Advantages of full spectrum flow cytometry

Abstract

A charge coupled device-based flow-cytometer for the measurement of full spectra was implemented and characterized. The spectral resolution was better than 1.5 nm and the coefficient of variation for fluorescence from flow check beads was 5% or better. Both cell and bead data were analyzed by fitting to measured component spectra. Separation of flow check and align flow beads, which have similar spectra, was nearly identical whether using a spectral analysis or a scatter analysis. After mixing, cells stained with ethidium bromide or propidium iodide were measured at different timepoints. The contribution of these 12 nm separated emission spectra could be separately quantified and the kinetic process of the samples becoming homogeneous due to fluorophor dissociation and rebinding was observed. Principle component analysis was used to reduce noise and alternating least squares (ALS) was used to analyze one set of noise-reduced cell data without knowledge of the component spectra. The component spectra obtained via ALS are very similar to the measured component spectra. The contributions of ethidium bromide and propidium iodide to the individual spectra are also similar to those obtained via the spectral fitting procedure.

Fig. 1

A schematic of the spectral flow cytometer. Light is represented by colored lines, analog signals by narrow black lines, TTL pulses by two narrowly separated black lines and USB cables by double thick black lines. Components are described in the text.

Fig. 2

(a) Spectra of ethidium bromide (dashed-blue) and propidium iodide (solid-red) bound to cells. These spectra are each averages of nearly 1000 cells and have been scaled to have the same maximum intensity in order to optimize visualization of spectral differences. (b) Spectra of free ethidium bromide (dashed-blue) and propidium iodide (solid-red). The spectrum of sheath, which is primarily the Raman spectrum of water is in black. Each spectrum is the average of hundreds of spectra.

Fig. 3

Cell spectra, their fits and the contributions of the basis spectra to the fits. (a) A cell with more EB than PrI. (b) A cell with more PrI than EB.

Fig. 4

Raw side scatter (a) and forward scatter (b) time traces from measurements of flow check fluorospheres. Traces from a single event have matching colors and line types.

Fig. 5

(a) The two black spectra are align flow beads while the two red spectra are flow check beads. The yellow (top) one is a doublet of flow check beads. (b) Averaged spectra of flow check and align flow beads and Raman/Sheath.

Fig. 6

(a) Forward scatter height versus side scatter height for a mix of flow check and align flow beads. Side scatter is a negative signal, so stronger side scatter is more negative. The flow check beads are the population in the upper left corner and the align flow beads are the population in the lower right corner. (b) Fit coefficient for the flow check beads versus the fit coefficient for the align flow beads.

Fig. 7

Density plots from four samples with the density of cells represented by the darkness of the colors. Cells stained with only EB are blue. Cells stained with only PrI are green. These two cell populations were washed and mixed. Measurements made $\sim 3\min$ after mixing are red. Measurements made $\sim 45\min$ after mixing are light blue.

Fig. 8

Results of a PCA analysis of the spectra of the sample measured 3 min after mixing cells individually stained with PrI and EB. (a) The first three principle components (b) The fourth principle component.

Fig. 9

(a) Comparison of a measured spectra with a PCA reconstructed spectra. (b) Results of fitting the measured data set compared with results of fitting the reconstructed data set.

Fig. 10

(a) The Gaussian starting spectra for the ALS analysis are shown in orange and green. After 12,000 ALS iterations, the ALS spectra are in solid red and blue. The measured basis spectra are shown in dotted red and blue lines. The measured sheath spectrum is black. (b) Comparison of fluorescence contributions from EB and PrI to individual spectra obtained from fitting the data to component spectra and from ALS. The black dots and red circles are the same data shown in Fig. 9(b), however, the scales have been changed.