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Raman Spectroscopy: Overview

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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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Real-Time, Two-Color Stimulated Raman Scattering Imaging of Mouse Brain for Tissue Diagnosis

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Two-Color, Two-Photon Imaging at Long Excitation Wavelengths Using a Diamond Raman Laser.

Johanna Trägårdh¹, Michelle Murtagh¹, Gillian Robb¹

¹1Centre for Biophotonics,Strathclyde Institute of Pharmacy and Biomedical Sciences,University of Strathclyde,161 Cathedral Street,Glasgow,G4 0RE,UK.

Microscopy and Microanalysis : the Official Journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada

|August 6, 2016

Summary

A novel diamond Raman laser efficiently excites red dyes for two-photon microscopy. This system enables simultaneous multi-color imaging, expanding possibilities for biological research and dye applications.

Keywords:

Raman lasers fluorescence microscopy fluorescent proteins nonlinear microscopy two-photon microscopy

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Area of Science:

Biophotonics
Laser Physics
Cellular Imaging

Background:

Two-photon excitation microscopy offers advantages for deep tissue imaging.
Excitation of red-emitting fluorophores typically requires longer wavelengths.
Femtosecond lasers are crucial for non-linear optical processes like two-photon excitation.

Purpose of the Study:

To demonstrate the efficacy of a diamond Raman laser for two-photon excitation of red-emitting dyes.
To explore the potential for simultaneous multi-color two-photon imaging using this laser system.
To showcase the versatility of the tunable laser system for various dye combinations.

Main Methods:

Utilizing the second-Stokes output of a diamond Raman laser, pumped by a femtosecond Ti:Sapphire laser.
Employing two-photon excitation at 1,080 nm and beyond to image HeLa cells and red fluorescent dyes (Texas Red, Mitotracker Red).
Using the residual pump beam for simultaneous excitation of green-emitting dyes (Alexa Fluor 488 and Alexa Fluor 568).

Main Results:

Efficient two-photon excitation of red-emitting dyes and red fluorescent protein in HeLa cells was achieved.
Successful simultaneous two-color, two-photon imaging of green and red fluorophores was demonstrated.
The diamond Raman laser system proved tunable from 680-1,200 nm, enabling excitation of a wide range of dyes.

Conclusions:

The diamond Raman laser is a powerful tool for efficient two-photon excitation of red dyes.
This laser system facilitates versatile and simultaneous multi-color two-photon imaging.
The tunable wavelength range significantly broadens the scope of applicable dyes for advanced microscopy techniques.