Improved Routine Bio-Medical and Bio-Analytical Online Fluorescence Measurements Using Fluorescence Lifetime Resolution

Abstract

Fluorescence techniques are widely used as sensitive detection methods in bio-analytics. The use of the bio-physical parameter fluorescence lifetime additional to the spectral characteristics of fluorescence has the potential to improve fluorescence-related detection methods in terms of selectivity in signal recognition, robustness against disturbing influences, and the accessibility of novel bio-chemical process parameters. This article describes the technical set up of a time-resolving instrument with either a fixed time-gated detection principle for improved evaluation of tissue metabolism by an online monitoring of the tissue autofluorescence or a direct fluorescence lifetime detection principle for lifetime-based fluorescent assays.

Autofluorescence spectroscopy and imaging: a tool for biomedical research and diagnosis

“… 56-58 In this relationship, technical set-ups for direct lifetime detection and monitoring of tissue AF were proposed, aiming to improve routine biomedical and bio-analytical online analyses. 59 Recently, a phasor approach has also been developed allowing an easier fluorescence lifetime data processing and interpretation, through a non-invasive, label-free, fit-free lifetime imaging microscopy technique, escaping the problems of exponential analysis to assess the presence of multiple fluorescing species. A graphical global view is given as an image, each pixel contributing as a point to the plot.…”

State-of-the Art Comparability of Corrected Emission Spectra.1. Spectral Correction with Physical Transfer Standards and Spectral Fluorescence Standards by Expert Laboratories

“…At present, the only fully standardized photoluminescence measuring techniques are colorimetry or surface fluorescence , and, in part, flow cytometry, , but new initiatives to improve the comparability and measurement uncertainties of photoluminescence measurements are in progress. Aside from the increasing number of publications and workshops dedicated to these topics, ,,,,,− ongoing activities include efforts by the International Union of Pure and Applied Chemistry (IUPAC; e.g., Project 2004-021-1-300) to develop technical notes for the characterization and validation of instruments and methods and a new guide from ASTM International, formerly known as the American Society for Testing and Materials, for fluorescence instrument qualification . Moreover, only recently, a first multinational, multilaboratory comparison was performed evaluating fluorescence lifetime standards for time and frequency domain fluorescence spectroscopy as well as a multinational, multilaboratory comparison on fluorescence immunoassays .…”

Fluorescent chameleon labels for bioconjugation and imaging of proteins, nucleic acids, biogenic amines and surface amino groups. a review

“…Bleaching and fluorescence decay times are hardly affected if the probe is internalized into cells. The sensitivity of the fluorescence decay time of proteins labelled Py-1 (=Chromeo P503) to interactions with other proteins was used in new timegated detection principle for improved evaluation of tissue metabolism and in affinity interactions [65]. The changes observed in the decay time of Py-labelled biomolecules on interaction with other species make such probes also viable candidates for use in fluorescence lifetime imaging (FLIM) and in time-correlated single photon counting [66,67].…”