On-line visualization of multicolor chemical images with stimulated Raman scattering spectral microscopy

Abstract

We demonstrate multicolor, on-line visualization in label-free biomedical microscopy based on stimulated Raman scattering (SRS). Fast data acquisition of SRS spectral images and subsequent image generation are achieved. The loading vectors for the blind separation of chemical components are predetermined by multivariate analysis at a certain field of view (FOV) and are applied to execute on-line visualization of chemical images at other FOVs. We also show that the response time can be shortened by reducing the number of spectral data points. This on-line visualization system is expected to increase the usability of the Raman imaging system and the analytical throughput for screening.

In situ visualization of intracellular morphology of epidermal cells using stimulated Raman scattering microscopy

“…The details of our SRS microscope have been described previously. 30,39 Briefly, a Ti:sapphire (Ti:S) laser at 790 nm and a wavelength-tunable Yb fiber (YbF) laser at 1014 to 1046 nm were used as light sources. The power of the Ti:S and YbF lasers at the input of the laser scanners was 120 mW each.…”

“…In contrast, coherent Raman scattering microscopy techniques, 22,23 including coherent anti-Stokes Raman scattering microscopy and stimulated Raman scattering (SRS) microscopy, [24][25][26] have enabled faster imaging of various functional groups of molecules. In particular, SRS microscopy has led to a variety of applications to biological samples [27][28][29][30][31][32][33][34][35][36][37][38][39][40] including skin, [41][42][43][44] taking advantage of the capability of SRS microscopy for high-speed image acquisition with vibrational contrast. However, it is still unclear whether SRS can visualize intracellular morphology of various types of epidermal cells and whether SRS can discriminate cell lineages and types in the epidermis.…”

Vibrational Fingerprint Mapping Reveals Spatial Distribution of Functional Groups of Lignin in Plant Cell Wall

“…To resolve overlapped fingerprint Raman bands for chemical identification, multiple groups have developed hyperspectral SRS imaging platforms − and multivariate image analysis methods, ,,− with applications to chemical histology, drug delivery, and lipid metabolism. , On the basis of either femtosecond pulse shaping or pulse chirping, several spectral scanning schemes have been demonstrated (for a review, see ref ). A remaining challenge for these techniques is the trade-off between excitation power and spectral resolution, both essential for SRS imaging of weak Raman bands in the congested fingerprint region.…”

High-speed Raman imaging of cellular processes

“…Camp et al developed a CARS imaging system using a super-continuum laser that covers the spectral range from 500 to 3500 cm -1 with spectral resolution of 10 cm -1 [35], allowing tissue imaging at a frame rate of ~315 seconds. Ozeki et al developed a hyper-spectral SRS imaging system with a wavelength-tunable laser, which covers from 2800 to 3100 cm -1 with spectral resolution of 3 cm -1 [36], enabling tissue imaging at a frame rate of ~30 seconds, and combined it with an on-line analytical system for increased throughput [37]. To further improve the imaging speed, Liao et al developed a system that modulates the intensity of broadband pump beam with different frequencies for each color.…”