Steady-state and time-resolved two-photon fluorescence microscopy: a versatile tool for probing cellular environment and function

Abstract

In the last decade, the two-photon fluorescence laser-scanning microscopy (TPLSM) has become an indispensable tool for the bioscientific and biomedical research. TPLSM techniques as well as their applications are currently experiencing a dramatic evolution and represent the focus of many biophysical research projects. In this work, we compare in detail two steady-state TPLSM techniques, i.e. single-beam scanning microscopy combined with point-detection (SB-PMT) and multi-beam scanning microscopy combined with synchronous detection (MB-CCD), as far as their technical characteristics relevant for the bioscientific research are concerned, i.e. optical performance and imaging speed. We demonstrate that the SB-PMT technique is more adequate for deep-tissue imaging (few 100 µm depth) than the MB-CCD technique, whereas only the MB-CCD technique enables high-speed imaging for characterizing the dynamics of fast biological phenomena. Novel applications of these techniques are additionally discussed. Moreover, we employ a time-resolved TPLSM technique, i.e. biexponential fluorescence lifetime imaging based on the cellular fluorescence of the nicotinamide pyridine dinucleotides NADH and NADPH, which allows us to probe for the first time the redox cellular metabolism of MIN6 cells (mutated insulin producing pancreatic β-cells) as well as to show the potential of this method for the specific and dynamic investigation of NADH-and NADPH-dependent cellular processes.

Applications of the time-resolved two-colour two-photon excitation of UV fluorophores using femtosecond laser pulses

“…Multi-photon excitation of fluorescence has long been used in molecular spectroscopy [5], but it was the invention of the two-photon microscope (TPM) in the early 1990s that introduced this technique to fluorescence microscopy [6]. It is superior for deep tissue imaging as well as for high-speed in vivo imaging [7]. The key to its success is the possibility of providing detailed and minimal invasive insight into the function of biological systems under genuine conditions.…”

Luminescence lifetime imaging of three-dimensional biological objects

“…1A). FLI methodologies can be integrated into traditional fluorescence imaging approaches, including widefield, confocal, multiphoton, super-resolution and light-sheet microscopy, as well as macroimaging, fluorescence molecular tomography (FMT) and other optical imaging-based approaches (Ishikawa-Ankerhold et al, 2012;Meyer-Almes, 2017;Sarder et al, 2015;Denicke et al, 2007;Becker et al, 2017;Le Marois and Suhling, 2017;Datta et al, 2020;Poudel et al, 2020;Wang et al, 1992). FLI can also be combined with more specialized approaches such as Förster resonance energy transfer (FRET), which enables sensing of nanoscale interactions.…”

Photophysical characterization and flow cytometry applications of cholylamidofluorescein, a fluorescent bile acid scaffold

“…In fact, lifetime determinations in cellular systems have been successfully applied to time-resolved fluorescence microscopy. [25][26][27][28][29][30] Moreover, triplet excited states can also be investigated in complex biological media, including cells; their longer lifetimes provide a wider dynamic range, which could be potentially exploited for the development of new analytical tools. 31 Hence, it makes sense to perform a full photophysical characterization of the fluorescent derivatives of bile acids employed for uptake studies.…”