Quenched coumarin derivatives as fluorescence lifetime phantoms for NADH and FAD

Abstract

Two-photon fluorescence lifetime imaging is a versatile laboratory technique in the field of biophotonics and its importance is also growing in the field of in vivo diagnostics for medical purposes. After years of experience in dermatology, endoscopic implementations of the technique are now posing new technical challenges. To develop, test, and compare instrumental solutions for this purpose suitable reference samples have been devised and tested. These reference samples can serve as reliable NADH-and FAD-mimicking optical phantoms for 2-photon fluorescence lifetime imaging, as they can be prepared relatively easily with reproducible and stable characteristics for this quite relevant diagnostic technique. The reference samples (mixtures of coumarin 1 and coumarin 6 in ethanol with suitable amounts of 4-hydroxy-TEMPO) have been tuned to exhibit spectral and temporal fluorescence characteristics very similar to those of NADH and FAD, the two molecules most frequently utilized to characterize cell metabolism.

Light-sheet autofluorescence lifetime imaging with a single-photon avalanche diode array

“…(b) The aggregated decay histogram of all pixels after timing skew correction was fit to a single-exponential decay model using an iterative reconvolution algorithm that performs a least-squares minimization of the residuals. A fluorescence lifetime of 2.51 ns is estimated which agrees with reported published values [62][63][64].…”

Development of a microstructured tissue phantom with adaptable optical properties for use with microscopes and fluorescence lifetime imaging systems

“…Figure 4 shows a comparison of the beads two‐photon fluorescence spectra with that of NADH (N8129, Sigma‐Alrich GmbH) and FAD (ALX‐480‐084‐M050, Enzo Life Sciences GmbH) solutions in 7.5 pH TRIS buffer (BU‐125S, TRIS 1 M pH 7.5, Jena Bioscience GmbH). The two‐photon spectra were recorded with a multiphoton FLIM microscope described previously 21 …”

“…The two-photon spectra were recorded with a multiphoton FLIM microscope described previously. 21 To fix the fluorescent beads on the steps, they are dispersed (1 µl of each fluorescent beads suspension) in Approximately 10 µl of the mixture is filled into the structures using a pipette, distributed to cover the whole structure and a small rim on the surface of the glass slide. The filled structure was then dried on a hot plate (RCT basic, IKA Labortechnik) at ≈60°C and subsequently wrapped in aluminium foil for light protection until further use.…”

“…Also, the emission spectra of NADH and FAD are sufficiently similar to those of their chosen artificial phantom analogues. 21 Literature on optical properties of human oral tissue in the relevant wavelength range proved to be sparse. Data was found for the human maxillary sinus as a graph 31 and for different oral and colon tissues at 855 and 1064 nm Values of μ ′ s at the relevant wavelengths 780 and 880 nm were therefore derived from Bashkatov et al 31 with a digitizer tool 33 and used for calculating the composition of the scattering suspension.…”

Strategic design of a 2,6-disubstituted pyridine-based probe having hard-soft centers: responsive divergence from one core

“…32 The quenching of the fluorescence intensity upon analyte addition is usually associated with the decrease in fluorescence lifetime as well; however, an increase in lifetimes can also be observed in many cases. 33,34 In our case, the pulse width of the Instrument Response Function (IRF) used for these studies is 0.9 nm, and with this pulse width the changes in the fluorescence lifetimes may be considered insignificant.…”