscopy also hinders the interpretation of imaging results. Thus we develop SR fluorescence-assisted diffraction computational tomography (SR-FACT), which combines label-free three-dimensional ODT with two-dimensional fluorescence Hessian structured illumination microscopy. The ODT module is capable of resolving mitochondria, lipid droplets, the nuclear membrane, chromosomes, the tubular endoplasmic reticulum and lysosomes. Using dual-mode correlated live cell imaging for prolonged period of time, we observe a novel subcellular structure named dark-vacuole bodies. These works demonstrate the unique capabilities of SR-FACT, which suggest its wide applicability in cell biology in general.
11656-27
Author(s): Jiaze Yin, Lu Lan, Yeran Bai, Celalettin Yurdakul, Haonan Zong, Ji-Xin Cheng, Boston Univ. (United States)
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Mid-infrared photothermal (MIP) microscopy overcomes the resolution and huge water background limits in conventional mid-infrared imaging by probing the mid-infrared absorption induced photothermal effect. However, to detect the subtle MIP signal, large probe power and lock-in detection are needed, which limit the imaging speed of current MIP systems. To overcome this limitation, we develop a single-pixel pump-probe camera that leverages the large well-depth capacity of photodiode to achieve high-speed wide-field MIP imaging. With compressive sensing applied, close to video-rate MIP imaging can be achieved, offering a powerful label-free chemical imaging tool to scrutinize the complex biological systems.
11656-28
Author(s): Haonan Zong, Celalettin Yurdakul, Yeran Bai, Meng Zhang, Selim Ünlü, Ji-Xin Cheng, Boston Univ. (United States)
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Mid-infrared photothermal microscopy (MIP) has been a promising chemical imaging technique for functionality characterization of biological and pharmaceutical specimens owing to its enhanced resolution and high-specificity. Recently developed wide-field MIP modalities drastically improved the imaging speed and thus enabled high-throughput imaging. However, the sensitivity of the wide-field MIP technique has been limited by shot-noise of background photons. Here, we demonstrate a dark-field MIP modality to allow 4-fold signal-to-noise ratio improvement. Our technique is based on selectively blocking the reflected light. Simulation and experimental results are both provided, and they are consistent with each other.
Session 3: Data Science in Chemical Microscopy I
11656-1
Author(s): Rohith K. Reddy, Chalapathi Gajjela, Rupali Mankar, David Mayerich, Univ. of Houston (United States)
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11656-2
Author(s): Francesco Saverio Pavone, LENS - Lab. Europeo di Spettroscopie Non-Lineari (Italy)
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In this talk, a pipeline to perform 3D digital histology of different tissue will be presented. Morphological characterization of tissue will be displayed using the combination of different clearing techniques and labeling in combination with high-resolution optical microscopy. Furthermore, particular attention will be devoted to the big data managing strategies adopted to extract some quantitative parameters, like cell census, by using some deep learning approaches
11656-3
Author(s): Sang Mok Park, Young L. Kim, Purdue Univ. (United States)
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Reflectance spectroscopy and hyperspectral imaging are extensively used to quantify biochemical composition, but often rely on bulky and costly optical components, limiting the development of compact, rapid, and cost-effective technologies. We have recently developed a spectral super-resolution framework that enables virtual transformation of the built-in camera (RGB sensor) into a hyperspectral imager for accurate and precise spectroscopic analyses. As an example, our mobile version of spectral super-resolution combines imaging of peripheral tissue and spectroscopic quantification of blood hemoglobin levels. Spectral super-resolution spectroscopy can also serve as an example that data-driven technologies minimize hardware complexity, facilitating the tempo of clinical translation.
11656-4
Author(s): Alejandro De la Cadena, Carlo Valensise, Federico Vernuccio, Dario Polli, Giulio N. Cerullo, Politecnico di Milano (Italy)
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We present innovative approaches to broadband coherent Raman scattering microscopy, both in the stimulated Raman scattering (SRS) and broadband coherent anti-Stokes Raman scattering (CARS) modalities. A convolutional neural network removes the unwanted non-resonant background from broadband CARS spectra. The deep-learning model processes experimental data in 100microseconds and correctly retrieves all the relevant vibrational peaks without any user intervention or independent background measurement. A multi-channel lock-in amplifier, in combination with in-line balanced detection and a broadband optical parametric oscillator, allows sensitive measurement of the SRS spectrum at 32 frequencies in parallel, with pixel dwell times as short as 40 microseconds.
