Autofluorescence from NADH Conformations Associated with Different

28 Apr 2015 - Department of Physics, Miami University, 500 East Spring Street, Oxford, Ohio 45056, United States. Anal. Chem. , 2015, 87 (10), pp 5117...
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Autofluorescence from NADH Conformations Associated with Different Metabolic Pathways Monitored Using Nanosecond-Gated Spectroscopy and Spectral Phasor Analysis Jeff Maltas, Lana Amer, Zac Long, Dylan Palo, Arthur Oliva, Jeff Folz, and Paul Urayama* Department of Physics, Miami University, 500 East Spring Street, Oxford, Ohio 45056, United States S Supporting Information *

ABSTRACT: Cellular NADH conformation is increasingly recognized as an endogenous optical biomarker and metabolic indicator. Recently, we reported a real-time approach for tracking metabolism on the basis of the quantification of UV-excited autofluorescence spectrum shape. Here, we use nanosecond-gated spectral acquisition, combined with spectrum-shape quantification, to monitor the long excited-state lifetime autofluorescence (usually associated with protein-bound NADH conformations) separately from the autofluorescence signal as a whole. We observe that the autofluorescence response induced by two NADH-oxidation inhibitorscyanide and ethanolare similar in Saccharomyces cerevisiae when monitored using timeintegrated detection but easily distinguished using time-gated detection. Results are consistent with the observation of multiple NADH conformations as assessed using spectral phasor analysis. Further, because well-known oxidation inhibitors are used, changes in spectrum shape can be associated with NADH conformations involved in the different metabolic pathways, giving bioanalytic utility to the spectral responses.

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the spectrum shape. Here, we demonstrate that autofluorescence spectrum shape can be used to identify changes in protein-bound NADH conformations associated with different metabolic pathways. In order to monitor autofluorescence from protein-bound conformations, we use time-gated detection after pulsed excitation to obtain nanosecond-resolved emission spectra. Analogous to an approach by Paul and Schneckenburger,20 this allows for long-lifetime conformations (usually associated with protein-bound NADH21) to be monitored separately from the autofluorescence signal as a whole. For the current study, we first characterize the temporal response of the spectroscopic system, demonstrating subnanosecond gating control. Next, we illustrate how long-lifetime emission can be monitored using time-gated spectroscopy by probing mixtures of reference fluorophores known to have similar emission spectra but a large difference in excited-state lifetime. We employ computational approaches for quantifying spectrum shape that are noniterative and that require minimal

ellular reduced nicotinamide adenine dinucleotide (NADH) conformation can be probed on the basis of many sources of optical contrast, including differences in the excited-state emission spectrum,1−5 excited-state lifetime,6−13 and anisotropy decay rate.14−16 Consistent with solution measurements of NADH and NADH/protein complexes,17 recent measurements on autofluorescence have identified multiple intracellular NADH conformations, significant because the cellular proportions of the various conformations respond differently to metabolic conditions.12−14 This suggests the often-used “free versus bound” description of NADH conformation can lead to ambiguities when identifying metabolic state. Because conformational information is useful as a metabolic indicator and biomarker,1,4,5,12−14,18 methods that precisely describe cellular NADH conformation have potential biotechnological and bioanalytical applications. Previously, we have shown that despite the small spectral difference between free and protein-bound NADH emission, the time-integrated autofluorescence spectrum shape sufficiently correlates with metabolic state to be useful for real-time monitoring.19 Nonetheless, because a two-component free-vsprotein-bound interpretation inadequately accounted for observed autofluorescence responses, we speculated that additional conformational information might be contained in © XXXX American Chemical Society

Received: November 24, 2014 Accepted: April 28, 2015

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Analytical Chemistry user intervention, including spectral phasor22 and spectrum shape19 analyses. Next, using solution measurements of NADH bound to dehydrogenase proteins, we demonstrate the ability to detect different protein-bound conformations using spectral phasor analysis. Finally, we apply the approach to the metabolic monitoring of Saccharomyces cerevisiae (baker’s yeast), discriminating between responses to the addition of cyanide23 and ethanol,24−26 two inhibitors of NADH oxidation pathways. We observe that responses are similar when sensed using timeintegrated detection, but easily distinguished when sensed using time-gated detection. Results confirm that different pathways are affected because changes in the population of proteinbound forms−sensed using time-gated detection−cannot be interpreted as two-state changes according to spectral phasor analysis. Because well-known metabolic inhibitors are used, optical signals correspond to emission from NADH conformations associated with specific metabolic pathways. This bioanalytical interpretation will contribute to the development of NADH as an endogenous biomarker and of methods for the precise optical assignment of metabolic state.

