Imaging Fibrosis and Separating Collagens using Second Harmonic Generation and Phasor Approach to Fluorescence Lifetime Imaging

Ranjit, Suman; Dvornikov, Alexander; Stakic, Milka; Hong, Suk Hyun; Levi, Moshe; Evans, Ronald M.; Gratton, Enrico

doi:10.1038/srep13378

Cited by 88 publications

(95 citation statements)

References 36 publications

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“…Collagens of different types and sources exhibit fluorescence emission which varies in spectral band shape, position of maximum and fluorescence lifetimes 44–47, 69, 70 . Ranjit et al .…”

Section: Resultsmentioning

confidence: 99%

Two-photon autofluorescence lifetime imaging of human skin papillary dermis in vivo: assessment of blood capillaries and structural proteins localization

Shirshin

Gurfinkel

Priezzhev

et al. 2017

Sci Rep

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The papillary dermis of human skin is responsible for its biomechanical properties and for supply of epidermis with chemicals. Dermis is mainly composed of structural protein molecules, including collagen and elastin, and contains blood capillaries. Connective tissue diseases, as well as cardiovascular complications have manifestations on the molecular level in the papillary dermis (e.g. alteration of collagen I and III content) and in the capillary structure. In this paper we assessed the molecular structure of internal and external regions of skin capillaries using two-photon fluorescence lifetime imaging (FLIM) of endogenous compounds. It was shown that the capillaries are characterized by a fast fluorescence decay, which is originated from red blood cells and blood plasma. Using the second harmonic generation signal, FLIM segmentation was performed, which provided for spatial localization and fluorescence decay parameters distribution of collagen I and elastin in the dermal papillae. It was demonstrated that the lifetime distribution was different for the inner area of dermal papillae around the capillary loop that was suggested to be due to collagen III. Hence, we propose a generalized approach to two-photon imaging of the papillary dermis components, which extends the capabilities of this technique in skin diagnosis.

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“…Collagens of different types and sources exhibit fluorescence emission which varies in spectral band shape, position of maximum and fluorescence lifetimes 44–47, 69, 70 . Ranjit et al .…”

Section: Resultsmentioning

confidence: 99%

Two-photon autofluorescence lifetime imaging of human skin papillary dermis in vivo: assessment of blood capillaries and structural proteins localization

Shirshin

Gurfinkel

Priezzhev

et al. 2017

Sci Rep

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“…Other imaging advancements that may benefit the analysis of in vitro fibrosis platforms include a novel pairing of SHG with fluorescence lifetime imaging microscopy (FLIM), which was able to distinguish between collagen type I and type III, the two main ECM markers of fibrosis [41••]. Meanwhile, the combination of SHG with spectral lifetime imaging microscopy (SLIM) provides the ability to image real-time changes in cellular metabolites in the context of ECM alterations [42,43].…”

Section: New Directions For the Evaluation Of In Vitro Fibrosis Platfmentioning

confidence: 99%

Engineering approaches to study fibrosis in 3-D in vitro systems

Porras

Hutson

Berger

et al. 2016

Current Opinion in Biotechnology

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Fibrotic diseases occur in virtually every tissue of the body and are a major cause of mortality, yet they remain largely untreatable and poorly understood on a mechanistic level. The development of anti-fibrotic agents has been hampered, in part, by the insufficient fibrosis biomimicry provided by traditional in vitro platforms. This review focuses on recent advancements toward creating 3-D platforms that mimic key features of fibrosis, as well as the application of novel imaging and sensor techniques to analyze dynamic extracellular matrix remodeling. Several opportunities are highlighted to apply new tools from the fields of biomaterials, imaging, and systems biology to yield pathophysiologically-relevant in vitro platforms that improve our understanding of fibrosis and may enable identification of potential treatment targets.