11656-5
Author(s): Weiru Fan, Tianrun Chen, Zhejiang Univ. (China); Vlad V. Yakovlev, Texas A&M Univ. (United States); Dawei Wang, Delong Zhang, Zhejiang Univ. (China)
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Optical phase microscopy is widely adopted for quantitative imaging of optical density in transparent cells and tissues that lack absorption contrast. Fundamentally, the phase information of the sample is contained in the wavefront of the probe beam, often detected by interferometry-based techniques. Here, a novel approach has been developed based on the phase-sensitive second harmonic signals that are generated after the sample. A deep learning algorithm is developed for efficient recovery of the original phase images. Inheriting the advantages of the second harmonic imaging, our second harmonic phase imaging is a label-free technique with a demonstrated phase sensitivity of 1/100 wavelength and high robustness against noises, facilitating applications in biological imaging and remote sensing.
11656-6
Author(s): Yi Sun, Stephen A. Boppart, Univ. of Illinois (United States); Chi Zhang, Purdue Univ. (United States)
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Hyperspectral stimulated Raman scattering (HSRS) is a powerful label-free microscopy technique to map chemical compositions of biological samples. However, HSRS imaging usually requires much longer acquisition time or much complicated optical instrumentation compared to the single-color SRS microscopy. We apply a generative adversarial network (GAN) to address this challenge and show a single-color SRS image of cells contains sufficient information to predict chemical maps conventionally only obtained from HSRS imaging. Using this GAN-aided approach, we can separate different organelles of living cells using only single-color SRS images. Our method enables high-speed chemical imaging and cytometry with highly simplified experimental configurations.
11656-7
Author(s): Haonan Lin, Yuying Tan, Hyeon Jeong Lee, Ji-Xin Cheng, Boston Univ. (United States)
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Hyperspectral stimulated Raman scattering (hSRS) is a label-free microspectroscopic modality that enables live-cell metabolic imaging with chemical specificity. Yet, hSRS in the CH region has low throughput and poor chemical specificity, which limits its application to a broader range of metabolic studies. We propose a high-content, high-throughput hSRS imaging method by a sparsity-driven spectral unmixing and active spectral sub-sampling. We unprecedently generate chemical maps of four major metabolic species (lipid, protein, nucleic acid and carbohydrate) in a Mia PaCa-2 cell using seven spectral frames in the CH region, improving the acquisition speed by over an order of magnitude.
Session 4: Raman/CARS/SRS II
11656-36
Author(s): Rayssa de Andrade, Technical Univ. of Denmark (Denmark); Hugo Kerdoncuff, DFM A/S (Denmark); Kirstine Berg-Sørensen, Tobias Gehring, Technical Univ. of Denmark (Denmark); Mikael Lassen, DFM A/S (Denmark); Ulrik L. Andersen, Technical Univ. of Denmark (Denmark)
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Stimulated Raman spectroscopy has become a powerful tool to study the spatio-dynamics of molecular bonds with high sensitivity, resolution, and speed. However, the sensitivity and speed of stimulated Raman spectroscopy are ultimately limited by the shot-noise of the light beam probing the Raman process. Here, we demonstrate an enhancement of the sensitivity of stimulated Raman spectroscopy by reducing the noise below the shot-noise limit by means of squeezed states of light. Our demonstration constitutes the first step towards a new generation of quantum-enhanced Raman microscopes.
11656-37
Author(s): Marcus T. Cicerone, Xavier Audier, Jessica Zahn, Wei-Wen Chen, Georgia Institute of Technology (United States)
Digital Forum: On-demand starting 6 March
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I will discuss instrumentation improvements in Fourier-transform broadband coherent Raman microscopy that improve instrument simplicity, speed and spectral resolution. Together, these advances allow us to routinely and rapidly acquire quality Raman spectroscopic micrographs of tissues and cells. I will also discuss results of cancer and Covid tissue imaging using this microscope.
11656-38
Author(s): Robert W. Oda, Keio Univ. School of Medicine (Japan); Jingwen Shou, The Univ. of Tokyo (Japan); Keiko Karasawa, Mutsuo Nuriya, Masato Yasui, Keio Univ. School of Medicine (Japan); Yasuyuki Ozeki, The Univ. of Tokyo (Japan)
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The ability to simultaneously visualize the organization and interactions of cells and molecules within the brain can help us to understand it comprehensively. Stimulated Raman scattering (SRS) and fluorescence imaging are two modalities that can image biomolecules within cells and tissues. Here we present a high-speed, integrated SRS and confocal fluorescence imaging platform to investigate the three-dimensional organization of biomolecules in the brain. We show simultaneous imaging of cellular components and organelles within brain tissue. This platform has the potential to be adapted to explore disease progression within the brain and potentially the development of therapeutic options for brain disease.
