FLIm and Raman Spectroscopy for Investigating Biochemical Changes of Bovine Pericardium upon Genipin Cross-Linking

Biomaterials used in tissue engineering and regenerative medicine applications benefit from longitudinal monitoring in a non-destructive manner. Label-free imaging based on fluorescence lifetime imaging (FLIm) and Raman spectroscopy were used to monitor the degree of genipin (GE) cross-linking of antigen-removed bovine pericardium (ARBP) at three incubation time points (0.5, 1.0, and 2.5 h). Fluorescence lifetime decreased and the emission spectrum redshifted compared to that of uncross-linked ARBP. The Raman signature of GE-ARBP was resonance-enhanced due to the GE cross-linker that generated new Raman bands at 1165, 1326, 1350, 1380, 1402, 1470, 1506, 1535, 1574, 1630, 1728, and 1741 cm−1. These were validated through density functional theory calculations as cross-linker-specific bands. A multivariate multiple regression model was developed to enhance the biochemical specificity of FLIm parameters fluorescence intensity ratio (R2 = 0.92) and lifetime (R2 = 0.94)) with Raman spectral results. FLIm and Raman spectroscopy detected biochemical changes occurring in the collagenous tissue during the cross-linking process that were characterized by the formation of a blue pigment which affected the tissue fluorescence and scattering properties. In conclusion, FLIm parameters and Raman spectroscopy were used to monitor the degree of cross-linking non-destructively.

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Study of bovine pericardium biochemical and biomechanical properties during collagenase degradation using fluorescence lifetime imaging (Conference Presentation)

Diagnostic and Therapeutic Applications of Light in Cardiology 2018 ◽

10.1117/12.2287487 ◽

2018 ◽

Author(s):

Cai Li ◽

Jeny Shklover ◽

Mojtaba Parvizi ◽

Benjamin E. Sherlock ◽

Alba Alfonso García ◽

...

Keyword(s):

Fluorescence Lifetime ◽

Biomechanical Properties ◽

Bovine Pericardium ◽

Fluorescence Lifetime Imaging ◽

Lifetime Imaging

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Raman Imaging and Fluorescence Lifetime Imaging Microscopy for Diagnosis of Cancer State and Metabolic Monitoring

Cancers ◽

10.3390/cancers13225682 ◽

2021 ◽

Vol 13 (22) ◽

pp. 5682

Author(s):

Lucas Becker ◽

Nicole Janssen ◽

Shannon L. Layland ◽

Thomas E. Mürdter ◽

Anne T. Nies ◽

...

Keyword(s):

Raman Spectroscopy ◽

Fluorescence Lifetime ◽

Drug Efficacy ◽

Raman Microspectroscopy ◽

Fluorescence Lifetime Imaging Microscopy ◽

Fluorescence Lifetime Imaging ◽

Lifetime Imaging ◽

Non Invasive

Hurdles for effective tumor therapy are delayed detection and limited effectiveness of systemic drug therapies by patient-specific multidrug resistance. Non-invasive bioimaging tools such as fluorescence lifetime imaging microscopy (FLIM) and Raman-microspectroscopy have evolved over the last decade, providing the potential to be translated into clinics for early-stage disease detection, in vitro drug screening, and drug efficacy studies in personalized medicine. Accessing tissue- and cell-specific spectral signatures, Raman microspectroscopy has emerged as a diagnostic tool to identify precancerous lesions, cancer stages, or cell malignancy. In vivo Raman measurements have been enabled by recent technological advances in Raman endoscopy and signal-enhancing setups such as coherent anti-stokes Raman spectroscopy or surface-enhanced Raman spectroscopy. FLIM enables in situ investigations of metabolic processes such as glycolysis, oxidative stress, or mitochondrial activity by using the autofluorescence of co-enzymes NADH and FAD, which are associated with intrinsic proteins as a direct measure of tumor metabolism, cell death stages and drug efficacy. The combination of non-invasive and molecular-sensitive in situ techniques and advanced 3D tumor models such as patient-derived organoids or microtumors allows the recapitulation of tumor physiology and metabolism in vitro and facilitates the screening for patient-individualized drug treatment options.

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