Multiplex coherent anti-Stokes Raman scattering (MCARS) for chemically sensitive, label-free flow cytometry

Abstract

Flow cytometry is an ever-advancing high-throughput multivariate analysis tool that natively provides size and morphological information. To obtain molecular information, however, typically requires the addition of fluorophores, which are limited by spectral overlap, nonspecific binding, available conjugation chemistries, and cellular toxicity. A complementary or alternative, label-free approach to molecular information is through multiplex coherent anti-Stokes Raman scattering (MCARS), which is a coherent, nonlinear optical method that provides a wealth of molecular information by probing the Raman energies within a molecule. In this work, we demonstrate the unique capability of our MCARS flow cytometer to distinguish flowing particles and discuss system performance capabilities and possibilities.

Lipid Desaturation Is a Metabolic Marker and Therapeutic Target of Ovarian Cancer Stem Cells

“…Combining this marker with Raman spectroscopic imaging could lead to a reliable and universal method for in situ detection of CSCs. Further refinement of Raman spectroscopic imaging based techniques, such as coherent Raman scattering flow cytometry (Camp et al, 2009; Camp et al, 2011; Wang et al, 2008), would allow fast, noninvasive detection and isolation of CSCs.…”

Broadband Coherent Raman Scattering Microscopy

“…The current implementation of PTS‐SRS, using an 80 kHz repetition‐rate laser, covers a spectral window of 500 cm −1 , in the C—H stretching region, with frequency resolution of ≈10 cm −1 and a sensitivity of 10 −3 over 400 µs and approaching 2 × 10 −5 for a 1 s acquisition time. These performances are already suitable for a number of applications; PTS could be exploited in the future for monitoring microfluidic flows, the onset of chemical reactions or characterizing solid‐state samples such as pharmaceutical products . Future envisaged developments of the technique are: i) broadening the OPA bandwidth, so as to allow the Stokes pulse to cover simultaneously the C─H stretching and the fingerprint region; in fact PTS‐SRS has the remarkable advantage that the acquisition speed does not depend on the covered spectral region; ii) increasing the laser repetition rate by two orders of magnitude to the 10 MHz range, allowing to improve both the acquisition speed and the detection sensitivity.…”

Continuous-wave stimulated Raman scattering (cwSRS) microscopy

“…The high spectral resolution of cwSRS microscopy enables imaging low-frequency Raman modes, such as the DNA double helix vibrational modes, and various low-frequency modes in protein molecules [19, 24, 25]. However, under current setup, cwSRS setup is unable to make multiplex Raman/CARS spectrum detections, such as spontaneous Raman scattering or Multiplex Coherent Anti-Stokes Raman Scattering (MCARS) [6, 26]. Comparing with ultrafast laser sources, the relatively narrower tunable range (<; 100 nm, or <; 1000 cm −1 ) for cw lasers is another drawback for cwSRS.…”

“…Vibrational spectroscopies, such as infrared absorption [1], spontaneous Raman scattering [2, 3], and coherent anti-Stokes Raman scattering (CARS) [4-6], have been widely used as a contrast mechanism providing characteristic information about various chemical bonds. However, infrared microscopy suffers from a relatively low spatial resolution, as it relies on a long operating wavelength.…”

“…Spontaneous Raman microscopy is limited to its sensitivity and, usually, is contaminated by a strong fluorescence background. CARS microscopy provides a much stronger signal and enables multiplex detection of the whole spectrum [6]. However, when extending CARS spectroscopy to point-of-care applications, its complicated experimental setup may be problematic.…”