Two-photon Excited Fluorescence Lifetime Imaging Research Articles

Far-UVC- and UVB-induced DNA damage depending on skin type.

Far-UVC radiation sources of wavelengths 222 nm and 233 nm represent an interesting potential alternative for the antiseptic treatment of the skin due to their high skin compatibility. Nevertheless, no studies on far-UVC-induced DNA damage in different skin types have been published to date, which this study aims for. After irradiating the skin with far-UVC of the wavelengths 222 and 233 nm as well as broadband UVB, the tissue was screened for cyclobutane pyrimidine dimer-positive (CPD+ ) cells using immunohistochemistry. The epidermal DNA damage was lower in dark skin types than in fair skin types after irradiation at 233 nm. Contrary to this, irradiation at 222 nm caused no skin type-dependent differences, which can be attributed to the decreased penetration depth of radiation. UVB showed the relatively strongest differences between light and dark skin types when using a suberythemal dose of 3 mJ/cm2 . As melanin is known for its photoprotective effect, we evaluated the ratio of melanin content in the stratum basale and stratum granulosum in samples of different skin types using two-photon excited fluorescence lifetime imaging (TPE-FLIM) finding a higher ratio up to skin type IV-V. As far-UVC is known to penetrate only into the upper layers of the viable skin, the aforementioned melanin ratio could explain the less pronounced differences between skin types after irradiation with far-UVC compared to UVB.

Two-Photon Excited Fluorescence Lifetime Imaging of Tetracycline-Labeled Retinal Calcification.

Deposition of calcium-containing minerals such as hydroxyapatite and whitlockite in the subretinal pigment epithelial (sub-RPE) space of the retina is linked to the development of and progression to the end-stage of age-related macular degeneration (AMD). AMD is the most common eye disease causing blindness amongst the elderly in developed countries; early diagnosis is desirable, particularly to begin treatment where available. Calcification in the sub-RPE space is also directly linked to other diseases such as Pseudoxanthoma elasticum (PXE). We found that these mineral deposits could be imaged by fluorescence using tetracycline antibiotics as specific stains. Binding of tetracyclines to the minerals was accompanied by increases in fluorescence intensity and fluorescence lifetime. The lifetimes for tetracyclines differed substantially from the known background lifetime of the existing natural retinal fluorophores, suggesting that calcification could be visualized by lifetime imaging. However, the excitation wavelengths used to excite these lifetime changes were generally shorter than those approved for retinal imaging. Here, we show that tetracycline-stained drusen in post mortem human retinas may be imaged by fluorescence lifetime contrast using multiphoton (infrared) excitation. For this pilot study, ten eyes from six anonymous deceased donors (3 female, 3 male, mean age 83.7 years, range 79-97 years) were obtained with informed consent from the Maryland State Anatomy Board with ethical oversight and approval by the Institutional Review Board.

Water-soluble iridium(III) complexes as multicolor probes for one-photon, two-photon and fluorescence lifetime imaging

Fluorescence microscopy imaging provides an indispensable way to visualize morphological details in tissue with subcellular resolution. Of the various imaging agents currently available, heavy-metal complex based probes offer a powerful tool for live cell imaging, primarily due to their excellent photophysical properties, including evident Stokes shifts, high photoluminescence efficiency and relatively long emission lifetime of phosphorescent signals. Herein, we have designed and synthesized seven water soluble cationic iridium(III) solvato complexes (1 − 7) for live cell imaging. The emission colors of these iridium(III) complexes can be tuned from green to red in PBS buffer (phosphate buffered saline). In particular, multicolor phosphorescent imaging for cytoplasm staining of living cells has been realized. More importantly, two-photon excited imaging and fluorescence lifetime imaging microscopy have also successfully been applied for complex 7.

