Laser particle activated cell sorting in microfluidics

Abstract

Laser particles providing bright, spectrally narrowband emission renders them suitable for use as cellular barcodes. Here, we demonstrate a microfluidic platform integrated with a high-speed spectrometer, capable of reading the emission from laser particles in fluidic channels and routing cells based on their optical barcodes. The sub-nanometer spectral emission of each laser particle enables us to distinguish individual cells labeled with hundreds of different laser colors in the near infrared. Furthermore, cells tagged with laser particles are sorted based on their spectral barcodes at a kilohertz rate by using a real-time field programmable gate array and 2-way electric field switch. We demonstrate several different flavors of sorting, including isolation of barcoded cells, and cells tagged with a specific laser color. We term this novel sorting technique laser particle activated cell sorting (LACS). This flow reading and sorting technology adds to the arsenal of single-cell analysis tools using laser particles.

Fig. 1. Readout of LP emission in the flow. a, Image frames showing a cell containing a single LP (arrow), as it traverses the pump laser focus (dashed ellipse). b, Recorded spectra at the corresponding time. A narrow lasing peak is observed on the spectrometer at the frame corresponding to the cell traversing this point. c, Collection of lasing spectra observed during a single experimental run. d, Histogram showing recordings of lasing wavelengths from a near half million LPs measured in a single flow experiment.

Fig. 2. The microfluidic system. a, Schematic of a microfluidic chip. Pump laser focus location shown in inset (red arrow). b, Schematic of the cell sorting setup. A spectrometer reads LP laser emission. Decision hardware in a field programmable gate array (FPGA) triggers the computer to send high voltage pulses to electrodes to deflect cell-containing droplets into the (+) outlet.

Fig. 3. Binary cell sorting to divide cells in populations with and without LPs. The cells collected from the (+) outlet were actively sorted. The cells collected from the (−) outlet were allowed to flow through the sorting junction without the application of a dielectrophoretic force. a, Image frames of multiple droplets flowing through the sorting junction. Three successive droplets (i)–(iii) are labelled of which (ii) contains an LP-tagged cell and is thus deflected into the (+) outlet. b, Recorded spectra of the three droplets as they traverse the detection zone.

Fig. 4. a and b, Representative bright-field images of HeLa cells after sorting as collected from the (−) and (+) outlets. c–e, Statistics of the fraction of cells that contain a specified number of disks, analyzed from >500 cells in all acquired images.

Fig. 5. Short-wavelength sorting. a, A negative cell (λ > 1450 nm) flowing into the (−) channel. b, The LP emission spectrum of the negative cell. The magenta box indicates the gating condition used. c, A positive cell (λ < 1450 nm) directed into the (+) channel. d, The LP spectrum of the positive cell.

Fig. 6. Band-pass sorting. a, A bright-field image of replated cells collected from the (−) outlet. b, The LP lasing emissions from the LPs labelled i, ii and ii. c and d, Image and emission spectra of cells collected from the (+) outlet, showing three LPs (iv–vi).