Spectral measurements of large particles by flow cytometry

Abstract

Flow cytometers designed to analyze large particles are enabling new applications in biology and chemistry. Similarly, flow spectroscopy approaches are extending the capabilities of the flow cytometry platform. Here, we report on the adaptation of a commercial large particle analyzer to measure fluorescence and Raman spectra of individual particles at high speeds. We modified a Union Biometrica COPAS Plus instrument to allow red excitation and optical fiber-based light collection and spectral analysis using a spectrograph and CCD array detector. These modifications did not compromise the ability of the instrument to resolve different sized particles based on their extinction and time of flight signals. The modified instrument has the sensitivity and spectral resolution to measure the fluorescence and Raman signals from individual particles with signal integration times of 10 usec. The high speed spectral analysis of individual particles in flow will enable new applications in biological and chemical analyses.

Figure 1. Large Particle Spectral Flow Cytometer

Schematic (right) and photo (left) of the spectral detection leg constructed on the open face of the COPAS flow cell. DM: dichroic mirror; L1: 80 mm spherical lens; L2: 6 mm cylindrical lens; FC: flow cell; L3, L4: 21 mm aspherical lenses; OF: optical fiber; ND: 0.8 OD neutral density filter.

Figure 2. Particle size resolution by extinction

A) One parameter histograms of extinction for particles of 55 um, 98 um, 134 um, and 222 um diameter measured with the Raman Flow Cytometer. B) Dependence of the extinction signal on particle diameter. Error bars are the standard deviation of the distributions.

Figure 3. Fluorescence spectral measurements

A) Fluorescence spectra of oxazine170. B) Average spectra of ~200 stained microspheres (black lines), +/− 1 SD (grey lines). C) Frequency histograms of integrated emission from CCD-based spectral measurements of fluorescent (open bars) and blank (filled bars) beads. D. Frequency histograms from PMT-based measurements of fluorescent (open) and blank (filled) beads.

Figure 4. Raman spectral measurements

Representative spectra from single beads (10 usec integration times) stained with Ag-oxazine170 (A) and Ag-rhodamine800 (B) SERS nanoparticles and the respective average spectra (C, D) from 100–200 individual bead measurements.