ns nominal pulse width at a repetition rate of 3 Hz for generating the excitation wavelength (337 nm) used in these studies. Spectra are acquired using a spectrograph (model MS 125, Spectra-Physics/Newport) coupled to a time-gated intensified CCD (ICCD) (model iStar 734, Andor) with a picosecond-resolution digital delay generator (DDG) for controlling gate delay. A constant-fraction optical discriminator (model OCF-401, Becker & Hickl) is used to sense the excitation pulse and serves as a low temporal-jitter reference for gate-delay timing. Because there is a minimum time between detector triggering and intensifier gate opening, a 25 m optical fiber (cat. no. 57-075, Edmund Optics) is used to delay the excitation pulse from arriving at the sample so that the sample’s emission falls within the range of gate delays accessible by the ICCD/DDG. Finally, excitation light from the delay fiber is focused into the sample using a converging lens. The excitation energy at the sample is 40 μJ/pulse, measured using a pyroelectric joulemeter (model J9LP, Coherent). Emission is collected at 90° from the excitation using a separate optical fiber, delivering light to the spectrograph/ICCD. The 1024 × 1024 pixel CCD chip was fully binned along one axis during readout, resulting in a 1024-spectral-channel output. The spectrograph utilized a 400-lp/mm grating, and the spectrograph-ICCD system was calibrated for wavelength using a mercury−argon lamp (cat. no. 6035, Newport). The measured spectral width of atomic lines was 2 nm, and the 1024 spectral channels covered a 250 nm wavelength range. The ICCD gate delay and gate width are specified by control software provided by the manufacturer, and a full spectrum was collected for each excitation pulse without the need for scanning, although spectra were averaged over five detector readouts in order to increase maximum-signal-to-noise ratio (SNR). The CCD was temperature-regulated, and the CCD dark current (measured by performing a CCD readout without use of the intensifier) was subtracted from measured spectra prior to analysis. Sample Preparation and Data Acquisition Protocols. For measurements using reference fluorophores, stock solutions of 9-cyanoanthracene (cat. no. 152765, SigmaAldrich) and 1,4-bis(5-phenyl-2-oxazolyl)benzene (POPOP, cat. no. P3754, Sigma-Aldrich) were prepared to a final concentration of 2.5 μM fluorophore in spectroscopic-grade ethanol. All fluorophore mixtures were at 2.5 μM total fluorophore concentration with measurement performed in a quartz spectroscopic cuvette at room temperature. Both 9:1 and 1:9 POPOP/9-cyanoanthracene mixtures were made by combining stock solutions in a 9:1 or 1:9 volume ratio prior to spectroscopic measurement. For the illustration of real-time monitoring, the cuvette was initially filled to 2.5 mL of a stock solution with 0.5 mL of the other fluorophore’s stock solution added twice sequentially. Solutions were pipetted directly into the stirred, open cuvette during the measurement run, resulting in mix ratios of 1:0, 3:1, and 3:2 for the two fluorophores. The protocol was then repeated for the opposite fluorophore combination. For protein-binding measurements, solutions of 50 μM NADH (cat. no. N8129, Sigma-Aldrich) or 50 μM NADPH (reduced nicotinamide adenine dinucleotide phosphate, cat. no. N20140, Research Products International) were prepared in 200 mM 3-morpholinopropane-1-sulfonic acid (MOPS) buffer, adjusted to pH 7.4 using sodium hydroxide. Ammonium-sulfate precipitates of malate dehydrogenase from porcine heart (MDH; cat. no. M1567, Sigma-Aldrich; manufacturer’s lot

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METHODS Instrumentation. Time gating refers to the rejection of signal outside a given time window. In our implementation (Figure 1a), the sample is excited using a pulsed source E(t),

