Label-Free Flow Cytometry Using Multiplex Coherent Anti-Stokes Raman Scattering (MCARS)
Flow cytometry is a multivariate analytical tool that incorporates optics and electronics to measure an ever-growing variety of sample phenotypes [1]. Flowing samples are interrogated by one or more lasers, and the elastically scattered photons are measured to ascertain morphological information. Acquiring molecular information, however, requires the addition of fluorescent labels, which although a powerful tool, present several challenges such as large emission spectra and cytotoxicity, which can alter cellular chemistries and perturb the experimental outcomes [1]. Additionally, the process of conjugating fluorophores and labeling cells can be time consuming; thus, reducing clinical turn-around times and affecting time-sensitive samples. Label-free technologies, such as coherent anti-Stokes Raman scattering (CARS) [2] and stimulated Raman scattering (SRS) [3], which have seen tremendous growth in the microscopy community, offer attractive possibilities for label-free flow cytometry [4,5].
In the Photonics Research Group (PRG), we have developed the first label-free flow cytometer that uses MCARS [6] to probe a broad band of Raman vibrational energy levels at high-speed. A schematic of the developed system is shown in Fig. 1. This system is capable of collecting over 100 MCARS spectra/s and measuring the elastically scattered photons in the forward (FSC) and lateral direction (i.e., side-scatter [SSC]) at 1 MHz. This unique combination of focused beam elastic scatter measurement and MCARS will provide information that may not be available using conventional flow cytometry.
Figure
1. Schematic of the label-free MCARS flow cytometer [5]. For schematic
clarity, the SSC detector has been omitted from the drawing. Microfluidic chip
image courtesy of Translume, Inc.
With this system we have successfully analyzed various bead assays, cultures of a variety of yeast species and strains, and cultures of diatoms that were not easily measurable with traditional techniques, such as microspectroscopic Raman and flow cytometry, due to their high autofluorescence– even with near-infrared (NIR) excitation. Fig. 2 is an example density scatter plot of the maximum FSC with respect to the minimum FSC. The larger subpopulation with the larger maximum FSC corresponds to yeast cells with high concentrations of scatterers (primarily lipid vacuoles). The smaller subpopulations contains fewer internal scatterers. Fig. 3(a) shows the MCARS spectra for these two respective subpopulations. One can see that the lipid-rich subpopulations shows a pronouced MCARS spectrum. To compare our results with those taken with a commercial microRaman, we applied a Kramers-Kronig (KK) algorithm to our raw data to remove the contributions of the so-called “nonresonant background” (NRB) [7]. Fig. 3(b) compared the KK-reconstructed spectra for the two subpopulations with the Raman spectrum of a lipid-rich yeast. One can see good spectral agreement for the scattering peaks at 2930 (CH-stretch), 1652 (C=C stretch), 1442 (CH2-def.), and 1328 cm-1.
Figure
2. (a) Density scatter plot of maximum FSC voltage versus minimum FSC voltage for S. cerevisiae. (b) shows two representative time-traces of FSC from within the two subpopulations in (a).
Figure
3. (a) Mean MCARS spectrum of the subpopulations of yeast in 2(a) with high (blue) and low (red) maximum FSC. Corresponding reconstructed Raman spectrum compared to the spontaneous Raman spectrum of
a lipid-rich yeast (black).
The developed label-free flow cytometer offers the attractive possibilities of probing the rich vibrational information within sample molecules at 100’s-1000’s of cells per second– all without the necessity of fluorescent labels. Additionally, this technique offers the possibility of integration into commercially-available flow cytometers as the technique does not exclude the use of advanced labeling technology for targeting specific molecules, such as surface proteins.
References
[1] H. M. Shapiro, Practical Flow Cytometry, 4th ed. (Wiley Liss, New York,
2003).
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[4] C. H. Camp Jr., et al., “Multiplex coherent anti-Stokes Raman scattering
(MCARS) for chemically sensitive, label-free flow cytometry,” Opt.
Express 17, 22879-22889 (2009).
[5] C. H. Camp Jr., et al., “Label-free flow cytometry using multiplex
coherent anti-Stokes Raman scattering (MCARS) for the analysis of
biological specimens,” Opt. Lett. 36, 2309-2311 (2011).
[6] M. Mueller and J. M. Schins, “Imaging the Thermodynamic State of Lipid
Membranes with Multiplex CARS Microscopy,” J. Phys. Chem. B, 106,
3715-3723 (2002).
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