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. 2020 Jan;25(1):1-16.
doi: 10.1117/1.JBO.25.1.014510.

Multiphoton FLIM imaging of NAD(P)H and FAD with one excitation wavelength

Ruofan Cao  1 , Horst Wallrabe  1 , Ammasi Periasamy  1   2
Affiliations

Multiphoton FLIM imaging of NAD(P)H and FAD with one excitation wavelength

Ruofan Cao et al. J Biomed Opt. 2020 Jan.

Abstract

Two-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to capture autofluorescence signals from cellular components to investigate dynamic physiological changes in live cells and tissues. Among these intrinsic fluorophores, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD)-essential coenzymes in cellular respiration-have been used as intrinsic fluorescent biomarkers for metabolic states in cancer and other pathologies. Traditional FLIM imaging for NAD(P)H, FAD, and in particular fluorescence lifetime redox ratio (FLIRR) requires a sequential multiwavelength excitation to avoid spectral bleed-through (SBT). This sequential imaging complicates image acquisition, may introduce motion artifacts, and reduce temporal resolution. Testing several two-photon excitation wavelengths in combination with optimized emission filters, we have proved a FLIM imaging protocol, allowing simultaneous image acquisition with a single 800-nm wavelength excitation for NADH and FAD with negligible SBT. As a first step, standard NADH and FAD single and mixed solutions were tested that mimic biological sample conditions. After these optimization steps, the assay was applied to two prostate cancer live cell lines: African-American (AA) and Caucasian-American (LNCaP), used in our previous publications. FLIRR result shows that, in cells, the 800-nm two-photon excitation wavelength is suitable for NADH and FAD FLIM imaging with negligible SBT. While NAD(P)H signals are decreased, sufficient photons are present for accurate lifetime fitting and FAD signals are measurably increased at lower laser power, compared with the common 890-nm excitation conditions. This single wavelength excitation allows a simplification of NADH and FAD FLIM imaging data analysis, decreasing the total imaging time. It also avoids motion artifacts and increases temporal resolution. This simplified assay will also make it more suitable to be applied in a clinical setting.

Keywords: FAD; FLIM; FLIRR; NADH; metabolic imaging.

