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Fluorescence Lifetime Imaging Microscopy (FLIM) is an advanced tool that enables the description of exponential decay rate distribution of fluorescent molecules in the samples. This technique has been broadly used in biomedicine, material science, chemistry, and other related research fields, due to its ability to illustrate both localization of specific fluorophores and fluorophores' local microenvironment, and it is superior to fluorescence intensity based imaging. However, the FLIM imaging speed is inherently limited due to the long exponential decay collecting process, which may not be proper for monitoring fast dynamic biological processes in tissue, not to mention at single protein level. Excellent fluorescence labeling techniques, advanced imaging techniques and e±cient analytical tools together enable faster FLIM imaging. As the application of FLIM in biological field progresses, new requirements for FLIM technique are proposed, such as protein–protein interaction, label-free detection, deep tissue imaging, and so on.
PDF全文 Journal of Innovative Optical Health Sciences, 2019年第12卷第5期 pp.1902003
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 e±cacy, 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 e±ciency 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.
PDF全文 Journal of Innovative Optical Health Sciences, 2019年第12卷第5期 pp.1930003
Fluorescence lifetime (FLT) of fluorophores is sensitive to the changes in their surrounding microenvironment, and hence it can quantitatively reveal the physiological characterization of the tissue under investigation. Fluorescence lifetime imaging microscopy (FLIM) provides not only morphological but also functional information of the tissue by producing spatially resolved image of fluorophore lifetime, which can be used as a signature of disorder and/or malignancy in diseased tissues. In this paper, we begin by introducing the basic principle and common detection methods of FLIM. Then the recent advances in the FLIM-based diagnosis of three different skin cancers, including basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and malignant melanoma (MM) are reviewed. Furthermore, the potential advantages of FLIM in skin cancer diagnosis and the challenges that may be faced in the future are prospected.
PDF全文 Journal of Innovative Optical Health Sciences, 2019年第12卷第5期 pp.1930004
Digestive tract tumors account for 15% and 19.3% of the cancer incidence and deaths, respectively. Early detection of digestive tract tumors is crucial to the reduction of global cancer burden. Two-photon excitation fluorescence lifetime imaging microscopy (TP-FLIM) allows noninvasive, label-free, three-dimensional, high-resolution imaging of living tissues with not only histological but also biochemical characterization ability in both qualitative and quantitative way. Benefiting from these advantages, this technology is promising for clinical diagnosis of digestive tract tumors. In recent years, many efforts have been made in this field and some remarkable progress has been achieved. In this paper, we overview the recent progress of TPFLIM-based researches on digestive tract tumor detection. Among them, our latest results on the gastric cancer and esophageal cancer are elaborately depicted. Finally, we outlook and discuss the potential advantages and challenges of TP-FLIM in future clinical applications.
PDF全文 Journal of Innovative Optical Health Sciences, 2019年第12卷第5期 pp.1930009
With the development of the new detection methods and the function of fluorescent molecule, researchers hope to further explore the internal mechanisms of organisms, monitor changes in the intracellular microenvironment, and dynamic processes of molecular interactions in cells. Fluorescence resonance energy transfer (FRET) describes the energy transfer process between donor fluorescent molecules and acceptor fluorescent molecules. It is an important means to detect protein-protein interactions and protein conformation changes in cells. Fluorescence lifetime imaging microscopy (FLIM) enables noninvasive measurement of the fluorescence lifetime of fluorescent particles in vivo. The FRET-FLIM technology, which is use FLIM to quantify and analyze FRET, enables real-time monitoring of dynamic changes of proteins in biological cells and analysis of protein interaction mechanisms. The distance between donor and acceptor and their respective fluorescent lifetime, which are of great importance for studying the mechanism of intracellular activity can be obtained by data analysis and algorithm fitting.
PDF全文 Journal of Innovative Optical Health Sciences, 2019年第12卷第5期 pp.1930010
Recently, photothermal therapy (PTT) has been proved to have great potential in tumor therapy. In the last several years, MoS2, as one novel member of nanomaterials, has been applied into PTT due to its excellent photothermal conversion e±cacy. In this work, we applied fluorescence lifetime imaging microscopy (FLIM) techniques into monitoring the PPTtriggered cell death under MoS2 nanosheet treatment. Two types of MoS2 nanosheets (single layer nanosheets and few layer nanosheets) were obtained, both of which exhibited presentable photothermal conversion e±cacy, leang to high cell death rates of 4T1 cells (mouse breast cancer cells) under PTT. Next, live cell images of 4T1 cells were obtained via directly labeling the mitochondria with Rodamine123, which were then continuously observed with FLIM technique. FLIM data showed that the fluorescence lifetimes of mitochondria targeting dye in cells treated with each type of MoS2 nanosheets significantly increased during PTT treatment. By contrast, the fluorescence lifetime of the same dye in control cells (without nanomaterials) remained constant after laser irradiation. These findings suggest that FLIM can be of great value in monitoring cell death process during PTT of cancer cells, which could provide dynamic data of the cellular microenvironment at single cell level in multiple biomedical applications.
