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Related Concept Videos

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

Attenuated Total Reflectance (ATR) Infrared Spectroscopy: Overview

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.
The ATR process begins by directing a beam...
Raman Spectroscopy Instrumentation: Overview01:26

Raman Spectroscopy Instrumentation: Overview

A conventional Raman spectrophotometer includes a laser source, a sample holding system, a wavelength selector, and a detector.
The monochromatic laser source, typically using visible or near-infrared radiation, generates a highly focused beam of light. This light interacts with the molecules of the sample, scattering some of the light. Liquid and gaseous samples are usually tested in ordinary glass capillaries, while solids can be analyzed as powders packed in capillaries or as potassium...
Raman Spectroscopy: Overview01:20

Raman Spectroscopy: Overview

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.
However, a small fraction of the scattered light exhibits a frequency shift due to the exchange of energy between the incident photons and the...
IR Spectrometers01:25

IR Spectrometers

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...
Infrared (IR) Spectroscopy: Overview01:09

Infrared (IR) Spectroscopy: Overview

When electromagnetic radiation passes through a material, atoms or molecules transition from a lower to a higher energy state by absorbing radiation corresponding to the energy difference between the two states. The absorption of infrared (IR) radiation causes transitions between vibrational energy levels in a molecule. Therefore, IR spectroscopy is a useful analytical tool for determining the molecular structure of molecules.
Different compounds display unique properties due to their...
IR Spectroscopy: Molecular Vibration Overview01:24

IR Spectroscopy: Molecular Vibration Overview

When Infrared (IR) radiation passes through a covalently bonded molecule, the bonds transition from lower to higher vibrational levels. The fundamental vibrational motions that result in infrared absorption can be classified as stretching or bending vibrations.
Stretching vibrations are vibrational motions that occur along the bond line, changing the bond length or distance between two bonded atoms. They are further distinguished as symmetric or asymmetric. In symmetric stretching, the...

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Related Experiment Video

Updated: May 15, 2026

Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy
09:57

Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy

Published on: July 25, 2022

Raster image correlation spectroscopy and number and brightness analysis.

Michelle A Digman1, Milka Stakic, Enrico Gratton

  • 1Department of Biomedical Engineering, University of California, Irvine, California, USA.

Methods in Enzymology
|January 2, 2013
PubMed
Summary

Raster Image Correlation Spectroscopy (RICS) and Number and Molecular Brightness (N&B) analyze molecular diffusion and complex formation in cells. These methods quantify molecular interactions and map their spatial distribution using fluorescence fluctuations.

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Direct Comparison of Hyperspectral Stimulated Raman Scattering and Coherent Anti-Stokes Raman Scattering Microscopy for Chemical Imaging
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Direct Comparison of Hyperspectral Stimulated Raman Scattering and Coherent Anti-Stokes Raman Scattering Microscopy for Chemical Imaging

Published on: April 28, 2022

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Last Updated: May 15, 2026

Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy
09:57

Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy

Published on: July 25, 2022

Direct Comparison of Hyperspectral Stimulated Raman Scattering and Coherent Anti-Stokes Raman Scattering Microscopy for Chemical Imaging
09:46

Direct Comparison of Hyperspectral Stimulated Raman Scattering and Coherent Anti-Stokes Raman Scattering Microscopy for Chemical Imaging

Published on: April 28, 2022

Area of Science:

  • Biophysics
  • Cell Biology
  • Microscopy

Background:

  • Complex biological environments like the cell interior present challenges for molecular analysis.
  • Understanding molecular diffusion, aggregation, and interactions is crucial for cell function.
  • Conventional microscopy methods may lack the resolution for dynamic molecular studies.

Purpose of the Study:

  • To detail the application of Raster Image Correlation Spectroscopy (RICS) and Number and Molecular Brightness (N&B) methods.
  • To explain the mathematical framework and data acquisition parameters for RICS and N&B.
  • To demonstrate the capability of these methods in analyzing molecular complexes within living cells.

Main Methods:

  • Utilizing fluorescence intensity fluctuations from confocal laser-scanning microscopy images.
  • Applying correlation analysis techniques inherent to RICS and N&B.
  • Employing simulations and real-cell examples for method validation.

Main Results:

  • RICS and N&B can measure molecular diffusion and detect aggregate formation.
  • These techniques establish aggregate stoichiometry and map mobile molecule numbers.
  • Quantification of molecular complexes and their spatial interactions is achievable.

Conclusions:

  • RICS and N&B are powerful tools for studying molecular dynamics in biological systems.
  • The methods provide quantitative insights into molecular complex formation and behavior.
  • Proper calibration and control experiments are essential for reliable results.