Tattoo Pigments Are Localized Intracellularly in the Epidermis and Dermis of Fresh and Old Tattoos: In vivo Study Using Two-Photon Excited Fluorescence Lifetime Imaging

Background: The knowledge about the location and kinetics of tattoo pigments in human skin after application and during the recovery is restricted due to the limitation of in vivo methods for visualizing pigments. Here, the localization and distribution of tattoo ink pigments in freshly and old tattooed human skin during the regeneration of the epidermis and dermis were investigated in vivo. Methods: Two-photon excited fluorescence lifetime imaging (TPE-FLIM) was used to identify tattoo ink pigments in human skin in vivo down to the reticular dermis. One subject with a freshly applied tattoo and 10 subjects with tattoos applied over 3 years ago were investigated in the epidermal and dermal layers in vivo. One histological slide of tattooed skin was used to localize skin-resident tattoo pigment using light microscopy. Results: The carbon black particles deposited around the incision have still been visible 84 days after tattoo application, showing delayed recovery of the epidermis. The TPE-FLIM parameters of carbon black tattoo ink pigments were found to be different to all skin components except for melanin. Distinction from melanin in the skin was based on higher fluorescence intensity and agglomerate size. Using TPE-FLIM in vivo tattoo pigment was found in 75% of tattoos applied up to 9 years ago in the epidermis within keratinocytes, dendritic cells, and basal cells and in the dermis within the macrophages, mast cells, and fibroblasts. Loading of highly fluorescent carbon black particles enables in vivo imaging of dendritic cells in the epidermis and fibroblasts in the dermis, which cannot be visualized in native conditions. The collagen I structures showed a higher directionality similar to scar tissue resulting in a greater firmness and decreased elasticity of the tattooed skin. Conclusions: Here, we show the kinetics and location of carbon black tattoo ink pigment immediately after application for the first time in vivo in human skin. Carbon black particles are located exclusively intracellularly in the skin of fresh and old tattoos. They are found within macrophages, mast cells, and fibroblasts in the dermis and within keratinocytes, dendritic cells, and basal cells in the continuously renewed epidermis even in 9-year-old tattoos in skin showing no inflammation.

Label-free imaging of M1 and M2 macrophage phenotypes in the human dermis in vivo using two-photon excited FLIM.

Macrophages (ΜΦs) are important immune effector cells that promote (M1 ΜΦs) or inhibit (M2 ΜΦs) inflammation and are involved in numerous physiological and pathogenic immune responses. Their precise role and relevance, however, are not fully understood for lack of noninvasive quantification methods. Here, we show that two-photon excited fluorescence lifetime imaging (TPE-FLIM), a label-free noninvasive method, can visualize ΜΦs in the human dermis in vivo. We demonstrate in vitro that human dermal ΜΦs exhibit specific TPE-FLIM properties that distinguish them from the main components of the extracellular matrix and other dermal cells. We visualized ΜΦs, their phenotypes and phagocytosis in the skin of healthy individuals in vivo using TPE-FLIM. Additionally, machine learning identified M1 and M2 MФs with a sensitivity of 0.88±0.04 and 0.82±0.03 and a specificity of 0.89±0.03 and 0.90±0.03, respectively. In clinical research, TPE-FLIM can advance the understanding of the role of MФs in health and disease.

Probing polyvinylpyrrolidone-passivated graphene oxide nanoflakes as contrast agents inside tissue-like phantoms via multimodal confocal microscopy

Beside attractive electrical, thermal and mechanical properties, graphene oxide (GO) exhibits visible and near-infrared (NIR) photoluminescence (PL) and well-defined fingerprint Raman bands which are remarkable optical signatures to implement GO as new contrast agent for the visualization of cells or tissue, including cancer tumors. However, the biomedical use of GO as optical contrast agent is to some extent hindered by the intrinsic low emission efficiency especially at neutral pH. Herein, we successfully modulate the PL of GO nanoflakes in acidic and neutral medium by passivating them with polyvinylpyrrolidone (PVP), an amphiphilic and biocompatible polymer, thus improving the PL at pH relevant for biomedical applications. We demonstrate the potential of as-fabricated PVP-GO nanocomposites to operate as dual Raman-PL contrast agents inside tissue-like agarose-phantoms via scanning confocal Raman microscopy (CRM) under excitation at 532 nm. Super-resolution re-scan confocal microscopy (RCM) was further employed to investigate the distribution of PVP-GO inside biological phantoms at 3D level under three excitation lines (405, 488, and 561 nm). Finally, two-photon excited fluorescence lifetime imaging microscopy (TPE-FLIM) at 810 nm excitation reveals the ability of PVP-GO to serve as NIR-activatable contrast agent inside tissue-like phantom. Notably, PVP coating empowers GO nanoflakes not only with enhanced optical signature, but also with excellent dispersibility inside biological phantoms, thus offering improved labeling performance of as-designed imaging contrast agent.

