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      Depth-resolved mid-infrared photothermal imaging of living cells and organisms with submicrometer spatial resolution

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          Abstract

          Photothermal measurement enabled infrared spectroscopic imaging of live cells and organisms with submicrometer resolution.

          Abstract

          Chemical contrast has long been sought for label-free visualization of biomolecules and materials in complex living systems. Although infrared spectroscopic imaging has come a long way in this direction, it is thus far only applicable to dried tissues because of the strong infrared absorption by water. It also suffers from low spatial resolution due to long wavelengths and lacks optical sectioning capabilities. We overcome these limitations through sensing vibrational absorption–induced photothermal effect by a visible laser beam. Our mid-infrared photothermal (MIP) approach reached 10 μM detection sensitivity and submicrometer lateral spatial resolution. This performance has exceeded the diffraction limit of infrared microscopy and allowed label-free three-dimensional chemical imaging of live cells and organisms. Distributions of endogenous lipid and exogenous drug inside single cells were visualized. We further demonstrated in vivo MIP imaging of lipids and proteins in Caenorhabditis elegans. The reported MIP imaging technology promises broad applications from monitoring metabolic activities to high-resolution mapping of drug molecules in living systems, which are beyond the reach of current infrared microscopy.

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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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            Photothermal imaging of nanometer-sized metal particles among scatterers.

            Ambient optical detection of labeled molecules is limited for fluorescent dyes by photobleaching and for semiconducting nanoparticles by "blinking" effects. Because nanometer-sized metal particles do not optically bleach, they may be useful optical labels if suitable detection signals can be found. We demonstrate far-field optical detection of gold colloids down to diameters of 2.5 nanometers with a photothermal method that combines high-frequency modulation and polarization interference contrast. The photothermal image is immune to the effects of scattering background, which limits particle imaging through Rayleigh scattering to diameters larger than 40 nanometers.
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              Selective blockade of 2-arachidonoylglycerol hydrolysis produces cannabinoid behavioral effects

              2-Arachidonoylglycerol (2-AG) and anandamide are endocannabinoids that activate cannabinoid receptors CB1 and CB2. Endocannabinoid signaling is terminated by enzymatic hydrolysis, a process that, for anandamide, is mediated by fatty acid amide hydrolase (FAAH) and, for 2-AG, is thought to involve monoacylglycerol lipase (MAGL). FAAH inhibitors produce a select subset of the behavioral effects observed with CB1 agonists, intimating a functional segregation of endocannabinoid signaling pathways in vivo. Testing this hypothesis, however, requires specific tools to independently block anandamide and 2-AG metabolism. Here, we report a potent and selective inhibitor of MAGL, JZL184, that, upon administration to mice, raises brain 2-AG by 8-fold without altering anandamide. JZL184-treated mice exhibited a broad array of CB1-dependent behavioral effects, including analgesia, hypothermia, and hypomotility. These data indicate that 2-AG endogenously modulates several behavioral processes classically associated with the pharmacology of cannabinoids and point to overlapping and unique functions for 2-AG and anandamide in vivo.
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                Author and article information

                Journal
                Sci Adv
                Sci Adv
                SciAdv
                advances
                Science Advances
                American Association for the Advancement of Science
                2375-2548
                September 2016
                28 September 2016
                : 2
                : 9
                : e1600521
                Affiliations
                [1 ]Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47907, USA.
                [2 ]Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA.
                [3 ]Department of Mechanical Engineering, Purdue University, West Lafayette, IN 47907, USA.
                [4 ]Jonathan Amy Facility for Chemical Instrumentation, Purdue University, West Lafayette, IN 47907, USA.
                [5 ]Purdue Institute of Inflammation, Immunology and Infectious Disease, Purdue University, West Lafayette, IN 47907, USA.
                Author notes
                [* ]Corresponding author. Email: jcheng@ 123456purdue.edu
                Author information
                http://orcid.org/0000-0002-9734-3573
                http://orcid.org/0000-0003-3311-9385
                http://orcid.org/0000-0002-2486-3471
                http://orcid.org/0000-0002-5607-6683
                Article
                1600521
                10.1126/sciadv.1600521
                5040478
                27704043
                37225648-fe12-4001-8f82-d07e89507169
                Copyright © 2016, The Authors

                This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

                History
                : 14 March 2016
                : 20 August 2016
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100000888, W.M. Keck Foundation;
                Award ID: ID0EK3CI
                Award Recipient :
                Categories
                Research Article
                Research Articles
                SciAdv r-articles
                Imaging
                Custom metadata
                Florcloven Cruz

                label-free imaging,infrared microscopy,infrared spectroscopy,live cell imaging,in vivo imaging

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