Figure 1. (a) F(λ) is the spectrum of the excited-state emission measured at gate delay tdelay and over a gate width Δtgate. (b) Setup for the time-gated spectroscopy system. Abbreviations: NL, nitrogen laser; ND, neutral-density filter; L, lens; DF, optical-delay fiber; S, sample; OD, constant-fraction optical discriminator; LP, long-pass filter; FO, fiber optic; SG, spectrograph; ICCD, intensified charge-coupled device; DDG, digital delay generator; C, control computer.

resulting in an excited-state emission of intensity I(t). The timegated spectrum F(λ) is measured at time tdelay, called the gate delay, over a time interval Δtgate, called the gate width. The configuration compares with time-gated fluorescence lifetime imaging microscopy (FLIM) systems,27−31 except with the time-gated detector coupled to a spectrograph and not a microscope. The system (Figure 1b) uses a nitrogen-gas discharge laser (model GL-3300, Photon Technology International) with a 1 B

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When monitoring long-lifetime emission separately from the emission as a whole, such as for the reference fluorophores and cellular suspensions, a two-gate protocol was used. At each data-acquisition time, two time-gated spectra were acquired one using a long gate width (Δtgate = 80 ns) measuring the time-integrated emission (henceforth, called the “integrated gate”) and the other using a short gate width (Δtgate = 5.0 ns) containing time-gated emission. The latter will be called the “late gate,” with tdelay set 15 ns (for reference-fluorophore data) or 4 ns (for cellular data) after the delay time for which the maximum emission intensity was observed. For protein-binding measurements, only the integrated gate was used. Spectrum Shape Quantification. Spectral phasor analysis22 is used for quantifying spectrum shape. The spectral phasor A is the first harmonic of the spectrum’s Fourier transform. For N spectral channels, the real and imaginary phasor components are

analysis: 7.8 mg/mL protein, 1090 units/mg protein) and lactate dehydrogenase from rabbit muscle (LDH; cat. no. L2500, Sigma-Aldrich; manufacturer’s lot analysis: 10.5 mg/mL protein, 942 units/mg protein) were used without additional purification and were kept refrigerated until just prior to use. The spectroscopic cuvette was initially filled with 3.0 mL of 50 μM NADH or NADPH solution. Protein solutions were pipetted directly into the open cuvette during the measurement run in the following sequence: 40 μL of MDH, 40 μL of MDH, and 40 μL of LDH. Assuming molecular weights of 70 kDa for MDH32 and 140 kDa for LDH,33 and using the manufactuer’s lot analysis for protein content, estimated protein concentrations are 1.5 μM MDH after the first addition, 2.9 μM MDH after the second addition, and 2.9 μM MDH/1.0 μM LDH after the third addition. Note that measurements were made under conditions of excess NADH or NADPH. Samples were continuously stirred to facilitate mixing and to prevent photobleaching. S. cerevisiae was prepared from dry powder (Fleischmann’s ActiveDry) and grown on YPD agar medium (cat. no. Y1000, TekNova) for 2 or 3 days. Petri dishes were covered but not airtight, and were kept inverted to avoid excess condensation. Cells were grown at room temperature. Prior to measurement, cells were triple washed in phosphate-buffered saline (PBS, cat. no. 20012, Life Technologies) to remove background emission coming from the growth medium. Cells were then resuspended in PBS. Cellular suspensions in PBS had a density of approximately 107 cells/mL, as determined from a measurement of optical density calibrated using a hemocytometer. Emission intensity was linear with cell concentration, and samples had an optical density of less than 1.4 at 600 nm wavelength. Samples were not temperature-regulated; room temperature was measured at 22 ± 2 °C. Samples were measured in a quartz cuvette and continuously stirred during all measurements to maintain oxygenation and to prevent photobleaching. Limiting spectral measurements to every 30 s during monitoring further minimized photobleaching. Cell viability prior to measurement was ∼90%, estimated via Trypan blue staining on a representative sample. Cellular responses were induced after the autofluorescence spectrum was observed to reach a steady state, approximately 30 min after resuspension in PBS. To induce a response, either potassium cyanide (cat. no. 60178, Sigma-Aldrich) in PBS or spectroscopic-grade ethanol (cat. no. 459828, Sigma-Aldrich) was added to the cuvette to a final concentration of 8 mM or 2 vol %, respectively. With regards to data acquisition, temporal responses were characterized as the emission intensity versus tdelay. Emission intensity was a computed spectral integration over all wavelengths. For measurement of the system’s temporal response, a 0.03-OD neutral density filter was placed at the sample location, with measurement made of the reflected signal and with the spectrograph aligned for 337 nm wavelength detection. The characterization was made for various ICCD gate widths, using a step size of 0.1 ns between subsequent gate delays. The system’s temporal response was characterized once and not prior to each experimental run. For the temporal response of reference fluorophores and cellular suspensions, Δtgate = 5 ns. Gate delays started 15 ns before the gate-delay time for which the maximum emission intensity was observed, with a step size of 1.0 ns between subsequent spectral acquisitions.