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“…It is well established that retinoids play a crucial role in stem cell differentiation and embryo development 18,19 and their concentration and gradients have been detected in vivo during zebrafish development 9,20 . Other intrinsic fluorophores such as porphyrin, collagen, elastin, keratin, lipofuscin and melanins have relevant roles in several physiological processes and diseases such as cancer and multiphoton microscopy can provide functional imaging in a non-invasive way [21][22][23][24] . While several advanced approaches have been developed to improve identification and quantification of endogenous fluorophores based on fluorescence lifetime imaging 9 , spectrally resolved detection 23,25 , and spectrally resolved FLIM [26][27][28] , multiphoton microscopy of endogenous fluorophores and its applications to live imaging are still limited by acquisition speed and challenging multicolor excitation.…”

mentioning

confidence: 99%

Multicolor Two-photon Imaging of Endogenous Fluorophores in Living Tissues by Wavelength Mixing

Stringari

Abdeladim

Mahou

et al. 2018

Biophotonics Congress: Biomedical Optics Congress 2018 (Microscopy/Translational/Brain/Ots)

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Two-photon imaging of endogenous fluorescence can provide physiological and metabolic information from intact tissues. However, simultaneous imaging of multiple intrinsic fluorophores, such as nicotinamide adenine dinucleotide(phosphate) (NAD(P)H), flavin adenine dinucleotide (FAD) and retinoids in living systems is generally hampered by sequential multi-wavelength excitation resulting in motion artifacts. Here, we report on efficient and simultaneous multicolor two-photon excitation of endogenous fluorophores with absorption spectra spanning the 750-1040 nm range, using wavelength mixing. By using two synchronized pulse trains at 760 and 1041 nm, an additional equivalent two-photon excitation wavelength at 879 nm is generated, and achieves simultaneous excitation of blue, green and red intrinsic fluorophores. This method permits an efficient simultaneous imaging of the metabolic coenzymes NADH and FAD to be implemented with perfect image coregistration, overcoming the difficulties associated with differences in absorption spectra and disparity in concentration. We demonstrate ratiometric redox imaging free of motion artifacts and simultaneous two-photon fluorescence lifetime imaging (FLIM) of NADH and FAD in living tissues. The lifetime gradients of NADH and FAD associated with different cellular metabolic and differentiation states in reconstructed human skin and in the germline of live C. Elegans are thus simultaneously measured. Finally, we present multicolor imaging of endogenous fluorophores and second harmonic generation (SHG) signals during the early stages of Zebrafish embryo development, evidencing fluorescence spectral changes associated with development.Multiphoton microscopy is a powerful tool for label-free and non-invasive functional imaging in small organisms and tissues 1, 2 . Pulsed near infrared excitation light allows in-depth imaging based on contrasts such as endogenous fluorescence 2 , second harmonic generation (SHG) 3 and third harmonic generation (THG) 4 . Endogenous fluorescence in living tissues arises from several intrinsic biomarkers that play important roles in physiological processes 2 . The primary intracellular sources are NAD(P)H and FAD, the two major cofactors of redox reactions in the cell and central regulators of energy production and metabolism 5,6 . Their fluorescence reports on the metabolic activity of cells allowing tissue physiology and processes such as stem cell differentiation, cancer development and neurodegenerative diseases to being non-invasively monitored [7][8][9][10][11][12] . The fluorescence lifetimes of NADH and FAD are different upon binding to the protein during the electron transport chain. FLIM provides sensitive measurements of the free and protein-bound NAD(P)H ratio and of the redox states (NADH/NAD + ) of cells, and can be used to distinguish glycolytic and oxidative phosphorylation metabolic states [13][14][15][16][17] . Monitoring lifetime of free and protein-bound FAD has also been exploited to quantify redox ratio FAD/FADH 2 , and used as a b...

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Imaging Fibrosis and Separating Collagens using Second Harmonic Generation and Phasor Approach to Fluorescence Lifetime Imaging

Cited by 88 publications

References 36 publications

Two-photon autofluorescence lifetime imaging of human skin papillary dermis in vivo: assessment of blood capillaries and structural proteins localization

Two-photon autofluorescence lifetime imaging of human skin papillary dermis in vivo: assessment of blood capillaries and structural proteins localization

Engineering approaches to study fibrosis in 3-D in vitro systems

Multicolor Two-photon Imaging of Endogenous Fluorophores in Living Tissues by Wavelength Mixing

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