Insight into redox regulation of apoptosis in cancer cells with multiparametric live-cell microscopy

Cellular redox status and the level of reactive oxygen species (ROS) are important regulators of apoptotic potential, playing a crucial role in the growth of cancer cell and their resistance to apoptosis. However, the relationships between the redox status and ROS production during apoptosis remain poorly explored. In this study, we present an investigation on the correlations between the production of ROS, the redox ratio FAD/NAD(P)H, the proportions of the reduced nicotinamide cofactors NADH and NADPH, and caspase-3 activity in cancer cells at the level of individual cells. Two-photon excitation fluorescence lifetime imaging microscopy (FLIM) was applied to monitor simultaneously apoptosis using the genetically encoded sensor of caspase-3, mKate2-DEVD-iRFP, and the autofluorescence of redox cofactors in colorectal cancer cells upon stimulation of apoptosis with staurosporine, cisplatin or hydrogen peroxide. We found that, irrespective of the apoptotic stimulus used, ROS accumulation correlated well with both the elevated pool of mitochondrial, enzyme-bound NADH and caspase-3 activation. Meanwhile, a shift in the contribution of bound NADH could develop independently of the apoptosis, and this was observed in the case of cisplatin. An increase in the proportion of bound NADPH was detected only in staurosporine-treated cells, this likely being associated with a high level of ROS production and their resulting detoxification. The results of the study favor the discovery of new therapeutic strategies based on manipulation of the cellular redox balance, which could help improve the anti-tumor activity of drugs and overcome apoptotic resistance.

Adaptive optics two-photon excited fluorescence lifetime imaging ophthalmoscopy of photoreceptors and retinal pigment epithelium in the living non-human primate eye.

Fluorescence lifetime imaging has demonstrated promise as a quantitative measure of cell health. Adaptive optics two-photon excited fluorescence (TPEF) ophthalmoscopy enables excitation of intrinsic retinal fluorophores involved in cellular metabolism and the visual cycle, providing in vivo visualization of retinal structure and function at the cellular scale. Combining these technologies revealed that macaque cones had a significantly longer mean TPEF lifetime than rods at 730 nm excitation. At 900 nm excitation, macaque photoreceptors had a significantly longer mean TPEF lifetime than the retinal pigment epithelium layer. AOFLIO can measure the fluorescence lifetime of intrinsic retinal fluorophores on a cellular scale, revealing differences in lifetime between retinal cell classes.

The Role of Plasma Membrane Viscosity in the Response and Resistance of Cancer Cells to Oxaliplatin.

Simple SummaryUnderstanding the role of the plasma membrane in the responses of cancer cells to chemotherapy is important because the cell membrane is directly involved in drug transport and the regulation of numerous biological processes. However, the role of the plasma membrane in cell resistance to platinum drugs like oxaliplatin is not fully understood. In this study we identified the changes to plasma membrane viscosity and lipid composition induced by oxaliplatin in responsive, cultured cancer cells and in mouse tumors. It was also found that the acquisition of chemoresistance is accompanied by modification of membrane lipids in ways that preserve the viscous properties unchanged upon further treatment. Therefore, new therapeutic approaches could be developed to reverse chemoresistance based on membrane lipid modifications and the de-stabilisation of membrane viscosity.Maintenance of the biophysical properties of membranes is essential for cell survival upon external perturbations. However, the links between a fluid membrane state and the drug resistance of cancer cells remain elusive. Here, we investigated the role of membrane viscosity and lipid composition in the responses of cancer cells to oxaliplatin and the development of chemoresistance. Plasma membrane viscosity was monitored in live colorectal cancer cells and tumor xenografts using two-photon excited fluorescence lifetime imaging microscopy (FLIM) using the fluorescent molecular rotor BODIPY 2. The lipid profile was analyzed using time-of-flight secondary ion mass spectrometry (ToF-SIMS). It was found that the plasma membrane viscosity increased upon oxaliplatin treatment, both in vitro and in vivo, and that this correlated with lower phosphatidylcholine and higher cholesterol content. The emergence of resistance to oxaliplatin was accompanied by homeostatic adaptation of the membrane lipidome, and the recovery of lower viscosity. These results suggest that maintaining a constant plasma membrane viscosity via remodeling of the lipid profile is crucial for drug resistance in cancer.