Re(A) =

j

Im(A) =

⎛ 2π ⎞ j⎟ N ⎠

∑ Fobserved(λj)cos⎜⎝

⎛ 2π ⎞ j⎟ N ⎠

∑ Fobserved(λj)sin⎜⎝ j

(1)

where j is the spectral channel. The spectrum Fobserved has been normalized to the integrated intensity. A spectral phasor plot of Im(A) versus Re(A) is useful for the analytical assessment of fluorophores in a sample because phasors calculated from spectra of similar shape tend to cluster, and because phasors from a two-component system lay along a line.22 A second approach, which we call spectrum shape analysis,19 is to quantify the intensity-weighted distribution of signal within a wavelength interval. The intensity-weighted average wavelength is calculated as ⟨λ⟩ =

∑ Fobserved(λj)λj j

(2)

The spectrum Fobserved has been normalized to the integrated intensity. The standard deviation σ of the distribution is the square root of the variance, which is calculated as σ2 =

∑ Fobserved(λj)λj 2 − ⟨λ⟩2 j

(3)

Analogous to a phasor plot, a plot of standard deviation versus average wavelength is useful when discriminating between fluorophores within a sample. Spectrum shape plots are helpful for discussing results in real-space (wavelength) units.

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RESULTS System’s Temporal Response. The ICCD gate width is estimated by measuring the system’s temporal response, and comparing with manufacturer’s values. Figure 2 shows several temporal response profiles (spectrally integrated intensity versus tdelay) for nominal gate widths of 1, 2, and 5 ns. Fullwidth-at-half-maximums (fwhm) are 2.7, 5.0, and 6.1 ns, respectively. Before comparing with manufacturer’s values, note that the temporal response is a convolution of the laser pulse and ICCD gate. Other factors (e.g., modal dispersion in the delay fiber) play a less significant role.31 Because we do not have an C

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Figure 2. System’s temporal response, plotted as the integrated intensity versus tdelay. Nominal Δtgate are 1 ns (red, square), 2 ns (green, circle), and 5 ns (blue, triangle). To facilitate visual comparison, intensities have been scaled to the fitted maximum intensity, and tdelay values are shifted so that the fitted maximum occurs at tdelay = 0 ns.

