SEMINARS IN BIOMEDICAL ENGINEERING
Integrated Raman and Angular Scattering of Single Biological Cells
Zach Smith
We have developed a multimodal microscope that is capable of non-invasively characterizing the chemistry and morphology of single, unprepared cells with no loss of cell viability or function through measurements of Raman spectra and angle-resolved elastic scattering patterns. This is in contrast to more destructive techniques such as staining with fluorescent markers. The microscope system was constructed by altering a traditional confocal Raman microscope to add an additional collection path that images the Fourier plane of the microscope objective onto a CCD, recording a map of elastically scattered intensity versus scattering angles. The system is capable of collecting either forward or backward scattered light. The benefits and drawbacks of forward versus backward scattering will be discussed. The system was validated using both single polystyrene beads as well as populations of beads as size and chemical standards. We have recently used our system to measure Raman and elastic scattering signatures from single cells, with the data allowing us to non-invasively: extract mean cell volumes for a population of bacteria, classify subpopulations of immune cells, and detect changes that occur upon immune cell activation without the need for exogenous dyes or labels. In particular, our measurements of activating immune cells show a heterogeneity of responses within a cellular population to a single activation stimulus, as well as a heterogeneity of responses between different stimuli. We will discuss these results as well as future prospects for this new instrument platform.
sponsored by
The Laser Microbeam and Medical Program (LAMMP)
a NIH biotechnology resource facility at the Beckman Laser Institute