Fluorescence lifetime imaging in biosciences: technologies and applications

Abstract

The biosciences require the development of methods, which allow a non-invasive and rapid investigation of biological systems. In this frame high-end imaging techniques allow an intravital microscopy in real-time, providing information on a molecular basis. Far-field fluorescence imaging techniques are some of the most adequate methods for such investigations. However, there are great differences between the common fluorescence imaging techniques, i.e. wide-field, confocal one-photon and two-photon microscopy, respectively, as far as their applicability in diverse bioscientific research areas is concerned. In the first part of this work, we briefly compare these techniques. Standard methods used in the biosciences, i.e. steady-state techniques based on the analysis of the total fluorescence signal originating from the sample, can successfully be employed in the study of cell, tissue and organ morphology as well as in monitoring the macroscopic tissue function. However, they are mostly inadequate for the quantitative investigation of the cellular function at molecular level. The intrinsic disadvantages of steady-state techniques are counteracted by using time-resolved techniques. Among these the fluorescence lifetime imaging (FLIM) is currently the most common. Different FLIM principles as well as applications of particular relevance for the biosciences, especially for fast intravital studies are discussed in this work.

Can new optical techniques for in vivo imaging and flow cytometry of the microcirculation benefit sickle cell disease research?

“…As an example, it is known that sickling of red cells can occur at high altitudes and in parts of the body where pH is low and dehydration contributes to an increase in blood viscosity (78). FLIM is capable of monitoring intracellular changes which are dependent on these factors (41). The oxygen saturation of the red blood cells plays an important role in the adhesion and gelation process in SCD.…”

“…Fluorescence lifetime imaging microscopy (FLIM) measures the time a molecule spends in an excited state and can be used to discriminate between multiple fluorophores. Measurement of the lifetime of exogenous and endogenous fluorophores within a cell strongly depends on the cellular environment, e.g., pH, viscosity, ion concentration (41). It can be used as a stand‐alone technique or in combination with others, such as multiphoton microscopy.…”

Two-photon excitation fluorescence lifetime imaging microscopy: A promising diagnostic tool for digestive tract tumors

“…10 Physically, it is de¯ned as the time that the°uorescence intensity decays to 1/e of its maximum. 12,15,24 For a given°u orophore, this process can be described as follows:…”

“…At least, the proportion of spontaneous emission pathway k f should be included. 12,15,24 There are abundant endogenous°uorophores in biological systems, such as NADH, oxidized°avin adenine dinucleotide (FAD), melanin, and tryptophan, whereas biomedical studies always require introducing exogenous°uorescent markers into cells or tissues. Therefore, the°uorescence decay process in biological samples generally resembles a multiexponential decay:…”

“…which is a linear combination of multiple single exponential decays, respectively, corresponding to various endogenous or exogenous°uorophores, where i is the°uorescence lifetime of the ith°u orophore and a i is its weight factor. 24…”