independent measure of the laser pulse, we estimate gate widths by assuming the laser and ICCD contributions to the system response add in quadrature as for the convolution of Gaussianshaped responses. For a 1 ns laser pulse width, estimated gate widths are Δtgate = 2.5, 4.9, and 6.0 ns, respectively, which compare reasonably with manufacturer’s values of 2.5, 4.6, and 5.6 ns. For this study, nominal values are used when Δtgate is specified. Reference Fluorophores. Here we illustrate how longlifetime emission can be monitored separately from the emission as a whole. Time-gated spectroscopy is used to probe mixtures of reference fluorophores, POPOP and 9cyanoanthracene, known to have similar emission spectra but a large difference in excited-state lifetimes. Figure 3a shows the temporal response of pure and mixed fluorophore solutions. The differences in excited-state lifetimes are detectable, with mixtures showing decay rates that are intermediate to the pure samples. Single-exponential fits to the temporal response curve give excited-state lifetimes of 1.33 ns for POPOP and 12.2 ns for 9-cyanoanthracene. (So that the excitation is not present, only data 2 ns after the maximum intensity are fitted.) Fitted lifetimes are in good agreement with literature values of 1.32 ns for POPOP and 11.9 ns for 9cyanoanthracene in ethanol, in equilibrium with air.34 Time-integrated spectra from pure POPOP and 9-cyanoanthracence solutions are shown in Figure 3b, and time-gated spectra from the two mixtures are shown in Figures 3c,d. The spectrum at gate delays corresponding to maximum intensity, and at 5 and 10 ns after maximum intensity evolve as expected, that is, emission spectra for later gate delays approach that of 9cyanoanthracene, the longer lifetime fluorophore. Note how the mixture with the greater POPOP content (Figure 3c) starts off similar to the pure POPOP spectrum, but it rapidly decays in intensity, as indicated by a decreasing SNR. The mixture with the greater 9-cyanoanthracene content (Figure 3d) shows a slower decrease in intensity, with the fastest decrease occurring at 420 nm wavelength associated with a POPOP spectral maximum. Next we illustrate monitoring of the long-lifetime subpopulation of fluorophores in a sample, i.e., 9-cyanoanthracene in this case, by starting with a one-component solution, then sequentially adding the other fluorophore. Both the addition of 9-cyanoanthracence to POPOP in ethanol, and vice versa, are considered. When analyzed using spectral phasors (Figure 4a), integrated-gate phasors start at locations corresponding to the pure samples but shift linearly as the other fluorophore is

Figure 3. Time-gated spectra of reference fluorophores and mixtures. (a) Temporal response (spectrally integrated intensity versus tdelay) of POPOP (blue, squares), 9-cyanoanthracene (red, circles), and 9:1 and 1:9 POPOP/9-cyanoanthracene mixtures (green, upright triangles; orange, inverted triangles). Intensities have been scaled to the maximum intensity. The gate-delay axis spans 50 ns but has been truncated to clarify behavior near the maximum intensity. (b) Integrated-gate spectra of pure POPOP (blue) and 9-cyanoanthracene (red) in ethanol. Spectra have been scaled to the maximum intensity. (c) Time-gated spectra for the 9:1 POPOP/9-cyanoanthracene mixture at different tdelay: at maximum intensity (red), 5 ns after maximum intensity (blue), and 10 ns after maximum intensity (green). Note the rapid decrease in signal-to-noise ratio with increasing time, as expected for a short-lifetime mixture, as well as the decrease in the spectral maximum at 420 nm wavelength, associated with POPOP. (d) Time-gated spectra for the 1:9 POPOP/9-cyanoanthracene mixture at different tdelay: at the maximum intensity (red), 5 ns after maximum intensity (blue), and 10 ns after maximum intensity (green). Note the slower decrease in signal-to-noise ratio with increasing time, as expected for a long-lifetime mixture. There is a decrease in the relative intensity at 420 nm wavelength, associated with the decay of the POPOP signal. For (c) and (d), the maximum-intensity spectrum was scaled to its maximum intensity, with all other spectra scaled to minimize least-squares differences.

added, as expected for a two-component system. By comparison, the late-gate phasor plot only shows the phasor for 9-cyanoanthracence. The addition of POPOP to 9cyanoanthracene cannot be detected in the late-gate signal, while the signal from a solution of pure POPOP is noise (data points are beyond the phasor plot range due to axis scaling), until 9-cyanoanthracene is added. Figure 4b (spectrum shape analysis) shows real-space behavior analogous to Figure 4a. Shifts in the average emission wavelength are readily observed using the integrated gate, but not using the late gate. NADH Binding to Proteins. To demonstrate the ability to detect different protein-bound NADH conformations, we monitor a solution of NADH as dehydrogenase proteins are added sequentially (Figure 5, left column). Each protein addition is observed as a shift in the phasor plot (Figure 5b, left). Shifts for the two additions of MDH are collinear, consistent with emission from a two-component system of free and MDH-bound NADH. The spectral phasor shift associated with LDH addition is not collinear with shifts due to MDH addition, indicating that LDH-bound NADH is distinguishable D

