Integrated Raman and angular scattering of single biological cells.

摘要

Raman, or inelastic, scattering and angle-resolved elastic scattering are two optical processes that have found wide use in the study of biological systems. Raman scattering quantitatively reports on the chemical composition of a sample by probing molecular vibrations, while elastic scattering reports on the morphology of a sample by detecting structure-induced coherent interference between incident and scattered light. We present the construction of a multimodal microscope platform capable of gathering both elastically and inelastically scattered light from a 38 mum2 region in both epi- and trans-illumination geometries. Simultaneous monitoring of elastic and inelastic scattering from a microscopic region allows noninvasive characterization of a living sample without the need for exogenous dyes or labels. A sample is illuminated either from above or below with a focused 785 nm TEM00 mode laser beam, with elastic and inelastic scattering collected by two separate measurement arms. The measurements may be made either simultaneously, if identical illumination geometries are used, or sequentially, if the two modalities utilize opposing illumination paths. In the inelastic arm, Stokes-shifted light is dispersed by a spectrograph onto a CCD array. In the elastic scattering collection arm, a relay system images the microscope's back aperture onto a CCD detector array to yield an angle-resolved elastic scattering pattern. Post-processing of the inelastic scattering to remove fluorescence signals yields high quality Raman spectra that report on the sample's chemical makeup. Comparison of the elastically scattered pupil images to generalized Lorenz-Mie theory yields estimated size distributions of scatterers within the sample.;In this thesis we will present validations of the IRAM instrument through measurements performed on single beads of a few microns in size, as well as on ensembles of sub-micron particles of known size distributions. The benefits and drawbacks of the epi- and trans-illumination modalities are also discussed. In addition, transilluminated Raman and elastic-scattering spectra were obtained from several biological test-cases, including Streptococcus pneumoniae, baker's yeast, and single human immune cells. Both the Raman and elastic-scattering channels extract information from these samples that are well in line with their known characteristics from the literature. Finally, we report on an experiment in which CD8+ T lymphocytes were stimulated by exposure to the antigens staphylococcal enterotoxin B and phorbol myristate acetate. Clear chemical and morphological differences were observed between the activated and unactivated cells, with the results correlating well to analysis performed on parallel samples using fluorescent stains and a flow cytometer.