11656-39
Author(s): Alejandro De la Cadena, Andrea Ragni, Giuseppe Sciortino, Federico Vernuccio, Carlo Valensise, Marco Sampietro, Giorgio Ferrari, Giulio N. Cerullo, Dario Polli, Politecnico di Milano (Italy)
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Stimulated Raman scattering (SRS) microscopy is a powerful technique for label-free identification of molecules based on their intrinsic vibrational spectrum. We present a novel approach to broadband SRS microscopy based on a recently developed balanced low-noise integrated-circuit multichannel lock-in amplifier with 10-μs integration time and 32 channels working in parallel, enabling multiplex SRS detection at speed faster than one hyperspectral frame per second. The system is powered by a narrowband Stokes pulse at 1040 nm and a broadband (approx. 500 cm^-1) pump pulse generated by a home-built low-noise optical parametric oscillator with up to 100-mW average power, covering the whole C-H stretching band. We measure stimulated Raman loss on the broadband pump pulse employing an in-line balanced detection approach to suppress the laser fluctuations and achieve close to shot-noise-limited sensitivity. We will show the system performances in high-resolution cell imaging.
11656-40
Author(s): Xiaowei Ge, Boston Univ. (United States); Fatima C. Pereira, Univ. Wien (Austria); Meng Zhang, Peng Lin, Boston Univ. (United States); Michael Wagner, Univ. Wien (Austria); Ji-Xin Cheng, Boston Univ. (United States)
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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.
11656-41
Author(s): Siddarth Shivkumar, Institut Fresnel (France); Dekel Raanan, Weizmann Institute of Science (Israel); Xavier Audier, Aix Marseille Univ. (France), CNRS (France), Ecole Centrale de Marseille (France); Maor Asher, Matan Menahem, Omer Yaffe, Weizmann Institute of Science (Israel); Nicolas Forget, Fastlite (France); Dan Oron, Weizmann Institute of Science (Israel); Hervé Rigneault, Aix Marseille Univ. (France), CNRS (France), Ecole Centrale de Marseille (France)
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Low frequency vibrations (< 200cm^(-1)) contain the structural information of molecules or the crystal lattice - making low frequency Raman imaging an ideal candidate to analyze heavy molecules, crystal formation etc. The longer integration times of spontaneous Raman spectroscopy, however, limits the study of dynamic structural changes. In our work, we have built a stimulated Raman scattering (SRS) pump probe scheme combining impulsive excitation with a fast acousto-optics delay line. With a pixel dwell time of 25μs, we have acquired, on a shot noise limited detection system, sub second low frequency (< 200cm^(-1)) hyper-spectral SRS images of various samples.
11656-42
Author(s): Yasunori Nawa, National Institute of Advanced Industrial Science and Technology (Japan), Osaka Univ. (Japan); Yasuaki Kumamoto, Osaka Univ. (Japan); Menglu Li, Satoshi Fujita, National Institute of Advanced Industrial Science and Technology (Japan), Osaka Univ. (Japan); Katsumasa Fujita, Osaka Univ. (Japan)
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Resonance Raman scattering is useful for improving a signal-to-noise ratio and a data acquisition speed in Raman imaging. However, the detection of non-resonance Raman scattering is often hindered by resonance signals and fluorescent background. To aid this dilemma in using resonance Raman scattering, we have developed a confocal Raman microscope with dual-wavelength excitation. Living HeLa cells were measured simultaneously at two different excitation wavelengths. At 532 nm excitation, cytochromes were detected by the resonance effect. At 660 nm excitation, non-resonance signals from proteins and lipids were obtained without any clear influence from cytochromes and fluorescent background.
Session 5: Translation to Clinic
11656-50
Author(s): Laura Marcu, Univ. of California, Davis (United States)
Digital Forum: On-demand starting 6 March
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This presentation overviews fluorescence lifetime spectroscopy and imaging techniques for label-free in vivo tissue diagnosis. Emphasis is placed on recently developed devices and methods enabling real-time characterization of tissue biochemical makeup during clinical interventions. I will present studies conducted in human patients demonstrating the ability of these techniques to provide a rapid in-situ evaluation of tissue biochemical properties, rapid classification of distinct tissue types, real-time augmentation of the tissue classifier on surgical field of view, and ultimately their potential to guide surgical procedures. Current results demonstrate that intrinsic fluorescence can provide useful contrast for intraoperative delineation of primary brain cancer at the resection margins and oral and oropharyngeal cancer.