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Figure 4. Monitoring the sequential additions of one reference fluorophore into the other reference fluorophore. For all figures, the sample corresponding to additions of POPOP into 9-cyanoanthracene is in red, and the sample corresponding to additions of 9-cyanthracene into POPOP is in blue. Samples are monitored in real time, with a spectral acquisition every 6 s. Squares are for the pure sample, circles are for times after the first addition, and triangles are for times after the second addition. (a) Spectral phasor plots for the integrated gate (left) and late gate (right) during the monitoring of fluorophore additions. For the integrated-gate plot, the red (or blue) line is a linear fit of red (or blue) phasor points. Note the collinearity of phasor points, as expected for a two-component system. For the late-gate plot, only phasor points corresponding to 9-cyanoanthracene are observed. Both plots are on the same scale in order to facilitate comparison. (b) Spectrum shape plots for the integrated gate (left) and late gate (right) during the monitoring of fluorophore additions. Again, for the late gate, only phasor points corresponding to 9-cyanoanthracene are observed. Both plots are on the same scale in order to facilitate comparison. For (a) and (b), analysis is performed over the first 400 pixels (400−500 nm wavelength range).

Figure 5. Spectral detection of NADH protein binding. Sequential additions of MDH, MDH, and LDH to solutions of NADH (left column) or NADPH (right column). In all plots, the color or symbol shape corresponds to times before protein is added (red, square), after adding MDH once (blue, circle), after adding MDH twice (orange, triangle), and after adding LDH (green, inverted triangle). (a) Integrated-gate spectra. The spectrum taken before protein addition was scaled to its maximum intensity, with all other spectra scaled to minimize least-squares differences. Fractional-difference spectra are calculated using the spectrum before protein addition as the reference spectrum. (b) Spectral phasor plots. Shifts due to MDH addition to the NADH solution (left) are collinear (best fit line is shown). The shift due to LDH addition is not collinear with MDH-induced shifts, evidencing that protein-bound NADH conformations are spectrally distinguishable. As a negative control, no shift is observed when proteins are added to the NADPH solution (right). (c) Spectrum shape plots for the same data as in (b). For (b) and (c), analysis is performed over the first 512 pixels (400−530 nm wavelength range).

from MDH-bound NADH. Figure 5c (left) shows the average wavelength shifts to shorter emission wavelength upon protein binding. Because NADH and NADPH have nearly identical emission properties, and because MDH and LDH exhibit preferential binding for NADH, the lack of change in emission spectra when adding MDH and LDH to a NADPH solution (Figure 5, right column) serves as a control to confirm NADH binding and to show the absence of optical artifacts. Metabolic Monitoring in Cellular Samples. Figure 6 shows a representative temporal response profile for a cellular sample, showing the autofluorescence spectrum at various tdelay. We use the two-gate protocol to monitor S. cerevisiae autofluorescence response to cyanide (Figure 7, left column) and ethanol (Figure 7, right column) additions. The autofluorescence response to cyanide addition as monitored using the integrated gate (Figure 7a, left) is consistent with previous results,19 showing an increase in intensity and a shift to longer emission wavelength. Fractional change in intensity is smaller for the late gate than for the integrated gate (Figure 7b, left). There is also a difference in the response duration, with the late-gate intensity reaching a steady value sooner than the integrated-gate intensity. Because long-lifetime conformations have a greater quantum efficiency for fluorescence than short-lifetime conformations,

the increase in concentration for long-lifetime forms is less than is suggested by the increase in emission intensity. Overall, there is a larger increase in the concentration of short-lifetime conformations as compared with long-lifetime conformations. Similar observations were made using measurements of anisotropy decay during transitions from normoxia to hypoxia in hippocampal slices from Sprague−Dawley rats;14 concentrations of free NADH and short-lifetime bound forms increased preferentially over that of other enzyme-bound NADH forms. Because cyanide blocks the use of oxygen by the electron transport chain, these observations appear consistent. The autofluorescence response to ethanol addition (Figure 7, right column) as monitored using the integrated gate shows an increase in intensity and a shift to longer emission wavelength, again consistent with previous results.19,35 Furthermore, E

