Fluorescence lifetime imaging microscopy (FLIM) is increasingly used in biomedicine, material science, chemistry, and other related research fields, because of its advantages of high specificity and sensitivity in monitoring cellular microenvironments, studying interaction between proteins, metabolic state, screening drugs and analyzing their efficacy, characterizing novel materials, and diagnosing early cancers. Understandably, there is a large interest in obtaining FLIM data within an acquisition time as short as possible. Consequently, there is currently a technology that advances towards faster and faster FLIM recording. However, the maximum speed of a recording technique is only part of the problem. The acquisition time of a FLIM image is a complex function of many factors. These include the photon rate that can be obtained from the sample, the amount of information a technique extracts from the decay functions, the efficiency at which it determines fluorescence decay parameters from the recorded photons, the demands for the accuracy of these parameters, the number of pixels, and the lateral and axial resolutions that are obtained in biological materials. Starting from a discussion of the parameters which determine the acquisition time, this review will describe existing and emerging FLIM techniques and data analysis algorithms, and analyze their performance and recording speed in biological and biomedical applications.
Fluorescence lifetime imaging microscopy (FLIM) is widely used in biomedical, materials and other fields. It not only has strong specificity and high sensitivity, but also has the capability of quantitative measurement because the fluorescence lifetime is not affected by the intensity of excitation, the concentration of fluorophores and photobleaching, and consequently is able to monitor the changes of microenvironment and reflecting the interaction between molecules. However, its application is limited to some extent by the complexity of data analysis. In order to make FLIM technology more suitable for fast analysis of high-throughput data, a variety of new algorithms for fluorescence lifetime analysis have emerged in recent years, such as phasor analysis, maximum likelihood estimation, first-order moment, Bayesian analysis, and compressed sensing. Among them, the phasor analysis (PA) method obtains the fluorescence lifetime by converting the fitting in the time domain to the direct calculation in the frequency domain. Compared with traditional least-square fitting method, it is not only simpler and faster, but also more suitable for the case of low photon counts. In addition, in the PA approach to FLIM, the fluorescence decay is directly converted into a phasor diagram by simple mathematics, where the phasor points originating from different pixels in the image are represented by the positions in the phasor plot, and thus the graphical representation obtained by PA method is convenient for data visualization and cluster analysis. Therefore, it has become a simple and powerful analysis method for FLIM, and is increasingly favored by researchers. In this paper, the basic principle of PA method and how we can use it are described in detail. And on this basis, the latest application research progress of the method in cell metabolism state measurement, protein interaction study, cell microenvironment measurement, auxiliary pathological diagnosis, and resolution improvement in super-resolution imaging are introduced and summarized. The advantages of PA method in these FLIM applications are focused on, providing useful reference for the research in related fields. Finally, the phasor analysis method for FLIM data analysis and the development trend of its application are prospected.
In the past decade, fluorescence lifetime imaging microscopy (FLIM) has been widely used in biomedical research and other fields. As the fluorescence lifetime is unaffected by probe concentration, excitation intensity and photobleaching, the FLIM has the advantages of high specificity, high sensitivity and capability of quantitative measurement in monitoring microenvironment changes and reflecting the intermolecular interactions. Despite decades of technical development, the FLIM technology still faces some challenges in practical applications. For example, its resolution is still difficult to overcome the diffraction limit and the trade-off among imaging speed, image quality and lifetime accuracy needs to be considered. In recent years, a great advance in FLIM and its application has been made due to the rapid development of hardware and software and their integration with other optical technologies. In this review, we first introduce the principle and characteristics of FLIM technology based on time domain and frequency domain. We then summarize the latest progress of FLIM technology:1) imaging speed enhancement based on hardware improvement such as optimized time-correlated single photon counting module, single photon avalanche diode array detector, and acousto-optic deflector scanner; 2) lifetime measurement accuracy improvement by the proposed algorithms such as maximum likelihood estimate, Bayesian analysis and compressed sensing; 3) imaging quality enhancement and spatial resolution improvement by integrating FLIM with other optical technologies such as adaptive optics for correcting the aberration generated in the optical path, special illumination for equipping wide-field FLIM with optical sectioning ability, and super-resolution techniques for exceeding the resolution limit. We then highlight some recent applications in biomedical studies such as signal transduction or plant cell growth, disease diagnosis and treatment in cancers, Alzheimer's disease and skin diseases, assessment for toxicity and treatment efficiency of nanomaterials developed in the past few years. Finally, we present a short discussion on the current challenges and provide an outlook of the future development of enhanced imaging performance for FLIM technology. We hope that our summary on the state-of-the-art FLIM, our commentary on future challenges, and some proposed avenues for further advances will contribute to the development of FLIM technology and its applications in relevant fields.
In contrast to single photon excitation fluorescence imaging, laser scanning confocal imaging, and wide-field imaging, the multi-photon imaging has advantages of minimal invasion and deeper penetration by using near-infrared (NIR) laser source. Moreover, it can carry out three-dimensional high-spatial-resolution imaging of biological tissues due to its natural optical tomography capability. Since its advent, multi-photon imaging has become a powerful tool in biomedicine and achieved a series of significant discoveries in cancer pathology, neurological diseases and brain functional imaging. In the past decade, as a major form of multi-photon imaging techonoogy, two-photon excited fluorescence microscopy imaging has a great potential in biomedical applications. In order to satisfy the practical biomedical applications, multi-photon imaging technologies have made significant breakthroughs in improving the deficiencies of traditional 2PEF in multi-color imaging, functional imaging, live imaging and imaging depth, such as multicolor two-photon excitation fluorescence microscopy, two-photon fluorescence lifetime imaging microscopy, two-photon fiber endoscopic imaging, and three-photon microscopy imaging technology. For example, multicolor two-photon excitation fluorescence microscopy is demonstrated to achieve simultaneous imaging of multiple fluorophores with multiple wavelenth excitation lasers or continuous spectrum. In addition, the two-photon fluorescence lifetime microscopic imaging provides a method to achieve high-resolution three-dimensional imaging of biological tissue with multi-dimensional information including fluorescence intensity and lifetime. In addition, two-photon optical fiber endoscopic imaging with small system size and mimal invasion is developed and used to image the tissue inside the deep organ. Finally, two-photon excitation fluorescence microscopy technique still has relatively strong scattering for brain functional imaging in vivo. Therefore, the imaging depth is limited by the signal-to-background ratio. Three-photon microscopic imaging technique can achieve higher imaging depth and a desired signal-to-noise ratio by extending the wavelength from 1600 nm to 1820 nm because the attenuation of the excitation light in this wavelenth range is much smaller. In this article, we briefly introduce the principles and applications of these multi-photon imaging technologies, and finally provide our view for their future development.
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