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Figures

Fig. 1
Fig. 1
Cell normalization and ROI selection in HeLa cells: (a) NAD(P)H intensity was used to isolate dominant mitochondrial morphology. (b) After zeroing the nuclear region, intensity images were normalized to compensate for varying intensities in a time course. (c) Pixel ROIs are generated by thresholding to ranges isolating mitochondrial morphology.
Fig. 2
Fig. 2
Emission spectrum of single NADH (150  μM) and FAD (100  μM) in solution. Emission spectrum of NADH (solid line) and FAD (dashed line). Optimized filters for NADH (dark gray band, 450/50 nm BP) and FAD (light gray band, 560/80 nm BP).
Fig. 3
Fig. 3
Normalized intensity levels measured at a range of two-photon excitation wavelengths (720 to 890 nm at 20-nm intervals): (a) NADH solution, 150  μM, showing normalized intensity levels in the NADH and FAD channels at indicated excitation wavelengths on the x-scale. (b) FAD solution, 100  μM, showing normalized intensity levels in the NADH and FAD channels at indicated excitation wavelengths on the x-scale. At the excitation range 780 to 800 nm, the NADH SBT into the FAD channel is leveling off, and likewise, FAD back-SBT into NADH channel is very low.
Fig. 4
Fig. 4
Normalized intensity levels measured at a range of two-photon excitation wavelengths (720 to 890 nm at 20-nm intervals). (a–c) HeLa, African-American/Caucasian LNCaP PCa cells at normalized intensity levels in the NADH and FAD channels at indicated excitation wavelengths on the x-scale. (d) Calibrated solution, a mix of NADH (150  μM) and FAD (100  μM), at normalized intensity levels in the NADH and FAD channels at indicated excitation wavelengths on the x-scale. Compared with single solutions, cells are subject to other variables (e.g., REDOX states, OXPHOS versus glycolysis balance), but generally follow the same trends in Fig. 4. The calibrated solution can therefore serve to mimic cellular conditions.
Fig. 5
Fig. 5
Emission spectrum of calibration solution (150  μM NADH+100  μM FAD) at different two-photon excitation wavelength. NADH emission filter 450/50 BP (dark gray band) and FAD 560/80 (light gray band). Calibration solution was excited from 720 to 860 nm in 20-nm steps. At 800 nm, a “sweet spot” becomes apparent, with virtually no back-SBT from FAD into the NADH channel and FAD emission of the calibration solution matching that of the single FAD solution, indicating negligible or no SBT contribution into the FAD channel (framed boxes, on the right and at the bottom left). While photon counts for NADH are visibly reduced, they are sufficient for FLIM fitting.
Fig. 6
Fig. 6
Average (τm) fluorescence lifetime results of single NADH and FAD solutions versus calibration solution at different two-photon excitation wavelengths: (a) NADH emission channel. In the absence of any meaningful FAD back-SBT, both, mixed calibration and NADH solutions show only the NADH lifetime values at one-component fitting. (b) FAD emission channel: here, the calibration solution is at two-component fitting, because of NADH-SBT; here τm consists of τ1 NADH lifetime and τ2 FAD lifetime. At 720- and 740-nm excitation, τm is almost equal to the NADH lifetime, indicating that the majority of the signal is contributed by the NADH-SBT. At 760 and 780 nm, increasingly the FAD fraction dominates τm as the NADPH-SBT fraction declines. At 800 nm, the FAD lifetime matches that of the single FAD solution with virtually no contribution from NADH SBT, an important observation for supporting the choice of this wavelength for both coenzymes.
Fig. 7
Fig. 7
Fluorescence lifetime parameter results at different excitation wavelengths in the NAD(P)H channel for AA and LNCaP PCa cells: mean data for AA-African-American PCa cells (solid bars) and Caucasian LNCaP PCa cells (light, patterned bars). (a) NAD(P)H-τ1, (b) NAD(P)H-τ2, (c) NAD(P)H-τm, (d) NAD(P)H-a2%. We suggest that after 800 nm, the photon count is too low for accurate fitting.
Fig. 8
Fig. 8
Fluorescence lifetime parameter results at different excitation wavelengths in the FAD channel for AA and LNCaP PCa cells. Mean data for AA-African-American PCa cells (solid bars) and Caucasian LNCaP PCa cells (light, patterned bars): (a) FAD-τ1, (a) FAD-τ2, (c) FAD-τm, and (d) FAD-a1%.
Fig. 9
Fig. 9
Comparison of FLIRR parameters NAD(P)H-a2% and FAD-a1% and their ratio at 800 nm versus 740/890-nm excitation in LNCaP PCa cells. (a) All data are based on single-excitation wavelength 800 nm for NAD(P)H and FAD. FLIRR is the ratio of NAD(P)H-a2%/FAD-a1%, a marker for cellular REDOX. (b) NAD(P)H-a2% based on 740-nm excitation, FAD-1% at 890-nm excitation and FLIRR as described above. Data from both wavelength approaches are from identical FOVs and pixel locations. While some differences exist in absolute values, still within broad statistical ranges, trends and conclusions drawn from the effects of treatment are the same.
Fig. 10
Fig. 10
NAD(P)H-a2% images at 800-nm versus 740-nm excitation in LNCaP PCa cells. (a) 800-nm excitation for NAD(P)H control and 40 min, 60 min after doxorubicin treatment; histogram displays the frequency distribution of the enzyme-bound fraction of NAD(P)H. (b) NAD(P)H-a2% at 740-nm excitation, and the respective frequency histogram.
Fig. 11
Fig. 11
FAD-a1% images at 800-nm versus 890-nm excitation in LNCaP PCa cells. (a) 800-nm excitation for FAD control and 40 min, 60 min after doxorubicin treatment; histogram displays the frequency distribution of quenched fraction of FAD. (b) FAD-a1% at 890-nm excitation, and the respective frequency histogram.
Fig. 12
Fig. 12
FLIRR images at 800-nm versus traditional excitation in LNCaP PCa cells. (a) 800-nm excitation for FLIRR control and 40 min, 60 min after doxorubicin treatment; histogram displays the frequency distribution of FLIRR. (b) FLIRR at 740-nm excitation for NAD(P)H and 890 for FAD, and the respective frequency histogram. Better FLIRR signal-to-noise image for single excitation 800 nm compared to two excitation wavelengths.
Fig. 13
Fig. 13
Fluorescence lifetime parameter results at different excitation wavelengths in the FAD channel for human PCa tissue section. (a) Mean data for NAD(P)H-τm and a2%; the low means at 860 and 890 nm are signals from second-harmonic generation. (b) Mean data for FAD-τm and a1%.
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