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.
Highlights
Fluorescence lifetime imaging microscopy (FLIM) is applied in broad areas of the life sciences and industrial fields for its ability to capture information from a smaller focal volume, inter alia being independent of fluorophore concentration, but sensitive to environmental changes such as pH and temperature and other advantages, which can all be exploited in scientific investigations.[1,2,3,4,5,6,7,8,9]
When FLIM is combined with multiphoton (MP) excitation, greater focal depth is achieved, important for thicker tissue specimens, and out-of-focus fluorescence is avoided with a smaller focal volume, without the need for a confocal pinhole
3.1 Testing Single Solutions of NADH and flavin adenine dinucleotide (FAD) Based on published experiments[54,55] of NADH and FAD in solution, we optimized the concentrations of the two coenzymes to 150 μM for NADH and to 100 μM for FAD to match cell intensities
Summary
Fluorescence lifetime imaging microscopy (FLIM) is applied in broad areas of the life sciences and industrial fields for its ability to capture information from a smaller focal volume, inter alia being independent of fluorophore concentration, but sensitive to environmental changes such as pH and temperature and other advantages, which can all be exploited in scientific investigations.[1,2,3,4,5,6,7,8,9] Fluorescence lifetime is of particular interest for quantitative studies in scattering and absorbing samples, such as tissue sections, where intensity-based methods are problematic.[10,11,12,13,14] When FLIM is combined with multiphoton (MP) excitation, greater focal depth is achieved, important for thicker tissue specimens, and out-of-focus fluorescence is avoided with a smaller focal volume, without the need for a confocal pinhole.
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