High‐Throughput Raman‐Activated Cell Sorting in the Fingerprint Region

Abstract

Cell sorting is the workhorse of biological research and medicine. [1] Cell sorters are commonly used to sort heterogeneous populations of cells based on their intrinsic features and have numerous applications in microbiology, immunology, virology, stem cell biology, and metabolic engineering. [2][3][4][5][6] While several schemes have been developed for sorting cells based on features such as size, [7] morphology, [8][9][10] viability, [11,12] and surface antigens, [13] those able to sort at high throughput for intracellular chemical content are limited to fluorescence-activated and Raman-activated cell sorting (FACS and RACS, respectively). [1,14] In both methods, sorting is accomplished by a stepwise automatic process: 1) measurement of chemical content with single-cell resolution, 2) analysis of the measurement data to identify cells of interest, and 3) mechanical separation of target cells from the original sample. [1] FACS machines commonly identify molecular targets by signals from attached exogenous fluorophores or coexpressed transgenic fluorescent proteins. This indirect detection scheme is often referred to as labeled detection. [1] Although fluorescent labeling is essential for microbiological research, the use of labels has inherrent drawbacks. These include reduced cellular vitality due to the cytotoxicity of labels themselves, nonspecific binding of labels, a lack of labels or transfection methods for many molecular targets and species, incompatibility with human-targeted cell therapies, and increased experimental complexity imposed by labeling protocols. [14] On the other hand, RACS machines directly identify target molecules in a label-free manner via Raman spectroscopy, which measures the inelastic scattering of incident photons by characteristic molecular vibrations. [14] The chemical specificity of this optical measurement contrasts with non-chemically-specific label-free methods such as impedance-based sorting for cell viability and image-based sorting for morphology or deformability. [10][11][12] To date, FACS technology is well developed, commercialized, and broadly adopted, while RACS is nascent and comprised of a small number of lab-based demonstrations. [14][15][16][17][18][19][20][21][22][23][24][25][26] A major hurdle in the broad deployment of RACS for biomedical applications is the weak signal produced by the Raman scattering process. This necessitates comparatively Cell sorting is the workhorse of biological research and medicine. Cell sorters are commonly used to sort heterogeneous cell populations based on their intrinsic features. Raman-activated cell sorting (RACS) has recently received considerable interest by virtue of its ability to discriminate cells by their intracellular chemical content, in a label-free manner. However, the broad deployment of RACS beyond lab-based demonstrations is hindered by a fundamental trade-off between throughput and measurement bandwidth (i.e., cellular information content). Here this trade-off is overcome and broadband RACS in the fingerprint reg...

Raman cell sorting for single-cell research

“…The acoustic-focusing piezoelectric transducer is utilized to focus the cells into a single stream, and the on-chip dual-membrane push–pull cell sorter is utilized for cell sorting. At the same time, this technique overcomes the trade-off problem of throughput versus measurement bandwidth and demonstrates broadband RACS in the fingerprint region (300–1,600 cm−1) with a record-high throughput of ≈50 cells per second ( Lindley et al, 2022 ).…”

“…(A) RIACS based on SRS technology, reproduced from Nitta et al (2020 ). (B) Raman-activated cell sorting (RACS) based on CARS technology, reproduced from Lindley et al (2022 ). (C) SERS-based flow cytometry, reproduced from Sebba et al (2009 ).…”

Label-free microfluidic cell sorting and detection for rapid blood analysis

“…While spontaneous Raman scattering limits the detection throughput, newer alternatives such as stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) significantly improve light–sample interaction for high throughput chemical fingerprinting of flowing cells 165,166 or Raman-activated cell sorting. 167 Zhang et al reported a multiplexed SRS flow cytometry platform based on broadband laser excitation and multiplex spectral detection. 168 The system can measure the chemical contents of single particles at high throughput (200 000 spectra per s and 11 000 particles per s) and help discriminate non-differentiated and differentiated 3T3-L1 cells by quantification of different chemical compositions accumulated in single cells (Fig.…”

High-throughput single-cell sorting by stimulated Raman-activated cell ejection

“…Both stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) have been combined with microfluidics for high-throughput cell imaging (32)(33)(34)(35)(36). Recently, two coherent Raman-activated cell sorting studies were reported, one in the C-H stretching region (37) and the other in the fingerprint region (400-1800 cm -1 ) by an FT-CARS spectrometer (38). Despite the high throughput, these microfluidic-based methods encounter challenges in handling smaller cells (<3 μm) (37), and unstable flow caused by bubbles and/or debris in the microfluidic channel (39).…”