Visible and near infrared fluorescence spectral flow cytometry

Abstract

There is a long standing interest in measuring complete emission spectra from individual cells in flow cytometry. We have developed flow cytometry instruments and analysis approaches to enable this to be done routinely and robustly. Our spectral flow cytometers use a holographic grating to disperse light from single cells onto a CCD for high speed, wavelength-resolved detection. Customized software allows the single cell spectral data to be displayed and analyzed to produce new spectra-derived parameters. We show that familiar reference and calibration beads can be employed to quantitatively assess instrument performance. We use microspheres stained with six different quantum dots to compare a virtual bandpass filter approach with classic least squares (CLS) spectral unmixing, and then use antibody capture beads and CLS unmixing to demonstrate immunophenotyping of peripheral blood mononuclear cells using spectral flow cytometry. Finally, we characterize and evaluate several near infrared (NIR) emitting fluorophores for use in spectral flow cytometry. Spectral flow cytometry offers a number of attractive features for single cell analysis, including a simplified optical path, high spectral resolution, and streamlined approaches to quantitative multiparameter measurements. The availability of robust instrumentation, software, and analysis approaches will facilitate the development of spectral flow cytometry applications.

Advantages of full spectrum flow cytometry

“…9 Individual quantum dot spectra were reportedly 'readily identified' in spectra from beads stained with six quantum dots. Spectral fits were not shown in the paper nor is correct identification quantified, but rather the reader is left to estimate it from the figures.…”

Fundamentals and application of magnetic particles in cell isolation and enrichment: a review

“…The MoFlo Astrios™ instrument has a sort purity of < 99% and a 90% of theoretical sort yield; viability was also shown to not be influenced by the sorting technique (Davies, 2012). Recently significant advances in optics, detectors, and software have allowed for a significantly larger number of colors to be analyzed (> 80 colors) (Nolan et al, 2013, Nolan et al, 2012, Nolan and Sebba, 2011). Although still one of the most highly-used cell isolation platforms, a serious limitation to FACS systems remains their price and complexity.…”

Microfluidic Approaches to Fluorescence In Situ Hybridization (FISH) for Detecting RNA Targets in Single Cells

“…Most examples of chip-based flow cytometry detectors that we are aware of are limited to one or two lasers, and 2-3 fluorescence and scatter channels. An alternative approach that enables more spectral channels is to replace the cascade of filters and photomultiplier tubes with a spectrograph (e.g., a diffraction grating and a multianode PMT or CCD detector), enabling collection of an entire fluorescence spectrum for each cell, with multivariate curve analysis to resolve individual spectral components [8,20]. A variety of cell sorting modalities based on optical trapping [24], valving [5], deflection with a piezoelectric transducer [3], or electrokinetics [34] have been coupled to chip-based flow cytometry, and these are expected to be compatible with FISH detection.…”