A flow cytometer for the measurement of Raman spectra

Abstract

Multiparameter measurements in flow cytometry are limited by the broad emission spectra of fluorescent labels. By contrast, Raman spectra are notable for their narrow spectral features. To increase the multiparameter analysis capabilities of flow cytometry, we investigated the possibility of measuring Raman signals in a flow cytometry-based system. We constructed a Raman Spectral Flow Cytometer, substituting a spectrograph and CCD detector for the traditional mirrors, optical filters, and photomultiplier tubes. Excitation at 633 nm was provided by a HeNe laser, and forward-angle light scatter is used to trigger acquisition of complete spectra from individual particles. Microspheres were labeled with nanoparticle surface enhanced Raman scattering (SERS) tags and measured using the RSFC. Fluorescence and Raman spectra from labeled microspheres were acquired using the Raman Spectral Flow Cytometer. SERS spectral intensities were dependent on integration time, laser power, and detector pixel binning. Spectra from particles labeled with one each of four different SERS tags could be distinguished by either a virtual bandpass approach using commercial flow cytometry data analysis software or by principal component analysis. Raman flow cytometry opens up new possibilities for highly multiparameter and multiplexed measurements of cells and other particles using a simple optical design and a single detector and light source. ' 2008International Society for Analytical Cytology

Surface-enhanced Raman scattering biomedical applications of plasmonic colloidal particles

“…However, a major limitation of this approach is that single particles usually do not provide sufficient field enhancement to raise the SERS signal up to detectable levels. This problem can however be overcome by encapsulation of controlled aggregates with either silica or polymer shells (which leads to heterogeneity in both shape and size; Lutz et al 2008;Watson et al 2008) or by confined epitaxial growth of gold or silver islands inside homogeneous, hollow silica capsules (Sanles-Sobrido et al 2009a). Regarding practical applicability, these materials show great versatility.…”

Hyperspectral cytometry at the single‐cell level using a 32‐channel photodetector

“…Very likely, faster CCD readout electronics would allow higher analysis rates without coincidence. In 2008, work carried out by John Nolan's group (La Jolla Bioengineering Institute) outlined the development of a Raman spectral flow cytometer (16). Raman spectroscopy, via surface-enhanced Raman (SERS), and FCM were brought together by substituting a dispersive-optic spectrograph with multichannel detector (CCD) in place of the traditional mirrors/beam splitters, filters, and photomultipliers (PMTs) of conventional flow cytometers.…”

Patch bandits

“…This method was recently applied to Raman spectroscopy in flow cytometry (19). This group presents an approach to use high-speed near-infrared spectroscopy to precisely size large particles and measure fluorescence as well as Raman signals from the same objects (20).…”