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Figure 6. Time-gated autofluorescence spectra. (a) Representative temporal response (spectrally integrated intensity versus tdelay) of autofluorescence prior to cyanide or ethanol addition. Intensities have been scaled to the maximum intensity. (b) Autofluorescence spectra at different tdelay: at maximum intensity (red), 5 ns after maximum intensity (blue), and 10 ns after maximum intensity (green). Spectra have been scaled to the maximum intensity of the first spectra. All axes have the same range as for Figure 3 to facilitate comparisons.

integrated-gate behavior of cyanide- and ethanol-responses is reasonably similar (e.g., compare the left and right columns of Figure 7a,b). By comparison, the late-gate behavior of the responses (Figure 7c,d) is markedly different; the late-gate spectrum for the cyanide response shifts to shorter emission wavelengths, while the shift for the ethanol response is toward a longer wavelength. The directions of the shift for each response are not collinear in the spectral phasor plot; this confirms that different pathways are involved because changes in the population of protein-bound forms, which is sensed using time-gated detection, cannot be interpreted as being due to the same sets of conformations. (While shifts in the time-integrated phasor plots are also not collinear, a similar conclusion cannot be made because we cannot rule out a possible role for free NADH.)

Figure 7. Monitoring autofluorescence response to cyanide (left column) and ethanol (right column) additions. In all figures, integrated-gate data are red and/or squares, and late-gate data are blue and/or circles. For panels (b)−(d), solid symbols correspond to times before the chemical addition, and open symbols correspond to times after the chemical addition. (a) Autofluorescence spectra before (thick line) and after (thin line) the chemical addition. Spectra have been scaled to the maximum intensity prior to chemical additions. (b) Spectrally integrated intensity versus time, normalized to the intensity prior to chemical addition. Note that spectral measurements were made until new steady values were reached (10−20 min), but have been truncated for clarity near the time of chemical addition. (c) Spectral phasor plots. (d) Spectrum shape plots. For (b)−(d), analysis is performed over the first 400 pixels (400−500 nm wavelength range). Results for each column have been reproduced on at least three independently prepared samples.

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DISCUSSION Before considering biological interpretations of autofluorescence responses, we discuss ways in which measurement parameters affect observations. An important parameter in quantifying spectrum shape is the wavelength interval used in eqs 1−3. As an example of its effects, Figure 8 shows the same protein-binding data as in Figure 5, calculated over different wavelength intervals. While the interval for Figure 5 (pixels 1− 512, 400−530 nm wavelength) is broad and centered on the maximum emission wavelength, Figure 8 calculations are made using three smaller intervals spanning wavelengths shorter than (pixels 1−256, 400−465 nm wavelength), centered on (pixels 128−384, 430−498 nm wavelength), and longer than (pixels 256−512, 465−530 nm wavelength) the integrated-gate maximum emission wavelength. The measurement contrast (i.e., the observed shift as compared with the spread in data points) varies with interval used. In this case, most of the

observed change in spectrum shape occurs at short emission wavelengths, although the shift due to LDH addition appears to not be collinear in each case. In general, the wavelength interval needs to be optimized by the user. In our experience, an interval centered on the spectrum and wide enough to include the majority of the integrated signal appears to provide the greatest contrast. F