Novel (Phenothiazinyl)Vinyl-Pyridinium Dyes and Their Potential Applications as Cellular Staining Agents.

We report here the synthesis and structural characterization of novel cationic (phenothiazinyl)vinyl-pyridinium (PVP) dyes, together with optical (absorption/emission) properties and their potential applicability as fluorescent labels. Convective heating, ultrasound irradiation and mechanochemical synthesis were considered as alternative synthetic methodologies proficient for overcoming drawbacks such as long reaction time, nonsatisfactory yields or solvent requirements in the synthesis of novel dye (E)-1-(3-chloropropyl)-4-(2-(10-methyl-10H-phenothiazin-3-yl)vinyl)pyridin-1-ium bromide 3d and its N-alkyl-2-methylpyridinium precursor 1c. The trans geometry of the newly synthesized (E)-4-(2-(7-bromo-10-ethyl-10H-phenothiazin-3-yl)vinyl)-1-methylpyridin-1-ium iodide 3b and (E)-1-methyl-4-(2-(10-methyl-10H-phenothiazin-3-yl)vinyl)pyridin-1-ium tetrafluoroborate 3a′ was confirmed by single crystal X-ray diffraction. A negative solvatochromism of the dyes in polar solvents was highlighted by UV-Vis spectroscopy and explanatory insights were supported by molecular modeling which suggested a better stabilization of the lowest unoccupied molecular orbitals (LUMO). The photostability of the dye 3b was investigated by irradiation at 365 nm in different solvents, while the steady-state and time-resolved fluorescence properties of dye 3b and 3a′ in solid state were evaluated under one-photon excitation at 485 nm. The in vitro cytotoxicity of the new PVP dyes on B16-F10 melanoma cells was evaluated by WST-1 assay, while their intracellular localization was assessed by epi-fluorescence conventional microscopy imaging as well as one- and two-photon excited confocal fluorescence lifetime imaging microscopy (FLIM). PVP dyes displayed low cytotoxicity, good internalization inside melanoma cells and intense fluorescence emission inside the B16-F10 murine melanoma cells, making them suitable staining agents for imaging applications.

Investigating tunneling nanotubes in ovarian cancer based on two-photon excitation FLIM-FRET.

Precise and efficient cell-to-cell communication is critical to the growth and differentiation of organisms, the formation of various organism, the maintenance of tissue function and the coordination of their various physiological activities, especially to the growth and invasion of cancer cells. Tunneling nanotubes (TNTs) were discovered as a new method of cell-to-cell communication in many cell lines. In this paper, we investigated TNTs-like structures in ovarian cancer cells and proved their elements by fluorescent staining, which showed that TNTs are comprised of natural lipid bilayers with microtubules as the skeleton that can transmit ions and organelles between adjacent cells. We then used fluorescence resonance energy transfer (FRET) based on two-photon excitation fluorescence lifetime imaging microscopy (FLIM) (TP-FLIM-FRET) to detect material transport in TNTs. The experimental results showed that the number of TNTs have an impact on the drug treatment of cancer cells, which provided a new perspective for TNTs involvement in cancer treatment. Our results also showed that TP-FLIM-FRET would potentially become a new optical method for TNTs study.

Label-free characterization of ischemic cerebral injury using intravital two-photon excitation fluorescence lifetime imaging microscopy

Characterizing an ischemic brain injury at its early stage is critical to biological research and the clinical diagnosis of cerebral ischemia-related diseases. However, approaches with intravital, label-free, and real-time characterization capabilities are scarce. Two-photon excitation fluorescence lifetime imaging microscopy (FLIM) can detect variations in energy metabolism based on the autofluorescence of reduced nicotinamide adenine dinucleotide (NADH). Using this unique feature, we proposed a novel approach for cerebral ischemia characterization. From investigating cell and animal models, the cerebral NADH fluorescence lifetime was observed to be sensitive to metabolic changes caused by ischemia and consistent with ischemic time. A comparison with standard blood flow imaging and neuronal injury assessment further suggests that, the two-photon FLIM, using NADH as an indicator, can characterize degrees of cerebral ischemia and related injuries, particularly at the early stage. These findings demonstrate that NADH FLIM is promising for providing intravital, label-free, and real-time assessment of cerebral ischemia and ischemic brain injury that will be significant to the study and diagnosis of related diseases.

