Fluorescence lifetime imaging with near-infrared dyes Fluoreszenz-Lifetime-Imaging mit Nahinfrarot-Farbstoffen (original) (raw)
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Fluorescence lifetime imaging with near-infrared dyes
SPIE Proceedings, 2013
Near-infrared (NIR) dyes are used as fluorescence markers in small animal imaging and in diffuse optical tomography. In these applications it is important to know whether the dyes bind to proteins or to other tissue constituents, and whether their fluorescence lifetimes depend on the targets they bind to. Unfortunately, neither the optical beam paths nor the detectors of commonly used in confocal and multiphoton laser scanning microscopes (LSMs) directly allow for excitation and detection of NIR fluorescence. This paper presents three ways of adapting existing LSMs with time-correlated single photon counting (TCSPC) fluorescence lifetime imaging (FLIM) systems for NIR FLIM: 1) confocal systems with wideband beamsplitters and diode laser excitation, 2) confocal systems with wideband beamsplitters and onephoton excitation by titanium-sapphire lasers, and 3) twophoton systems with optical parametric oscillator (OPO) excitation and non-descanned detection. A number of NIR dyes are tested in biological tissue. All of them show clear lifetime changes depending on the tissue structures they are bound to. We therefore believe that NIR FLIM can deliver supplementary information about the tissue composition and on local biochemical parameters.
Fluorescence lifetime imaging microscopy using near-infrared contrast agents
Journal of Microscopy, 2012
Although single-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to image molecular processes using a wide range of excitation wavelengths, the captured emission of this technique is confined to the visible spectrum. Here, we explore the feasibility of utilizing near-infrared (NIR) fluorescent molecular probes with emission >700 nm for FLIM of live cells. The confocal microscope is equipped with a 785 nm laser diode, a red-enhanced photomultiplier tube, and a timecorrelated single photon counting card. We demonstrate that our system reports the lifetime distributions of NIR fluorescent dyes, cypate and DTTCI, in cells. In cells labelled separately or jointly with these dyes, NIR FLIM successfully distinguishes their lifetimes, providing a method to sort different cell populations. In addition, lifetime distributions of cells coincubated with these dyes allow estimate of the dyes' relative concentrations in complex cellular microenvironments. With the heightened interest in fluorescence lifetime-based small animal imaging using NIR fluorophores, this technique further serves as a bridge between in vitro spectroscopic characterization of new fluorophore lifetimes and in vivo tissue imaging.
In Vivo Imaging with Near-infrared Fluorescence Lifetime Contrast
Proceedings of SPIE - The International Society for Optical Engineering
Fluorescence imaging is a mainstay of biomedical research, allowing detection of molecular events in both fixed and living cells, tissues and whole animals. Such high resolution fluorescence imaging is hampered by unwanted signal from intrinsic background fluorescence and scattered light. The signal to background ratio can be improved by using extrinsic contrast agents and greatly enhanced by multispectral imaging methods. Unfortunately, these methods are insufficient for deep tissue imaging where high contrast and speedy acquisition are necessary. Fluorescence lifetime (FLT) is an inherent characteristic of each fluorescent species that can be independent of intensity and spectral properties. Accordingly, FLT-based detection provides an additional contrast mechanism to optical measurements. This contrast is particularly important in the near-infrared (NIR) due to relative transparency of tissue as well as the broad absorption and emission spectra of dyes that are active in this reg...
1 Brief history of fluorescence lifetime imaging
Multiphoton Microscopy and Fluorescence Lifetime Imaging, 2018
This review gives an overview of the history of fluorescence lifetime imaging (FLIM) in life sciences. FLIM microscopy based on an ultrafast laser scanning microscope and time-correlated single photon counting (TCSPC) was introduced in Jena/ Germany in 1988/89. FLIM images of porphyrin-labeled live cells and live mice were taken with an unique ZEISS confocal picosecond laser microscope. Five years later, the first in vivo FLIM on human volunteers started with time-gated cameras to detect dental caries based on one-photon wide-field pulsed laser excitation of autofluorescent bacteria. Another five years later, two-photon FLIM of autofluorescent skin was performed on a volunteer with a lab microscope in the frequency domain. The first clinical non-invasive optical, two-photon 3D FLIM biopsies were obtained fifteen years ago in patients with dermatological disorders using a certified clinical multiphoton tomograph based on a tunable femtosecond titanium:sapphire laser and TCSPC. A current major FLIM application in cell biology is the study of protein-protein interactions in transfected cells by FLIM-FRET microscopy. Clinical FLIM applications are still on a research level and include preliminary studies on (i) one-photon FLIM autofluorescence microscopy of patients with ocular diseases using picosecond laser diodes, (ii) time-gated imaging in brain surgery using a nanosecond nitrogen laser, and (iii) two-photon clinical FLIM tomography of patients with skin cancer and brain tumors with near-infrared femtosecond lasers and TCSPC.