11656-51
Author(s): Mischa Bonn, Max-Planck-Institut für Polymerforschung (Germany); Sapun Parekh, The Univ. of Texas at Austin (United States)
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Fibrin is the polymerized protein responsible for the stabilizing mesh in blood coagulation. Its superior mechanical properties of being stiff or elastic if needed result from a hierarchical structure, including semiflexible single fibers as well as constituent protein restructuring. Here, we stretch and shear fibrin hydrogels to a regime where unfolding transitions of α-helical structures to β-sheet are induced and observe protein structural changes with spatially-resolved coherent Raman microscopy. We confirm the theoretically predicted orthogonal orientation of helices and sheets in strained networks. Spatially resolved structure protein maps reveal that the extent of structural transition changes with gel composition and becomes highly heterogeneous at large strain, indicative of substantial load-bearing inhomogeneity .
11656-52
Author(s): David Punihaole, Univ. of Vermont (United States); Craig Van Bruggen, Renee Frontiera, Theresa Reineke, Univ. of Minnesota, Twin Cities (United States)
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Polymer-based vehicles that controllably deliver therapeutic nucleic acids to cells show great potential to develop safe and effective gene therapies. Realizing this potential, however, is limited by the lack of understanding of how polymeric vehicles unpackage and release their cargo in the cell. We address this problem by utilizing a novel quinine-containing polymeric vehicle that shows exceptional gene delivery activity. A key aspect of this platform is that the quinine serves as a reporter for DNA binding, which allows us to track cargo release inside the cell using chemical imaging. We find that proteins dominate the unpackaging of DNA encapsulated by these quinine polymers inside cells. This trackable delivery system should be broadly applicable to study gene delivery mechanisms, as well as be used for clinical therapy applications.
11656-53
Author(s): Ghazal Azarfar, Univ. of Illinois (United States), Beckman Institute for Advanced Science and Technology (United States), Carle Foundation Hospital (United States); Kianoush Falahkheirkhah, Univ. of Illinois (United States); Meera Gopu, Jennifer Pfister, Univ. of Illinois (United States), Beckman Institute for Advanced Science and Technology (United States); Sergey V. Brodsky, Tibor Nadasdy, The Ohio State Univ. Wexner Medical Ctr. (United States); Georgina Cheng, Carle Foundation Hospital (United States); Anjali A. Satoskar, The Ohio State Univ. Wexner Medical Ctr. (United States); Rohit Bhargava, Univ. of Illinois (United States), Beckman Institute for Advanced Science and Technology (United States), Carle Foundation Hospital (United States)
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Applications of machine learning in pathology is an active research area in modern medicine. Here, we presented a classifier for label-free renal histopathology. Three frequently encountered categories of monoclonal gammopathy-associated kidney disease were studied, which included light chain amyloidosis, monoclonal light chain disease deposition (MIDD) and myeloma cast nephropathy. Biopsies with diabetic nephropathy and normal baseline transplant biopsies were used as control. The samples are imaged using a FTIR hyperspectral microscope. More than three million infrared spectra are adopted for the training and evaluation of the computational model. The model recognizes the pixels associated with the glomerulus, and diagnoses the disease based on infrared absorption features.
11656-54
Author(s): Xiaona Wen, Vanderbilt Univ. (United States); Rizia Bardhan, Iowa State Univ. of Science and Technology (United States)
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Rapid and accurate response to targeted therapies is critical to differentiate tumors that are resistant to treatment early in the regimen. In this work, we demonstrate a rapid, noninvasive, and label-free approach to evaluate treatment response to molecular inhibitors in breast cancer (BC) cells with Raman spectroscopy (RS). Metabolic reprograming in BC was probed with RS and multivariate analysis was applied to classify the cells into responsive or nonresponsive groups as a function of drug dosage, drug type, and cell type. Metabolites identified with RS were then validated with mass spectrometry. Our findings support that oncometabolites identified with RS will ultimately enable rapid drug screening in patients ensuring patients receive the most effective treatment at the earliest time point.
11656-55
Author(s): Shachi Mittal, Rohit Bhargava, Beckman Institute for Advanced Science and Technology (United States)
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Spectroscopic imaging offers a potential path to all-digital molecular pathology by relating the spectral data to histopathological details of tissue. In the different statistical approaches applied to spectral data, there are gaps in the appropriate sample size estimation for a significant statistical power and accurate model assessment especially for multiclass problems. Underestimation of the sample size can lead to statistically insignificant diagnostic tests while an overestimation can greatly increase experimental costs and time frames.Since the receiver operating characteristic (ROC) curve is designed primarily for a binary test, there are no straightforward approaches to use it for multiple classes in a typical pathology image. In this study, we have described sample size estimation (power analysis) and multiclass diagnostic ROC curve generation for hyperspectral datasets.
