Hybrid Rayleigh, Raman and two-photon excited fluorescence spectral confocal microscopy of living cells

• Published in: Journal of Raman spectroscopy Vol. 41; no. 6; pp. 599 - 608
• Main Authors: Pully, Vishnu Vardhan, Lenferink, Aufried, Otto, Cees
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 01.06.2010
• Subjects: Confocal; confocal Raman imaging; Fluorescence; hybrid microspectroscopy; Image contrast; Imaging; living human stem cells; Microscopy; Platforms; Rayleigh scatter imaging; Spectra; two-photon excited fluorescence microscopy
• ISSN: 0377-0486; 1097-4555
• DOI: 10.1002/jrs.2501

A hybrid fluorescence–Raman confocal microscopy platform is presented, which integrates low‐wavenumber‐resolution Raman imaging, Rayleigh scatter imaging and two‐photon fluorescence (TPE) spectral imaging, fast ‘amplitude‐only’ TPE‐fluorescence imaging and high‐spectral‐resolution Raman imaging. This multi‐dimensional fluorescence–Raman microscopy platform enables rapid imaging along the fluorescence emission and/or Rayleigh scatter dimensions. It is shown that optical contrast in these images can be used to select an area of interest prior to subsequent investigation with high spatially and spectrally resolved Raman imaging. This new microscopy platform combines the strengths of Raman ‘chemical’ imaging with light scattering microscopy and fluorescence microscopy and provides new modes of correlative light microscopy. Simultaneous acquisition of TPE hyperspectral fluorescence imaging and Raman imaging illustrates spatial relationships of fluorophores, water, lipid and protein in cells. The fluorescence–Raman microscope is demonstrated in an application to living human bone marrow stromal stem cells. Copyright © 2009 John Wiley & Sons, Ltd. Hybrid confocal microscopy integrates low‐wavenumber‐resolution Raman imaging, Rayleigh scatter imaging and two‐photon fluorescence (TPE) spectral imaging with fast, ‘amplitude‐only’ TPE‐fluorescence imaging and high‐spectral‐resolution Raman imaging using a single continuous wave (cw) excitation light source. The cw TPE fluorescence imaging enables rapid selection of the cells of interest followed by Rayleigh and Raman imaging to obtain the chemical information of the molecular distribution.