Basis: Brief history of fluorescence lifetime imaging
Multiphoton Microscopy and Fluorescence Lifetime Imaging: Applications in Biology and Medicine, 2018
This review gives an overview of the history of fluorescence lifetime imaging (FLIM) in life sciences. FLIM microscopy based on an ultrafast laser scanning microscope and time-correlated single photon counting (TCSPC) was introduced in Jena/ Germany in 1988/89. FLIM images of porphyrin-labeled live cells and live mice were taken with an unique ZEISS confocal picosecond laser microscope. Five years later, the first in vivo FLIM on human volunteers started with time-gated cameras to detect dental caries based on one-photon wide-field pulsed laser excitation of autofluorescent bacteria. Another five years later, two-photon FLIM of autofluorescent skin was performed on a volunteer with a lab microscope in the frequency domain. The first clinical non-invasive optical, two-photon 3D FLIM biopsies were obtained fifteen years ago in patients with dermatological disorders using a certified clinical multiphoton tomograph based on a tunable femtosecond titanium:sapphire laser and TCSPC. A current major FLIM application in cell biology is the study of protein-protein interactions in transfected cells by FLIM-FRET microscopy. Clinical FLIM applications are still on a research level and include preliminary studies on (i) one-photon FLIM autofluorescence microscopy of patients with ocular diseases using picosecond laser diodes, (ii) time-gated imaging in brain surgery using a nanosecond nitrogen laser, and (iii) two-photon clinical FLIM tomography of patients with skin cancer and brain tumors with near-infrared femtosecond lasers and TCSPC.
Fluorescence lifetime imaging by multi-dimensional time correlated single photon counting
Medical Photonics, 2015
Fluorescence lifetime imaging (FLIM) techniques for biological imaging have to unite several features, such as high photon efficiency, high lifetime accuracy, resolution of multi-exponential decay profiles, simultaneous recording in several wavelength intervals and optical sectioning capability. The combination of multi-dimensional time-correlated single photon counting (TCSPC) with confocal or two-photon laser scanning meets these requirements almost ideally. Multi-dimensional TCSPC is based on the excitation of the sample by a high repetition rate laser and the detection of single photons of the fluorescence signal. Each photon is characterised by its arrival time with respect to the laser pulse and the coordinates of the laser beam in the scanning area. The recording process builds up a photon distribution over these parameters. The result can be interpreted as an array of pixels, each containing a full fluorescence decay curve. More parameters can be added to the photon distribution, such as the wavelength of the photons, the time from a stimulation of the sample, or the time with respect to an additional modulation of the laser. In this review, the application of the technique will be described for the measurement of molecular environment parameters within a sample, protein interaction experiments by Förster resonance energy transfer (FRET), autofluorescence measurements of cells and tissue, and in-vivo imaging of human skin and the fundus of the human eye.
Fluorescence lifetime imaging system for in vivo studies
Molecular imaging
In this article, a fluorescence lifetime imaging system for small animals is presented. Data were collected by scanning a region of interest with a measurement head, a linear fiber array with fixed separations between a single source fiber and several detection fibers. The goal was to localize tumors and monitor their progression using specific fluorescent markers. We chose a near-infrared contrast agent, Alexa Fluor 750 (Invitrogen Corp., Carlsbad, CA). Preliminary results show that the fluorescence lifetime for this dye was sensitive to the immediate environment of the fluorophore (in particular, pH), making it a promising candidate for reporting physiologic changes around a fluorophore. To quantify the intrinsic lifetime of deeply embedded fluorophores, we performed phantom experiments to investigate the contribution of photon migration effects on observed lifetime by calculating the fluorescence intensity decay time. A previously proposed theoretical model of migration, based on...
Fluorescence lifetime imaging in biosciences: technologies and applications
Frontiers of Physics in China, 2008
The biosciences require the development of methods that allow a non-invasive and rapid investigation of biological systems. In this aspect, high-end imaging techniques allow 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, 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 the molecular level. The intrinsic disadvantages of steadystate techniques are countered by using time-resolved techniques. Among these 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
Combined fluorescence and phosphorescence lifetime imaging
APPLIED PHYSICS LETTERS, 2016
We present a lifetime imaging technique that simultaneously records the fluorescence and phosphorescence lifetime images in confocal laser scanning systems. It is based on modulating a high-frequency pulsed laser synchronously with the pixel clock of the scanner, and recording the fluorescence and phosphorescence signals by multidimensional time-correlated single photon counting board. We demonstrate our technique on the recording of the fluorescence/phosphorescence lifetime images of human embryonic kidney cells at different environmental conditions.
Biomedical Optics Express
We demonstrate for the first time that an ultra-broadband 7 femtosecond (fs) fewcycle laser can be used for multicolor nonlinear imaging in a single channel detection geometry, when employing a time-resolved fluorescence detection scheme. On a multichromophore-labelled cell sample we show that the few-cycle laser can efficiently excite the multiple chromophores over a >400 nm two-photon absorption range. By combining the fewcycle laser excitation with time-correlated single-photon counting (TCSPC) detection to record two-photon fluorescence lifetime imaging microscopy (FLIM) images, the localization of different chromophores in the cell can be identified based on their fluorescence decay properties. The novel SyncRGB-FLIM multi-color bioimaging technique opens the possibility of real-time protein-protein interaction studies, where its single-scan operation translates into reduced laser exposure of the sample, resulting in more photoprotective conditions for biological specimens.