Session 6: IR and Photothermal Chemical Imaging
11656-8
Author(s): Rohit Bhargava, Beckman Institute for Advanced Science and Technology (United States)
Digital Forum: On-demand starting 6 March
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Infrared (IR) spectroscopic imaging has tremendous potential to provide new and useful data in a variety of fields. Instrument design and performance, however, need to be understood from a first principles approach to realize these advantages. Here we provide several examples, from microscopy to nanoscopy, of the development of theoretical understanding and performance enhancements in IR spectroscopic imaging. The increase in measurement capability is illustrated with biomedical samples.
11656-9
Author(s): Devon S. Jakob, Xiaoji G. Xu, Lehigh Univ. (United States)
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Multimodal measurements of chemical composition, electrical properties, mechanical properties, and topography by scanning probe microscopy (SPM) deliver correlations across properties at the nanoscale, and provide clues to the structure-function relationship of materials. In the past, measurements with these modalities are operated separately with different operational modes of SPM. At the conference, we will present our invention of peak force infrared-Kelvin probe force microscopy (PFIR-KPFM), which is an integrated SPM mode that can simultaneously provide chemical, surface potential, mechanical, and topographic imaging at < 10 nm spatial resolution under the ambient conditions. As an initial demonstration, we measured amyloid fibrils and observed the correlations between surface potentials and infrared response. The residual charges of the fibrils are associated with the anti-parallel beta stacking.
11656-10
Author(s): Mustafa Kansiz, Photothermal Spectroscopy Corp. (United States); Alice Spadea, The Univ. of Manchester (United Kingdom); Jayakrupakar Nallala, Univ. of Exeter (United Kingdom); Cassio Lima, Howbeer Muhamadali, Univ. of Liverpool (United Kingdom); Joanna Denbigh, Biomedical Research Ctr., Univ. of Salford (United Kingdom); Jayne Lawrence, The Univ. of Manchester (United Kingdom); Gorkem Bakir, Univ. of Manitoba (Canada); Peter Gardner, The Univ. of Manchester (United Kingdom); Nick Stone, Univ. of Exeter (United Kingdom); Roy Goodacre, Univ. of Liverpool (United Kingdom); Kathleen Gough, Univ. of Manitoba (Canada); Oxana Klementieva, Lund Univ. (Sweden)
Digital Forum: On-demand starting 6 March
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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, correlative IR+Raman microscopy. These unique and exciting synergistic capabilities are now spawning interest in life science application [1-2]. A broad range of life science applications, otherwise impossible with traditional FTIR/QCL microscopy, will be presented, ranging from live cell imaging in water, to ultra-high resolution images of breast tissue calcifications, amyloid aggregates in neurons (neurites and dendritic spines), individual collagen fibrils with polarized IR and individual isotopically labelled bacterial cells and more.
11656-11
Author(s): Ilia M. Pavlovetc, Kirill Kniazev, Junyeol Kim, Kyle Doudrick, Masaru Kuno, Univ. of Notre Dame (United States)
Digital Forum: On-demand
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Infrared (IR) imaging and spectroscopy is an important technique for identifying chemicals and elucidating their structure. Despite its advantages, IR imaging is mainly hindered by its poor spatial resolution on the order of 3-10 microns. This impedes applications of IR imaging and spectroscopy in nanoscience. This presentation highlights recent advances in optical super-resolution, mid-IR photothermal imaging and spectroscopy. It will illustrate several applications where infrared photothermal super-resolution capabilities has led to notable achievements in nano- and materials science. The presentation will also suggest an outlook for developing, super-resolution mid-IR imaging techniques as well as their applications in bio- and materials sciences.
11656-12
Author(s): Yamuna Phal, Kevin L. Yeh, Rohit Bhargava, Beckman Institute for Advanced Science and Technology (United States)
Digital Forum: On-demand starting 6 March
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Infrared polarimetry is a powerful label-free diagnostic tool to study the molecular alignment and organization in biological tissues and cells. Similar to absorbance images which capture intensity information, polarimetric imaging is essential for capturing the polarization states of the light intensity. Recent advancements in the development of Quantum Cascade Lasers (QCL) sources have opened new avenues in IR imaging with high spatial and spectral resolution while enabling drastic increases in imaging speeds than a corresponding FT-IR approach. We demonstrate improved performance in terms of fast and comprehensive polarimetric image acquisition using a custom-built QCL microscope with point mapping design.
