Raman Microspectroscopy:  A Comparison of Point, Line, and Wide-Field Imaging Methodologies

Abstract

Three different Raman microspectroscopic imaging methodologies using a single experimental configuration are compared; namely, point and line mapping, as representatives of serial imaging approaches, and direct or wide-field Raman imaging employing liquid-crystalline tunable filters are surveyed. Raman imaging data acquired with equivalent low-power 514.5-nm laser excitation and a cooled CCD camera are analyzed with respect to acquisition times, image quality, spatial resolution, intensity profiles along spatial coordinates, and spectral signal-to-noise ratios (SNRs). Point and line mapping techniques provide similar SNRs and reconstructed Raman images at spatial resolutions of approximately 1.1 microm. In contrast, higher spatial resolution is obtained by direct, global imaging (approximately 313 nm), allowing subtle morphological features on test samples to be resolved.

Label‐free biochemical characterization of stem cells using vibrational spectroscopy

“…In scanning mode, the diffraction limited laser spot is raster scanned in small incremental steps over the sample, collecting Raman signals at each spatial position. The beam could also be reshaped into a line [12,19], for example, which is then raster scanned in one direction across the cell sample and imaged through the slit of a spectrometer. The acquired spectra can be averaged to obtain an overall spectrum that is representative of the entire cell.…”

Recent applications of Chemical Imaging to pharmaceutical process monitoring and quality control

“…However, Schlucker et al [62] reported that higher spatial resolution is obtained from global Raman Imaging than point or map Raman Imaging, and this enables subtle morphological features on test samples to be imaged. Moreover, Raman line and point mapping experiments can be painfully time consuming (up to 20 h).…”

Raman Assessment of Bone Quality

“…Although theory supports the use of similar bands originally developed by bone infrared spectroscopists to assess nonreducible to reducible collagen crosslink ratios (approximately 1660/1690 cm À1 ratios) [55], there are differences in the spectra and instrumentation that needs to be taken into consideration. First, FTIR microspectrometers yield spectra with superior signal-tonoise (S/N) ratios of approximately 1000:1 compared with approximately 30-80:1 obtained by Raman spectrometers [40,63]. The low Raman S/N ratio is attributed to the weak scattering that occurs at 785 nm, less than one-fifth of the intensity at 532 nm [27].…”