In Situ Analysis of Living Embryonic Stem Cells by Coherent Anti-Stokes Raman Microscopy

Abstract

Embryonic stem cells (ESC), derived from preimplantation embryos, are defined by their ability to both self-renew and differentiate into all of the cells and tissues of a mature animal. Efforts to develop methods for in vitro culture of ESC for research or eventual therapeutic applications are hampered by the lack of rapid, nondestructive assays for distinguishing ESC from other (differentiated) cells within a growing culture. Coherent anti-Stokes Raman scattering (CARS) microscopy is shown here to be a sensitive and nondestructive method for identifying mouse ESC based on selective observation of specific molecular vibrations believed to be spectroscopic markers indicating the differentiated vs undifferentiated states of such cells. The nonlinear nature of CARS also permits imaging with subcellular resolution, potentially offering a means by which chemical changes accompanying the early stages of differentiation may be associated with certain intracellular compartments (e.g., nucleus, cytoplasm, membranes). A novel exposure/collection configuration is described, which yields high collection efficiency and low interference from nonresonant background components.

Label‐free biochemical characterization of stem cells using vibrational spectroscopy

“…In addition, the eventual use of this technology in future clinical cell-based transplantations for purifying and enriching cell populations would obviously require similar cell sorting instrumentation. It is anticipated that optical tweezers [42][43][44] for cell manipulation and advanced nonlinear Raman techniques such as CARS [45,46] and stimulated Raman scattering (SRS) to enhance the weak Raman scattering signals will be important components in future Raman-based cell sorting systems. A study using CARS for stem cell characterization has been reported [46], the results of which were in agreement with the Raman studies discussed above.…”

“…It is anticipated that optical tweezers [42][43][44] for cell manipulation and advanced nonlinear Raman techniques such as CARS [45,46] and stimulated Raman scattering (SRS) to enhance the weak Raman scattering signals will be important components in future Raman-based cell sorting systems. A study using CARS for stem cell characterization has been reported [46], the results of which were in agreement with the Raman studies discussed above. Recent studies have shown the proofof-principle concept of a novel Raman flow cytometer based on a laser tweezers Raman approach [47] and a coherent anti-Stokes Raman scattering (CARS) scheme [48] in combination with microfluidics to improve the speed and efficiency of analyzing the cells.…”

Molecular pathology via IR and Raman spectral imaging

“…Cells are selected under a microscope for data acquisition. This mode of data acquisition lends itself to the analysis of large numbers of cells, and has been used widely [40][41][42]51]. However, in the context of this chapter, another imaging based method for Ra-SCP data acquisition will be introduced.…”

“…This method is more labor intensive, but yields in relatively short time hundreds of spectra of selected cellular features that show enormous sensitivity toward many biochemical processes, such as the metabolic activity in live yeast cells [38,39]. For example, this approach has been used very successfully to follow stem cell differentiation [40][41][42], photochemical changes in melanosomes [43] or for the detection of circulating tumor cells in flow cytometry [16,17]. Finally, due to the very high spatial resolution of Ra-SCP, spectral information from individual bacterial cells can be collected [44,45].…”

Simultaneous forward and epi-CARS microscopy with a single detector by timecorrelated single photon counting

“…Konorov et al [10] also capitalized on the reflected F-CARS signal by placing their samples on mirrored cover slips to overlap the forward and backward scattered signals. Both groups attributed an enhancement of the detected signal in the epi-direction to the additional contributions from back-reflected F-CARS photons.…”