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

Cao, Ruofan; Wallrabe, Horst; Periasamy, Ammasi

doi:10.1117/1.jbo.25.1.014510

Cited by 42 publications

(35 citation statements)

References 55 publications

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“…In Figure 7, the decreasing trend due to self-quenching was successfully observed. The detected value of the autofluorescence lifetime of FAD was within the range of literature values (2.7-2.9 ns) [3,25,26].…”

Section: Bio-mimicking Phantomsupporting

confidence: 86%

Development of a Robust Autofluorescence Lifetime Sensing Method for Use in an Endoscopic Application

Ito

Hashimoto

Taguchi

2020

Sensors

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Endoscopic autofluorescence lifetime imaging is a promising technique for making quantitative and non-invasive diagnoses of abnormal tissue. However, motion artifacts caused by vibration in the direction perpendicular to the tissue surface in a body makes clinical diagnosis difficult. Thus, this paper proposes a robust autofluorescence lifetime sensing technique with a lens tracking system based on a laser beam spot analysis. Our optical setup can be easily mounted on the head of an endoscope. The variation in distance between the optical system and the target surface is tracked by the change in the spot size of the laser beam captured by the camera, and the lens actuator is feedback-controlled to suppress motion artifacts. The experimental results show that, when using a lens tracking system, the standard deviation of fluorescence lifetime is dramatically reduced. Furthermore, the validity of the proposed method is experimentally confirmed by using a bio-mimicking phantom that replicates the shape, optical parameters, and chemical component distribution of the cancerous tissue.

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Section: Bio-mimicking Phantomsupporting

confidence: 86%

Development of a Robust Autofluorescence Lifetime Sensing Method for Use in an Endoscopic Application

Ito

Hashimoto

Taguchi

2020

Sensors

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“…For fluorescence excited at 402 nm a rather high fluorescence intensity of the cells both in control and UV irradiated samples was observed. In the control sample, the fluorescence signal was mainly located in the region of the cytoplasm, where at this excitation wavelength NAD(P)H and flavins can be readily excited [4,34], while nuclei had a negligible fluorescence signal (Figure 4a). Irradiated sample demonstrated radical changes of fluorescence signal.…”

Section: Uv-irradiation Induced Changes In Af Of Keratinocytes: Flim and Confocal Microscopy Resultsmentioning

confidence: 99%

The Oxidation-Induced Autofluorescence Hypothesis: Red Edge Excitation and Implications for Metabolic Imaging

et al. 2020

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Endogenous autofluorescence of biological tissues is an important source of information for biomedical diagnostics. Despite the molecular complexity of biological tissues, the list of commonly known fluorophores is strictly limited. Still, the question of molecular sources of the red and near-infrared excited autofluorescence remains open. In this work we demonstrated that the oxidation products of organic components (lipids, proteins, amino acids, etc.) can serve as the molecular source of such red and near-infrared excited autofluorescence. Using model solutions and cell systems (human keratinocytes) under oxidative stress induced by UV irradiation we demonstrated that oxidation products can contribute significantly to the autofluorescence signal of biological systems in the entire visible range of the spectrum, even at the emission and excitation wavelengths higher than 650 nm. The obtained results suggest the principal possibility to explain the red fluorescence excitation in a large class of biosystems—aggregates of proteins and peptides, cells and tissues—by the impact of oxidation products, since oxidation products are inevitably presented in the tissue. The observed fluorescence signal with broad excitation originated from oxidation products may also lead to the alteration of metabolic imaging results and has to be taken into account.

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“…6 Furthermore, one wavelength has been used to excite the two intrinsic fluorophores, NAD(P)H and FAD. 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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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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Multiphoton FLIM imaging of NAD(P)H and FAD with one excitation wavelength

Cited by 42 publications

References 55 publications

Development of a Robust Autofluorescence Lifetime Sensing Method for Use in an Endoscopic Application

Development of a Robust Autofluorescence Lifetime Sensing Method for Use in an Endoscopic Application

The Oxidation-Induced Autofluorescence Hypothesis: Red Edge Excitation and Implications for Metabolic Imaging

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

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