title: Dark field Raman microscopy.
abstract: Confocal Raman microscopy is often used for optical sectioning but is problematic when the sample plane of interest has a weak Raman cross-section/signal relative to areas that are out-of-focus. This is especially true for clinical samples in pathology, which consist of a thin tissue (approximately 5 microm) sample placed on a thick glass slide. Here, we recognize that the problem is the result of the extent of the illumination at the confocal plane being larger than the size of the sample and propose a dark field illumination scheme to efficiently reject substrate signals. The ability of several optical configurations in rejecting out-of-plane signal is investigated for two model systems: SU-8 photo resist over Teflon and SU-8 photo resist over polystyrene. The proposed reflective dark field approach, in which excitation converged to a focal point slightly above the focal plane of the collection optics, was found to be most effective in recording data from the sample. The proposed approach is validated by the rejection of substrate response (fluorescence) in spectra acquired from approximately 4 microm of breast tissue on a glass microscope slide. The proposed approach is easy to implement on existing confocal systems, has a straightforward optimization in acquiring data, and is not expected to result in loss of lateral resolution in mapping experiments.
authors.affiliation: Department of Bioengineering and Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, 405 N. Mathews Ave. Urbana, Illinois 61801, USA.
authors.forename: Matthew V
authors.initials: MV
authors.lastname: Schulmerich
authors.forename: Rohith
authors.initials: R
authors.lastname: Reddy
authors.forename: Anil K
authors.initials: AK
authors.lastname: Kodali
authors.forename: Laura Jane
authors.initials: LJ
authors.lastname: Elgass
authors.forename: Krishnarao
authors.initials: K
authors.lastname: Tangella
authors.forename: Rohit
authors.initials: R
authors.lastname: Bhargava
mesh_terms: D004852:Epoxy Compounds / Q000737:chemistry; D018613:Microscopy, Confocal / Q000295:instrumentation* / Q000379:methods; D011108:Polymers / Q000737:chemistry; D011137:Polystyrenes / Q000737:chemistry; D011138:Polytetrafluoroethylene / Q000737:chemistry; D013059:Spectrum Analysis, Raman
chemical_list: D004852:Epoxy Compounds; D011108:Polymers; D011137:Polystyrenes; C520895:SU-8 compound; D011138:Polytetrafluoroethylene