Gold nanoclusters performing as contrast agents for non-invasive imaging of tissue-like phantoms via two-photon excited fluorescence lifetime imaging.

Recently, gold nanoclusters (AuNCs) have received considerable scientific interest due to their ability to generate intrinsic photoluminescence (PL), making them suitable for a wide range of applications, such as sensing, biolabeling and bioimaging. Fluorescence lifetime imaging microscopy (FLIM) is an extremely promising technique when it comes to tissue imaging, especially once combined with near-infrared two-photon excitation (TPE) due to deep tissue penetration and improved spatial resolution. In this paper, we carried out an innovative study on the ability of bovine serum albumin stabilized gold nanoclusters (BSA-AuNCs) to perform as reliable label-free contrast agents for the visualization of tissue-like agarose phantoms via TPE-FLIM. We prove that BSA-AuNCs exhibit uniform and reproducible TPE PL in the first biological window, when embedded in phantoms, under 820 nm excitation provided by a Ti:Sapphire pulsed laser. The two-photon origin of the emission signal inside the phantom is demonstrated by the quadratic dependence of the PL intensity on the excitation power. Moreover, we focused on the evaluation of BSA-AuNCs' potential as contrast agents at different concentrations inside phantoms, simulating an ex vivo environment, at three NIR excitation wavelengths, in view of defining the optimal experimental conditions for future real-tissue imaging assays. The present study aims at translating our previous results on the successful performance of BSA-AuNCs as contrast agents for in vitro FLIM imaging, using visible light, towards non-invasive ex vivo NIR imaging applications. Besides the advantageous use of the combined techniques TPE-FLIM, the novelty of our work consists of demonstrating for the first time the capacity of BSA-AuNCs to perform as bright contrast agents inside cancer-tissue mimicking phantoms. We prove that BSA-AuNCs show great promise as fluorescent contrast agents for TPE-FLIM towards image-assisted tumor surgery.

In vivo non-invasive staining-free visualization of dermal mast cells in healthy, allergy and mastocytosis humans using two-photon fluorescence lifetime imaging

Mast cells (MCs) are multifunctional cells of the immune system and are found in skin and all major tissues of the body. They contribute to the pathology of several diseases including urticaria, psoriasis, atopic dermatitis and mastocytosis where they are increased at lesional sites. Histomorphometric analysis of skin biopsies serves as a routine method for the assessment of MC numbers and their activation status, which comes with major limitations. As of now, non-invasive techniques to study MCs in vivo are not available. Here, we describe a label-free imaging technique to visualize MCs and their activation status in the human papillary dermis in vivo. This technique uses two-photon excited fluorescence lifetime imaging (TPE-FLIM) signatures, which are different for MCs and other dermal components. TPE-FLIM allows for the visualization and quantification of dermal MCs in healthy subjects and patients with skin diseases. Moreover, TPE-FLIM can differentiate between two MC populations in the papillary dermis in vivo—resting and activated MCs with a sensitivity of 0.81 and 0.87 and a specificity of 0.85 and 0.84, respectively. Results obtained on healthy volunteers and allergy and mastocytosis patients indicate the existence of other MC subpopulations within known resting and activated MC populations. The developed method may become an important tool for non-invasive in vivo diagnostics and therapy control in dermatology and immunology, which will help to better understand pathomechanisms involving MC accumulation, activation and degranulation and to characterize the effects of therapies that target MCs.

Optical changes in THP-1 macrophage metabolism in response to pro- and anti-inflammatory stimuli reported by label-free two-photon imaging.

