Fluorescence lifetime shifts of NAD(P)H during apoptosis measured by time-resolved flow cytometry

Abstract

Autofluorescence from the intracellular metabolite, NAD(P)H, is a biomarker that is widely used and known to reliably screen and report metabolic activity as well as metabolic fluctuations within cells. As a ubiquitous endogenous fluorophore, NAD(P)H has a unique rate of fluorescence decay that is altered when bound to coenzymes. In this work we measure the shift in the fluorescence decay, or average fluorescence lifetime (1-3 ns), of NAD(P)H and correlate this shift to changes in metabolism that cells undergo during apoptosis. Our measurements are made with a flow cytometer designed specifically for fluorescence lifetime acquisition within the ultraviolet to violet spectrum. Our methods involved culture, treatment, and preparation of cells for cytometry and microscopy measurements. The evaluation we performed included observations and quantification of the changes in endogenous emission owing to the induction of apoptosis as well as changes in the decay kinetics of the emission measured by flow cytometry. Shifts in NAD(P)H fluorescence lifetime were observed as early as 15 min post-treatment with an apoptosis inducing agent. Results also include a phasor analysis to evaluate free to bound ratios of NAD(P)H at different time points. We defined the free to bound ratios as the ratio of 'short-to-long' (S/L) fluorescence lifetime, where S/L was found to consistently decrease with an increase in apoptosis. With a quantitative framework such as phasor analysis, the short and long lifetime components of NAD(P)H can be used to map the cycling of free and bound NAD(P)H during the early-to-late stages of apoptosis. The combination of lifetime screening and phasor analyses provides the first step in high throughput metabolic profiling of single cells and can be leveraged for screening and sorting for a range of applications in biomedicine. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Keywords: NAD(P)H; apoptosis; endogenous fluorescence; flow cytometry; fluorescence lifetime; label-free; metabolic mapping; metabolism; phasor.

Figure 1

(A) Annexin V‐FITC Propidium Iodide (PI) binding assay for evaluation of apoptosis. 1 μM staurosporine (STS) was used to induce apoptosis in HeLa cells. Dot plot graphs generated from flow cytometric analysis (488 nm excitation) show percentage apoptotic cells in stained cells with no STS treatment (control), cells treated with STS for 1 h and cells treated with STS for 3 h. X‐axis represents fluorescence collected from channel with a 530/30 nm bandpass filter corresponding to Annexin V‐FITC. Y‐axis represents fluorescence collected from channel with a 585/40 nm bandpass filter corresponding to PI. Quadrant gates were generated using unstained HeLa cells as universal negative control. (B) Fluorescence microscopy of HeLa cells. Fluorescence images were obtained from unstained cells treated with STS to capture NADH fluorescence intensity. A 335–383 nm excitation filter and 420–470 nm emission filter were used. Bright‐field and fluorescence channels are overlaid. The scale bar for micrographs is 20 μm. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

Fluorescence decay curves from cuvette measurements of HeLa cells. Untreated cell sample (control, black squares) along with cells treated with STS measured after 15 min (red dots) and 30 min (blue triangles) show a shift in the decay kinetics. A 393‐nm peak wavelength laser diode was used for excitation and emission was captured using a 448/20 nm bandpass filter. The time calibration for this instrument was equivalent to 2.64078e‐2 ns/channel. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

Schematic of fluorescence lifetime flow cytometer (FLFC) built for improved sensitivity to capture dim autofluorescence signals. A 375 nm excitation laser is modulated at RF frequency to excite the cells passing through the flow cell. Light after passing through different focusing lenses and dichroic mirrors is collected as side scattered light, captured at 90° angle using 376/6 nm band pass filter and fluorescence is captured using 448/20 nm band pass filter. Both signals are amplified and directed to a data acquisition system which digitizes the signal at 50 MSPS. Digitized signals are saved as comma separated files to be processed in MATLAB®. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4

Fluorescence intensity and fluorescence lifetime histograms for glacial blue fluorescent microspheres. (A) 1‐D fluorescence histogram and dot plot (scatter vs. fluorescence). The side scatter and fluorescence are the amplitude of the modulated signals after Fourier transformation. The glacial blue amplitude measured with the FLFC resulted in a CV of 13% indicating optimal alignment and performance of the flow cytometer. (B) Fluorescence lifetime histogram for 2,000 events of glacial blue microspheres. The mean fluorescence lifetime is 0.98 ns with standard deviation of 0.3 ns.

Figure 5

Flow cytometric fluorescence lifetime measurements of HeLa cells before and after induction of apoptosis. Mean fluorescence lifetimes of cell samples (n ~3000) are plotted for three independent experimental repeats (purple, blue, and orange circles). Fluorescence lifetimes were calculated using the phase difference between a correlated side scatter and fluorescence modulated waveform signal for each cell. Data points shown are untreated HeLa cells (control), HeLa cells treated with 1 μM STS for 15 min, 30 min, 60 min, and 180 min. Mean of the three mean fluorescence lifetime values are plotted as a square with error bars showing standard error of means. Tukey post hoc test showed a significant difference in the fluorescence lifetime between control and cells treated with STS for 15 min, with P‐value <10e‐5. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 6

FLFC‐derived phasor plots. (A) Phasor graphs showing populations of untreated cells and cells treated with STS to trigger apoptosis. A MATLAB routine permitted extraction of the phase angle and demodulation depth from each set of correlated cytometric waveforms at 6.25 MHz. Plots were generated in R (r‐project.org) and represent individual cells by density regions where the regions of more cells are represented by warmer colors (red). Inset shows magnified view of overlaid cell populations with the median phasor points (black dots) for each sample. (B) Phasor plot with highlighted (colored) areas used to evaluate the fractional contributions. Short lifetime (S) of 0.3 ns represents the free NAD(P)H lifetime. Long lifetime (L) of 7 ns represents the bound NAD(P)H lifetime. Maximum lifetime component is chosen as the third vertex of the triangle which encompasses the cell populations collected herein. Fractional contribution of free and bound NAD(P)H can be estimated using vector algebra in phasor space as well as visualized by the total area of the triangle opposite of the vertex that specifies a short, long, or maximum lifetime (e.g., the size of the cyan colored region—opposite triangle to short lifetime vertex—is proportional to the total contribution of a short lifetime component). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 7

Ratio of fractional contribution of short and long lifetime components (S/L ratio) for the control cell and each time point post‐apoptosis induction. S/L ratios of each sample calculated using phasor analysis, are plotted for three independent experimental repeats (colored circles). Error bars represents standard error of the mean. [Color figure can be viewed at wileyonlinelibrary.com]