11656-13
Author(s): Kevin L. Yeh, Univ. of Illinois (United States); Yamuna Phal, Univ. of Illinois (United States); Rohit Bhargava, Univ. of Illinois (United States)
Digital Forum: On-demand starting 6 March
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Infrared spectroscopic imaging is an analytical approach that can reveal important molecular information without the need for substantial sample processing. These instruments can provide objective and automated evaluations to aid pathologists improve diagnostic accuracy. The quantum cascade laser, allows for a discrete frequency approach, increasing imaging speeds with superior spatial and spectral resolution. We present our recent progress toward developing new instruments capable of diffraction limited performance at all fingerprint-region wavelengths across the entire field of view. We demonstrate high throughput imaging of tissue sections and tissue microarrays and evaluate the advantages in data quality obtained from a well-corrected system.
11656-14
Author(s): Yeran Bai, Zhongyue Guo, Ji-Xin Cheng, Boston Univ. (United States)
Digital Forum: On-demand
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We developed a versatile mid-infrared photothermal (MIP) modality that enabled study of metabolic activities in living cells coupled with stable isotope probing. Performance of laser and light emitting diode were compared and system detection limit was demonstrated for different stable isotope probes. We treated the cancer and bacteria cells with deuterium, 13C and 15N labelled nutrients, and acquired the multi-spectral images with the MIP microscope. We observed the red shift in the infrared spectrum, indicating the incorporation of heavy atoms into cell metabolism. Sub-cellular spatial distribution of metabolites including carbohydrates, nucleic acids, proteins and lipids were profiled with high chemical specificity, sub-micrometer spatial resolution and high throughput.
Session 7: Raman/CARS/SRS III
11656-43
CANCELED: Coherent Raman polarization-resolved generation inside scattering tissues
Digital Forum: On-demand starting 6 March
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Coherent Raman scattering (CRS) imaging and its polarization resolved version, are promising optical contrasts to assess specific vibrational bonds in tissues. CRS imaging deep into scattering biological tissues is however challenging since after a few hundreds of micron depths, optical paths are randomized and both coherence and nonlinear generation are lost. We demonstrate coherent anti Stokes Raman scattering (CARS) wavefront optimization inside a mouse spinal cord at such depth, using a high number of degrees of freedom from a spatial light modulator. The intrinsic coherent manipulation allows fields of view above the expected memory range, as well as polarization preservation.
11656-44
Author(s): Dan Fu, Univ. of Washington (United States)
Digital Forum: On-demand
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The brain is an enormously complex organ that consumes a substantial amount of body energy. Understanding how brain function requires the ability to not only map out the cellular structure but also probe functional processes. Multiphoton fluorescence microscopy has played a crucial role in current investigations due to the wide variety of synthetic dyes and proteins available for imaging brain cells and neuronal activities at high spatial and temporal resolution. However, it has limited capability to resolve multiple features simultaneously. We combine multiphoton fluorescence with label-free nonlinear imaging techniques including transient absorption microscopy and stimulated Raman scattering microscopy to expand the structural and functional features that can be imaged simultaneously. With this platform, we demonstrate the reconstruction of axonal features and microvascular networks together with the mapping of cellular organization.
11656-45
Author(s): Xiaoqi Lang, Kevin Welsher, Duke Univ. (United States)
Digital Forum: On-demand
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The structure and dynamics of intracellular water constitute the cornerstone for understanding all aspects of cellular function. However, direct visualization of subcellular solvation heterogeneity has remained elusive. To explore this question, we have demonstrated a vibrational-shift imaging approach to probe solvation at the microscopic level by combining spectral-focusing hyperspectral stimulated Raman scattering (hsSRS) with an environmentally-sensitive nitrile probe. When applied to quantitatively measure the spatial variation of solvation in live cells, this new method reveals significantly reduced solvation in the cytoplasm compared to the nuclear compartment and bulk water! This work sheds light on heterogenous solvation at the subcellular level and opens up new avenues to explore solvation variance in complex systems.
