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      High-resolution, high-contrast mid-infrared imaging of fresh biological samples with ultraviolet-localized photoacoustic microscopy

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          Abstract

          Mid-infrared (MIR) microscopy provides rich chemical and structural information about biological samples, without staining. Conventionally, the long MIR wavelength severely limits the lateral resolution owing to optical diffraction; moreover, the strong MIR absorption of water ubiquitous in fresh biological samples results in high background and low contrast. To overcome these limitations, we propose a method that employs photoacoustic detection highly localized with a pulsed ultraviolet (UV) laser on the basis of the Grüneisen relaxation effect. For cultured cells, our method achieves water-background suppressed MIR imaging of lipids and proteins at UV resolution, at least an order of magnitude finer than the MIR diffraction limits. Label-free histology using this method is also demonstrated in thick brain slices. Our approach provides convenient high-resolution and high-contrast MIR imaging, which can benefit diagnosis of fresh biological samples.

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          Most cited references63

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          Optical Constants of Water in the 200-nm to 200-μm Wavelength Region

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            Using Fourier transform IR spectroscopy to analyze biological materials.

            IR spectroscopy is an excellent method for biological analyses. It enables the nonperturbative, label-free extraction of biochemical information and images toward diagnosis and the assessment of cell functionality. Although not strictly microscopy in the conventional sense, it allows the construction of images of tissue or cell architecture by the passing of spectral data through a variety of computational algorithms. Because such images are constructed from fingerprint spectra, the notion is that they can be an objective reflection of the underlying health status of the analyzed sample. One of the major difficulties in the field has been determining a consensus on spectral pre-processing and data analysis. This manuscript brings together as coauthors some of the leaders in this field to allow the standardization of methods and procedures for adapting a multistage approach to a methodology that can be applied to a variety of cell biological questions or used within a clinical setting for disease screening or diagnosis. We describe a protocol for collecting IR spectra and images from biological samples (e.g., fixed cytology and tissue sections, live cells or biofluids) that assesses the instrumental options available, appropriate sample preparation, different sampling modes as well as important advances in spectral data acquisition. After acquisition, data processing consists of a sequence of steps including quality control, spectral pre-processing, feature extraction and classification of the supervised or unsupervised type. A typical experiment can be completed and analyzed within hours. Example results are presented on the use of IR spectra combined with multivariate data processing.
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              Characterization of polydimethylsiloxane (PDMS) properties for biomedical micro/nanosystems.

              Polydimethylsiloxane (PDMS Sylgard 184, Dow Corning Corporation) pre-polymer was combined with increasing amounts of cross-linker (5.7, 10.0, 14.3, 21.4, and 42.9 wt.%) and designated PDMS1, PDMS2, PDMS3, PDMS4, and PDMS5, respectively. These materials were processed by spin coating and subjected to common micro-fabrication, micro-machining, and biomedical processes: chemical immersion, oxygen plasma treatment, sterilization, and exposure to tissue culture media. The PDMS formulations were analyzed by gravimetry, goniometry, tensile testing, nano-indentation, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and X-ray photoelectron spectroscopy (XPS). Spin coating of PDMS was formulation dependent with film thickness ranging from 308 microm on PDMS1 to 171 microm on PDMS5 at 200 revolutions per minute (rpm). Ultimate tensile stress (UTS) increased from 3.9 MPa (PDMS1) to 10.8 MPa (PDMS3), and then decreased down to 4.0 MPa (PDMS5). Autoclave sterilization (AS) increased the storage modulus (sigma) and UTS in all formulations, with the highest increase in UTS exhibited by PDMS5 (218%). PDMS surface hydrophilicity and micro-textures were generally unaffected when exposed to the different chemicals, except for micro-texture changes after immersion in potassium hydroxide and buffered hydrofluoric, nitric, sulfuric, and hydrofluoric acids; and minimal changes in contact angle after immersion in hexane, hydrochloric acid, photoresist developer, and toluene. Oxygen plasma treatment decreased the contact angle of PDMS2 from 109 degrees to 60 degrees. Exposure to tissue culture media resulted in increased PDMS surface element concentrations of nitrogen and oxygen.
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                Author and article information

                Journal
                101283276
                34862
                Nat Photonics
                Nat Photonics
                Nature photonics
                1749-4885
                1749-4893
                10 April 2019
                13 May 2019
                September 2019
                13 November 2019
                : 13
                : 609-615
                Affiliations
                [1 ]Caltech Optical Imaging Laboratory, Andrew and Peggy Cherng Department of Medical Engineering, Department of Electrical Engineering, California Institute of Technology, Pasadena, CA 91125, USA
                [2 ]Optical Imaging Laboratory, Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO 63130, USA
                [3 ]Present address: Translational and Advanced Bioimaging Laboratory, Department of Chemical and Biological Engineering, Hong Kong University of Science and Technology, Hong Kong, China
                [4 ]Applied Physics Division, National Institute of Standards and Technology, 325 Broadway Street, Boulder, CO 80305, USA
                Author notes

                Author contributions

                J.S., K.M, L.V.W. designed the experiment. J.S., T.W., Y.H., R.Z. contributed to the system construction. J.S., T.W. prepared the brain slices. Y.H. prepared the cell culture. C.Y. and J.H. designed and prepared the CNT-pattern on a MgF 2 substrate. L.L. helped with LFB staining. J.S., K.M., T.W., Y.H., L.L. involved in some discussions. J.S. performed the experiment and data analysis. L.V.W supervised the project. All authors are involved in the manuscript preparation.

                [* ]Correspondence should be addressed to L.V.W. ( LVW@ 123456caltech.edu ).
                Article
                NIHMS1526731
                10.1038/s41566-019-0441-3
                6705424
                31440304
                1dd2aa5c-4a4a-44a9-a6dd-97b2dac6bc8b

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                Optical materials & Optics
                Optical materials & Optics

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