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Attenuated Total Reflectance (ATR) Infrared Spectroscopy: Overview01:13

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Attenuated total reflectance (ATR) infrared spectroscopy is a powerful analytical technique used to study the composition of materials. It is widely employed in chemistry, materials science, forensic science, and other fields where sample characterization is required. ATR has several advantages over traditional transmission IR spectroscopy, including the requirement of little to no sample preparation and the ability to analyze a wide range of samples.
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Raman Spectroscopy: Overview01:20

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The underlying principle of Raman spectroscopy is based on the interaction between light and matter, specifically molecules' inelastic scattering of photons. When a monochromatic beam of light, typically from a laser source, interacts with a sample, most scattered light has the same frequency as the incident light. This is known as Rayleigh scattering.
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A conventional Raman spectrophotometer includes a laser source, a sample holding system, a wavelength selector, and a detector.
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IR Spectrometers

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There are two main infrared (IR) spectrophotometers: dispersive IR spectrometers and Fourier transform infrared (FTIR) spectrometers. In a dispersive IR spectrometer, a beam of infrared radiation produced by a hot wire is divided into two parallel equal-intensity beams using mirrors. One beam passes through the sample, while another is a reference beam. The beams then move through the monochromator, which separates the radiations into a continuous spectrum of different frequencies. The...
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Applications of IR Spectroscopy: Overview01:11

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The non-destructive nature and ability to provide valuable chemical information make IR spectroscopy a versatile technique with broad applications in various scientific and industrial fields. IR spectroscopy is commonly used to identify and characterize organic and inorganic compounds. It provides information about the functional groups present in a molecule and the bonding between atoms. This helps in the structural elucidation of compounds during organic synthesis, pharmaceutical research,...
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2D NMR: Overview of Heteronuclear Correlation Techniques01:18

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Heteronuclear correlation spectroscopy is an analytical technique that investigates the coupling between different types of nuclei, often a proton and an X-nucleus, such as carbon-13 or nitrogen-15. This method is commonly used in nuclear magnetic resonance (NMR) spectroscopy to gain insights into complex chemical compounds' structural and compositional aspects. A typical heteronuclear correlation spectrum displays X-nucleus chemical shifts on one axis and a proton spectrum on the other...
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Related Experiment Video

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Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy
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Arbitrary-Region Raster Image Correlation Spectroscopy.

Jelle Hendrix1, Tomas Dekens2, Waldemar Schrimpf3

  • 1Laboratory for Photochemistry and Spectroscopy, Division of Molecular Imaging and Photonics, KU Leuven, Leuven, Belgium.

Biophysical Journal
|October 21, 2016
PubMed
Summary
This summary is machine-generated.

A new arbitrary-region image correlation spectroscopy (ARICS) method enables detailed molecular analysis in complex cellular environments. This advanced technique overcomes limitations of square regions, allowing for precise measurements of diffusion and concentration in cells.

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Area of Science:

  • Biophysics
  • Cell Biology
  • Microscopy and Imaging

Background:

  • Raster Image Correlation Spectroscopy (RICS) analyzes confocal laser-scanning microscopy (CLSM) images to determine molecular diffusion, concentration, and interactions.
  • Traditional RICS is limited to square regions-of-interest (ROIs), hindering analysis in complex cellular areas.
  • Region-specific analysis of molecular mobility within cells is challenging with existing methods.

Purpose of the Study:

  • To develop a generalized image correlation spectroscopy algorithm capable of analyzing arbitrarily shaped regions (ARICS).
  • To demonstrate the utility of ARICS for simultaneous measurement of molecular mobility in different cellular compartments.
  • To enable investigation of molecular mobility near dynamic subcellular structures using dynamic ROIs.

Main Methods:

  • Development of a generalized image correlation spectroscopy algorithm for arbitrary-shaped ROIs (ARICS).
  • Image series preprocessing for enhanced ARICS analysis.
  • Application of ARICS to simultaneously measure molecular mobility in cell membranes and cytosol, and near dynamic structures.

Main Results:

  • The ARICS algorithm successfully analyzes arbitrarily shaped ROIs, overcoming limitations of square ROIs in RICS.
  • Simultaneous measurement of molecular mobility in distinct cellular locations (membrane and cytosol) was achieved.
  • ARICS enabled the investigation of molecular mobility dynamics near subcellular structures by using dynamic ROIs.
  • Diffusion and concentration pseudo-maps were derived using the ARICS method.

Conclusions:

  • ARICS is a powerful advancement in image correlation spectroscopy, expanding its applicability to complex cellular analyses.
  • The method allows for precise extraction of biophysical parameters like diffusion constants and concentrations from CLSM images.
  • ARICS has the potential to become a new standard for quantitative analysis of molecular dynamics in fluorescence microscopy.