PDF全文 Journal of Innovative Optical Health Sciences, 2019年第12卷第5期 pp.1940002
Inorganic quantum dots (QDs) have excellent optical properties, such as high fluorescence intensity, excellent photostability and tunable emission wavelength, etc., facilitating them to be used as labels and probes for bioimaging. In this study, CdSe@ZnS QDs are used as probes for Fluorescence lifetime imaging microscope (FLIM) and stimulated emission depletion (STED) nanoscopy imaging. The emission peak of CdSe@ZnS QDs centered at 526 nm with a narrow width of 19 nm and the photoluminescence quantum yield (PLQY) was 64%. The QDs presented excellent anti-photobleaching property which can be irradiated for 400 min by STED laser with 39.8mW. The lateral resolution of 42.0 nm is demonstrated for single QDs under STED laser (27.5mW) irradiation. Furthermore, the CdSe@ZnS QDs were for the first time used to successfully label the lysosomes of living HeLa cells and 81.5nm lateral resolution is obtained indicating the available super-resolution applications in living cells for inorganic QD probes. Meanwhile, Eca-109 cells labeled with the CdSe@ZnS QDs was observed with FLIM, and their fluorescence lifetime was around 3.1 ns, consistent with the in vitro value, suggesting that the QDs could act as a satisfactory probe in further FLIM-STED experiments.
PDF全文 Journal of Innovative Optical Health Sciences, 2019年第12卷第5期 pp.1940003
Simultaneous metabolic and oxygen imaging is promising to follow up therapy response, disease development and to determine prognostic factors. FLIM of metabolic coenzymes is now widely accepted to be the most reliable method to determine cellular bioenergetics. Also, oxygen consumption has to be taken into account to understand treatment responses. The phosphorescence lifetime of oxygen sensors is able to indicate local oxygen changes. For phosphorescence lifetime imaging (PLIM) dyes based on ruthenium (II) coordination complexes are useful, in detail TLD1433 which possesses a variety of different triplet states, enables complex photochemistry and redox reactions. PLIM is usually reached by two photon excitation of the drug with a femtosecond (fs) pulsed Ti:Sapphire laser working at 80 MHz repetition rate and (time-correlated single photon counting) (TCSPC) detection electronics. The interesting question was whether it is possible to follow up PLIM using faster repetition rates. Faster repetition rates could be advantageous for the induction of specific photochemical reactions because of similar light doses used normally in standard CW light treatments. For this, a default 2p-FLIM–PLIM system was expanded by adding a second fs pulsed laser (“helixx") which provides 50 fs pulses at a repetition rate of 250MHz, more than 2.3W average power and tunable from 720 nm to 920 nm. The laser beam was coupled into the AOM instead of the default 80MHz laser. We demonstrated successful applications of the 250MHz laser for PLIM which correlates well with measurements done by excitation with the conventional 80 MHz laser source.
PDF全文 Journal of Innovative Optical Health Sciences, 2019年第12卷第5期 pp.1940004
Ben Zhong Tang
Compared with visible light, near-infrared (NIR) light has deeper penetration in biological tissues. Three-photon fluorescence microscopy (3PFM) can effectively utilize the NIR excitation to obtain high-contrast images in the deep tissue. However, the weak three-photon fluorescence signals may be not well presented in the traditional fluorescence intensity imaging mode. Fluorescence lifetime of certain probes is insensitive to the intensity of the excitation laser. Moreover, fluorescence lifetime imaging microscopy (FLIM) can detect weak signals by utilizing time-correlated single photon counting (TCSPC) technique. Thus, it would be an improved strategy to combine the 3PFM imaging with the FLIM together. Herein, DCDPP-2TPA, a novel aggregation-induced emission luminogen (AIEgen), was adopted as the fluorescent probes. The three-photon absorption cross-section of the AIEgen, which has a deep-red fluorescence emission, was proved to be large. DCDPP-2TPA nanoparticles were synthesized, and the three-photon fluorescence lifetime of which was measured in water. Moreover, in vivo three-photon fluorescence lifetime microscopic imaging of a craniotomy mouse was conducted via a home-made optical system. High contrast cerebrovascular images of different vertical depths were obtained and the maximum depth was about 600 μm. Even reaching the depth of 600 μm, tiny capillary vessels as small as 1.9 μm could still be distinguished. The three-photon fluorescence lifetimes of the capillaries in some representative images were in accord with that of DCDPP-2TPA nanoparticles in water. A vivid 3D reconstruction was further organized to present a wealth of lifetime information. In the future, the combination strategy of 3PFM and FLIM could be further applied in the brain functional imaging.
PDF全文 Journal of Innovative Optical Health Sciences, 2019年第12卷第5期 pp.1940005
The endogenous fluorophores such as reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) in cells and tissues can be imaged by fluorescence lifetime imaging microscopy (FLIM) to show the tissue morphology features, as well as the biomolecular changes in microenvironment. The two important coenzymes in cellular metabolism, NAD(P)H and FAD, can be used to monitor the cellular metabolic status. This work proposed a novel method to study the uterine metabolism at the adjacent site of healthy cervix. It was found that the benign uterine tumors such as leiomyomas and adenomyosis with abnormal cell growth can be detected by measuring the fluorescence lifetime of NAD(P)H and FAD in adjacent healthy cervical tissues. This method opened a novel strategy for afflicted women to undergo the cervical biopsies instead of hysterectomies for detecting tumors, which can preserve the fertility of patients. The FLIM studying on NAD(P)H and FAD indicated the correlation between metabolism and some diseases, including diabetes, hyperthyroidism and obesity. It was also suggested that the metabolic level might be quite different for a patient with a malignant tumor history.
PDF全文 Journal of Innovative Optical Health Sciences, 2019年第12卷第5期 pp.1940006