11656-46
Author(s): Prabuddha Mukherjee, Univ. of Illinois (United States); Aneesh Alex, GlaxoSmithKline (United States), Univ. of Illinois (United States); Edita Aksamitiene, Derek Shi, Univ. of Illinois (United States); Darold R. Spillman, Univ. of Illinois (United States); Marina Marjanovic, Univ. of Illinois (United States); Michael Fazio, Punit Seth, Ionis Pharmaceuticals, Inc. (United States); Kendal Frazier, GlaxoSmithKline (United States); Steve R. Hood, GlaxoSmithKline (United States), Univ. of Illinois (United States); Stephen A. Boppart, Univ. of Illinois (United States)
Digital Forum: On-demand
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Antisense oligonucleotides (ASOs) are single stranded negatively charged molecules which downregulate the translation of specific target messenger RNA (mRNA). Chemically modified ASOs with phosphorothioate (PS) linkages have been extensively studied as research tools and as clinical therapeutics and nine oligonucleotide-based drugs have been approved by regulatory agencies. While several cell surface proteins that bind PS-ASOs and mediate their cellular uptake have been identified, the mechanisms leading to productive internalization of PS-ASOs are not well understood. We demonstrate the potential of hyperspectral CARS imaging to detect the intracellular presence of ASOs in a label-free manner.
11656-47
Author(s): Jiajun Du, Lu Wei, Caltech (United States)
Digital Forum: On-demand
11656-48
Author(s): Nan Wang, Feng Ren, Xu Nie, Xinyi Xu, Qi Zeng, Yonghua Zhan, Shouping Zhu, Xueli Chen, Xidian Univ. (China)
Digital Forum: On-demand
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In this study, aiming at the interference of scattering medium for detecting the drug in pharmaceutical dosage forms, the traditional methods will cause damage to the samples and the Raman spectroscopy based on the Gaussian beam is difficult to implement in vivo and deep imaging. We proposed a new scheme of using the Bessel beam as the excitation light for Raman spectroscopy, which can provide solution for in vivo and deep imaging. The feasibility and effectiveness of the proposed Bessel beam-based RS system for detecting the drug in different pharmaceutical dosage forms were verified by a group of experiments.
11656-49
Author(s): Kun Miao, Lu Wei, Caltech (United States)
Digital Forum: On-demand
Session 8: IR and Photothermal Chemical Imaging & New Methods for Chemical Imaging
11656-15
Author(s): Dave Knez, Richard C. Prince, Adam M. Hanninen, Dmitry Fishman, Univ. of California (United States); Eric O. Potma, Univ. of California, Irvine (United States)
Digital Forum: On-demand
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Vibrational microscopy based on the mid-infrared (MIR) absorption of molecular modes is a proven strategy for label-free imaging. Yet, as a tool for cellular imaging, MIR vibrational imaging suffers from several shortcomings., among which the low spatial resolution and the unavailability of affordable imaging cameras with high pixel densities. In this presentation, we will discuss how nonlinear optical (NLO) interactions can be employed to improve MIR imaging considerably. First, we introduce a new NLO method for capturing MIR images on a regular Si-based camera. Second, we highlight how NLO interactions can be used to improve the resolution in MIR microscopy.
11656-16
Author(s): Takuro Ideguchi, The Univ. of Tokyo (Japan)
Digital Forum: On-demand
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We develop a chemically-selective quantitative phase imaging technique based on mid-infrared photothermal effect, with which we can simultaneously measure a morphologically sensitive quantitative phase image and a molecularly sensitive mid-infrared photothermal phase image. We show that the wide-field label-free dual-modal microscopy technique can be implemented with digital holography (2D) and optical diffraction tomography (3D). We also demonstrate a new technique to improve the sensitivity of the mid-infrared photothermal phase imaging via a dynamic-range expanded quantitative phase imaging, called adaptive dynamic range shift quantitative phase imaging (ADRIFT-QPI).
11656-17
Author(s): Garth J. Simpson, Chen Li, Changqin Ding, Purdue Univ. (United States)
Digital Forum: On-demand
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Spatially patterning the polarization state of a beam enables a novel approach for phase contrast microscopy, in which two foci (sample and reference) are offset along the optical axis. Polarization wavefront shaping is achieved through the addition of two custom microretarter array (RA) 25mm optics, which serve as selective converging/diverging lenses focusing right and left circular polarization components onto different focal planes. Positioning of a matched RA in the transmission path recombines the two polarization components, with phase differences manifesting and rotations in the polarization state. This approach combines benefits of Nomarski and Zernike phase contrast strategies, while minimizing common artifacts associated with each. In addition, polarization wavefront shaping provides a straightforward route for depth of field extension through addition of a single fixed 1” optic.
