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Achieving Molecular Selectivity in Imaging Using Multiphoton Raman Spectroscopy Techniques
Author(s) -
Holtom Gary R.,
Thrall Brian D.,
Chin BeekYoke,
Wiley H. Steven,
Colson Steven D.
Publication year - 2001
Publication title -
traffic
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.677
H-Index - 130
eISSN - 1600-0854
pISSN - 1398-9219
DOI - 10.1034/j.1600-0854.2001.21106.x
Subject(s) - photobleaching , microscopy , raman scattering , fluorescence , raman spectroscopy , two photon excitation microscopy , fluorescence lifetime imaging microscopy , live cell imaging , confocal , materials science , fluorescence microscope , optics , biophysics , chemistry , biology , physics , cell , biochemistry
In the case of most optical imaging methods, contrast is generated either by physical properties of the sample (Differential Image Contrast, Phase Contrast), or by fluorescent labels that are localized to a particular protein or organelle. Standard Raman and infrared methods for obtaining images are based upon the intrinsic vibrational properties of molecules, and thus obviate the need for attached fluorophores. Unfortunately, they have significant limitations for live‐cell imaging. However, an active Raman method, called Coherent Anti‐Stokes Raman Scattering (CARS), is well suited for microscopy, and provides a new means for imaging specific molecules. Vibrational imaging techniques, such as CARS, avoid problems associated with photobleaching and photo‐induced toxicity often associated with the use of fluorescent labels with live cells. Because the laser configuration needed to implement CARS technology is similar to that used in other multiphoton microscopy methods, such as two‐photon fluorescence and harmonic generation, it is possible to combine imaging modalities, thus generating simultaneous CARS and fluorescence images. A particularly powerful aspect of CARS microscopy is its ability to selectively image deuterated compounds, thus allowing the visualization of molecules, such as lipids, that are chemically indistinguishable from the native species.