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for about 2 ns after the maximum, we select the late-gate delay to be 4 ns after the maximum emission intensity. This allows emission from short-lifetime forms to decay prior to gate opening, resulting in a preferential enhancement of the longlifetime signal. As to whether the autofluorescence response is an artifact of the chemical addition, we showed previously that autofluorescence from heat-treated, nonviable S. cerevisiae samples do not exhibit a change in time-integrated autofluorescence spectrum shape when cyanide or ethanol is added, confirming that changes in spectrum shape are physiological.19 Because the autofluorescence response to cyanide and to ethanol are not artifactual, we consider their biophysical origins in terms of the underlying physiological response. Cyanide and ethanol are both inhibitors of NADH oxidation, hence the increase in autofluorescence intensity when these chemicals are added (Figure 7). Cyanide inhibits the aerobic oxidative pathway,23 while multiple pathways of inhibition are believed to exist for ethanol action.24−26 Nonetheless, because inhibition pathways are not identical, we hypothesize that the redistribution of NADH conformations due to the physiological responses is also distinct and that it is the emission from these sets of protein-bound conformations that results in the observed change in spectrum shape. The evidence is strong that these sets are not the same because the late-gate phasor shifts for the responses are not collinear (Figure 7c). Studies leading to a more complete physiological description using bioanalytical approaches presented here are ongoing. That being said, it is also not known how spectral shifts might appear for other cell types, although it is of interest to know whether analogous responses in the autofluorescence spectrum shape exist for mammalian cells. Although it is difficult to calculate NADH conformation from emission spectrum shape, our interpretation appears consistent with other recent studies in addition to the one by Vishwasrao et al.14 discussed previously. For example, high-resolution excited-state lifetime fits on in vivo cerebral autofluorescence in rats showed four lifetime components exhibiting distinct responses during brief periods of anoxia.13 It was suggested that each component might be useful as a biomarker for specific molecular pathways related to oxidative metabolism. In another study using pixel histograms of excited-state lifetimes derived from two-photon excitation FLIM, subpopulations of NADH were identified in cellular cancer models and found to show distinct responses to metabolic state.12 Each indicates that there are subpopulations of NADH that respond differently to metabolic conditions, as proposed to have been observed here. Other endogenous fluorophores, including NADPH and various flavins, are also possible sources of autofluorescence. It was shown previously that flavins are not a significant contribution to changes in autofluorescence shape at this excitation wavelength.19 On the other hand, it is possible that NADPH, having nearly identical absorption and emission properties as NADH, contributes to the observed autofluorescence responses. Although signal from NADPH is not insignificant, the biochemical roles of NADH and NADPH are distinct and their pools are relatively uncoupled. In their analysis, Vishwasrao et al. concluded that NADPH represents a fluorescence background that is constant with respect to metabolic perturbations.14 Given the results and discussion presented, spectrum-shape quantification, combined with time-gated sensing of UV-excited autofluorescence provides bioanalytical information regarding

Figure 8. Measurement contrast depends on wavelength interval used in calculating spectral phasors. Shown is the same protein-binding data as in Figure 5 but calculated over wavelength intervals shorter than (top row), centered on (middle row), and longer than (bottom row) the maximum emission wavelength. Symbol colors and shapes are the same as for Figure 5. The average phasor position for each sequence in the protein addition is shown as a black open circle. To assess whether the phasor shift due to LDH addition is collinear with shifts due to MDH addition, linear fits (solid lines) to the first three open circles in each plot are shown. Note that plots do not use the same axes range and have been scaled to facilitate comparison of measurement contrast.

Another factor to consider in the spectral phasor analysis is that previous studies involved the identification of fluorophores with large spectral differences, covering a large portion of phasor space.22,36 By comparison, the phasor space covered in the current study is small, involving detection of conformations of a single fluorophore. While small spectral resolution and high SNR are expected to improve measurement contrast, we have not investigated this systematically. It is not known, for example, what system parameters would yield sufficient measurement contrast for single-cell measurements. We note that SNR in the range of 5 to 10 produced standard deviations in the average wavelength that were comparable to the spectral resolution of the system (2 nm wavelength)(Figure 7d). Additional examples and discussion of how measurement parameters affect observed results are found in previous studies.19,22 Next, the monitoring approach presented here uses a twogate protocol. In order for the late gate to exclude the shortlifetime signal, the gate delay needs to be late enough for the short-lifetime component to decay, while early enough for the long-lifetime component to be detectable. The excited-state lifetimes of NADH are about 0.4 ns for the free form and up to several nanoseconds for protein-bound forms.21,34 Because from Figure 2, the trailing edge of the system response persists G

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Analytical Chemistry

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NADH conformation. Development of this approach may lead to new and useful approaches for the label-free detection of metabolic state and metabolic response, and to biophysical investigations regarding the real-time dynamics of energyrelated biochemical processes.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ac504386x.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (513) 529-9274. Fax: (513) 529-5629. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by funds from Miami University’s College of Arts and Science and Miami University’s Office for the Advancement of Research and Scholarship’s Undergraduate Summer Scholars program. This material is based upon work supported by the National Science Foundation under Grant No. 0957675. We thank Morgan McGrath for her assistance on this project.

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REFERENCES

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DOI: 10.1021/ac504386x Anal. Chem. XXXX, XXX, XXX−XXX