11656-18
Author(s): Miguel A. Pleitez, Asrar Ali Khan, Alice Solda, Andriy Chmyrov, Josefine Reber, Markus Seeger, Francesca Gasparin, Benedikt Schätz, Marcel Scheideler, Stephan Herzig, Helmholtz Zentrum München GmbH (Germany); Vasilis Ntziachristos, Helmholtz Zentrum München (Germany), Technical Univ Munich (TUM) (Germany)
Digital Forum: On-demand
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Imaging modalities based on vibrational spectroscopy (Raman or mid-IR imaging) have demonstrated high label-free chemical specificity for different biomolecules. Nevertheless, conventional mid-IR microscopy have been limited mostly to dry tissues and fixed cells due to the strong mid-IR absorption of water and due to the use of conventional negative-contrast detection. We introduce positive-contrast Mid-infraRed Optoacoustic Microscopy (MiROM) for label-free metabolic imaging in living cells. We showcase the unique capabilities of MiROM in living cells by monitoring the spatiotemporal distribution of carbohydrates, lipids, and proteins in adipocytes during lipogenesis as well as monitoring the lipid-protein dynamics in brown and white adipocytes during lipolysis. We discuss how MiROM yields unique label-free metabolic imaging abilities for a broader range of bioanalytical studies in living cells and its potential for analytical histology in unprocessed tissues.
11656-19
Author(s): Peng Lin, Hongli Ni, Huate Li, Yuying Tan, Nicholas A. Vickers, Thomas Bifano, Ji-Xin Cheng, Boston Univ. (United States)
Digital Forum: On-demand
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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.
11656-21
Author(s): Christian H. Allen, Carleton Univ. (Canada); Ben Hansson, Dalhousie Univ. (Canada); Olivia Raiche-Tanner, Sangeeta Murugkar, Carleton Univ. (Canada)
Digital Forum: On-demand
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Silicon photomultipliers are a promising new type of detector for low light detection applications. In the field of multiphoton imaging, photomultiplier tubes have been the traditional detector of choice. Apart from their robustness, low cost, and low-voltage requirements, one key advantage SiPMs have over PMTs is their compact size. This allows for the creation of compact signal collection paths that will benefit the development of compact multimodal multiphoton microscopes. In this ongoing work, we demonstrate the acquisition of coherent anti-Stokes Raman scattering, second harmonic generation, and two-photon excitation fluorescence signals for multiphoton microscopy imaging of various samples using a compact SiPM detector and photomultiplier tube simultaneously for visual comparison. We discuss the relative advantages and disadvantages of using either detector, particularly to how it pertains to developing compact microscopes and probes.
Front Matter: Volume 11656
Conference Chair
Boston Univ. (United States)
Conference Chair
Columbia Univ. (United States)
Conference Chair
Purdue Univ. (United States)
Program Committee
Univ. of Illinois (United States)
Program Committee
Univ. of Illinois (United States)
Program Committee
Institut Fresnel (France)
Program Committee
Korea Univ. (Korea, Republic of)
Program Committee
Georgia Institute of Technology (United States)
Program Committee
Ecole Normale Supérieure (France)
Program Committee
Wellman Ctr. for Photomedicine (United States)
Program Committee
Hanieh Fattahi
Max-Planck-Institut für Quantenoptik (Germany)
Program Committee
Univ. of Washington (United States)
Program Committee
Osaka Univ. (Japan)
Program Committee
Univ. of Oxford (United Kingdom)
Program Committee
National Univ. of Singapore (Singapore)
Program Committee
Fudan Univ. (China)
Program Committee
Vanderbilt Univ. (United States)
Program Committee
Univ. of Exeter (United Kingdom)
Program Committee
The Univ. of Tokyo (Japan)
Program Committee
The Univ. of Texas at Austin (United States)
Program Committee
Univ. of Virginia (United States)
Program Committee
Politecnico di Milano (Italy)
Program Committee
Leibniz-Institut für Photonische Technologien e.V. (Germany)
Program Committee
Univ. of California, Irvine (United States)
Program Committee
Univ. of Houston (United States)
Program Committee
Institut Fresnel (France)
Program Committee
Univ. of California, San Diego (United States)
Program Committee
National Taiwan Univ. (Taiwan)
Program Committee
Meng Wang
Baylor College of Medicine (United States)
Program Committee
Duke Univ. (United States)
Program Committee
Colorado State Univ. (United States)
Program Committee
Peking Univ. (China)
Program Committee
Xiaoji G. Xu
Lehigh Univ. (Canada)
Program Committee
Beihang Univ. (China)
Additional Information
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Post-Deadline Submission Portal