Metabolic imaging by simultaneous FLIM of NAD(P)H and FAD

Significance: Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to distinguish the unique molecular environment of fluorophores. FLIM measures the time a fluorophore remains in an excited state before emitting a photon, and detects molecular variations of fluorophores that are not apparent with spectral techniques alone. FLIM is sensitive to multiple biomedical processes including disease progression and drug efficacy. Aim: We provide an overview of FLIM principles, instrumentation, and analysis while highlighting the latest developments and biological applications. Approach: This review covers FLIM principles and theory, including advantages over intensitybased fluorescence measurements. Fundamentals of FLIM instrumentation in time-and frequencydomains are summarized, along with recent developments. Image segmentation and analysis strategies that quantify spatial and molecular features of cellular heterogeneity are reviewed. Finally, representative applications are provided including high-resolution FLIM of cell-and organelle-level molecular changes, use of exogenous and endogenous fluorophores, and imaging protein-protein interactions with Förster resonance energy transfer (FRET). Advantages and limitations of FLIM are also discussed. Conclusions: FLIM is advantageous for probing molecular environments of fluorophores to inform on fluorophore behavior that cannot be elucidated with intensity measurements alone. Development of FLIM technologies, analysis, and applications will further advance biological research and clinical assessments.

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“…157 In addition, two single-photon wavelengths have been temporally interleaved to alternately excite NAD(P)H and FAD. 158…”

Section: Confocal and Multiphoton Microscopesmentioning

confidence: 99%

Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications

et al. 2020

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“…Some investigators have applied an expensive solution to the problem, using two parallel lasers, cutting down the laser wavelength switch-over time. 51,52 A one-excitation wavelength has been proposed 53 to excite both NAD(P)H and FAD at the same time. However, there is a need for a comprehensive study to cover aspects of intensity spectrum and fluorescence lifetime.…”

Section: Introductionmentioning

confidence: 99%

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

2020

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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.

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“…[35][36][37] Further work could include imaging endogenous fluorophores including other molecules of interest such as flavin adenine dinucleotide and nicotinamide adenine dinucleotide to measure for metabolic activity to identify cancerous cells. 38,39 Additional system improvements will involve imaging larger FOVs and moving toward faster image acquisition. Currently, the presented UV-PARS and CLSM system can produce 0.5 mm × 0.5 mm FOV images in around 45 s. However, faster scanning will be needed for practical imaging of larger FOVs.…”

Section: Confocal Laser Scanning Microscopy and Uv-parsmentioning

confidence: 99%

Multimodal 3D photoacoustic remote sensing and confocal fluorescence microscopy imaging

et al. 2021

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. Significance: Complementary absorption and fluorescence contrast could prove useful for a wide range of biomedical applications. However, current absorption-based photoacoustic microscopy systems require the ultrasound transducers to physically touch the samples, thereby increasing contamination and limiting strong optical focusing in reflection mode. Aim : We sought to develop an all-optical system for imaging cells and tissues using the three combined imaging modalities: photoacoustic remote sensing (PARS), epifluorescence, and confocal laser scanning microscopy (CLSM). Approach: A PARS subsystem with ultraviolet excitation was used to obtain label-free absorption-contrast images of nucleic acids in ex vivo tissue samples. Co-integrated epifluorescence and CLSM subsystems were used to verify the 2D and 3D nuclei distribution. Results : Complementary absorption and fluorescence contrast were demonstrated in phantom imaging experiments and subsequent cell and tissue imaging experiments. Lateral and axial resolution of ultraviolet-PARS (UV-PARS) is shown to be 0.39 and , respectively, with 266-nm light. CLSM lateral and axial resolution was measured as 0.97 and , respectively. This resolution is sufficient to image individual cell layers with fine optical sectioning. UV-PARS images of cell nuclei are validated in thick tissue using CLSM. Conclusions : Multimodal absorption and fluorescence contrast are obtained with a non-contact all-optical microscopy system for the first time and utilized to obtain images of cells and tissues with subcellular resolution.

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Metabolic imaging by simultaneous FLIM of NAD(P)H and FAD

Cited by 9 publications

References 15 publications

Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications

Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications

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

Multimodal 3D photoacoustic remote sensing and confocal fluorescence microscopy imaging

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