.Temporal changes in macrophage metabolism are likely crucial to their role in inflammatory diseases. Label-free two-photon excited fluorescence (TPEF) and fluorescence lifetime imaging microscopy are well suited to track dynamic changes in macrophage metabolism. We performed TPEF imaging of human macrophages following either pro- or an anti-inflammatory stimulation. Two endogenous fluorophores, NAD(P)H and FAD, coenzymes involved in key metabolic pathways, provided contrast. We used the corresponding intensity images to determine the optical redox ratio of FAD to FAD + NAD(P)H. We also analyzed the intensity fluctuation patterns within NAD(P)H TPEF images to determine mitochondrial clustering patterns. Finally, we acquired NAD(P)H TPEF lifetime images to assess the relative levels of bound NAD(P)H. Our studies indicate that the redox ratio increases, whereas mitochondrial clustering decreases in response to both pro- and anti-inflammatory stimuli; however, these changes are enhanced in pro-inflammatory macrophages. Interestingly, we did not detect any significant changes in the corresponding NAD(P)H bound fraction. A combination of optical metabolic metrics could be used to classify pro- and anti-inflammatory macrophages with high accuracy. Contributions from alterations in different metabolic pathways may explain our findings, which highlight the potential of label-free two-photon imaging to assess nondestructively macrophage functional state.

Interrogation of tumor metabolism in tissue samples ex vivo using fluorescence lifetime imaging of NAD(P)H

Exploring metabolism in human tumors at the cellular level remains a challenge. The reduced form of metabolic cofactor NAD(P)H is one of the major intrinsic fluorescent components in tissues and a valuable indicator of cellular metabolic activity. Fluorescence lifetime imaging (FLIM) enables resolution of both the free and protein-bound fractions of this cofactor, and thus, high sensitivity detection of relative changes in the NAD(P)H-dependent metabolic pathways in real time. However, the clinical use of this technique is still very limited. The applications of metabolic FLIM could be usefully expanded by probing cellular metabolism in tissues ex vivo. For this, however, the development of appropriate tissue preservation protocols is required in order to maintain the optical metabolic characteristics in the ex vivo sample in a state similar to those of the tumor in vivo. Using mouse tumor models of different histological types—colorectal cancer, lung carcinoma and melanoma—we tested eight different methods of tissue handling by comparing NAD(P)H fluorescence decay parameters ex vivo and in vivo as measured with two-photon excited FLIM microscopy. It was found that the samples placed in 10% BSA on ice immediately after excision maintained the same fluorescence lifetimes and free/bound ratios as measured in vivo for at least 3 hours. This protocol was subsequently used for metabolic assessments in fresh postoperative samples from colorectal cancer patients. A high degree of inter- and intra-tumor heterogeneity with a trend to a more oxidative metabolism was detected in T3 colorectal tumors in comparison with normal tumor-distant colon samples. These results suggest that the methodology developed on the basis of FLIM of NAD(P)H in tissues ex vivo show promise for interrogating the metabolic state of patients’ tumors.

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

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 non-invasive, 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 TP-FLIM-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.

Digital staining through the application of deep neural networks to multi-modal multi-photon microscopy.

Deep neural networks have been used to map multi-modal, multi-photon microscopy measurements of a label-free tissue sample to its corresponding histologically stained brightfield microscope colour image. It is shown that the extra structural and functional contrasts provided by using two source modes, namely two-photon excitation microscopy and fluorescence lifetime imaging, result in a more faithful reconstruction of the target haematoxylin and eosin stained mode. This modal mapping procedure can aid histopathologists, since it provides access to unobserved imaging modalities, and translates the high-dimensional numerical data generated by multi-modal, multi-photon microscopy into traditionally accepted visual forms. Furthermore, by combining the strengths of traditional chemical staining and modern multi-photon microscopy techniques, modal mapping enables label-free, non-invasive studies of in vivo tissue samples or intravital microscopic imaging inside living animals. The results show that modal co-registration and the inclusion of spatial variations increase the visual accuracy of the mapped results.

Investigating Natural Red Lakes with Two-Photon Excited Fluorescence Lifetime Imaging Microscopy

Investigating Natural Red Lakes with Two-Photon Excited Fluorescence Lifetime Imaging Microscopy

Improved label-free diagnostics and pathological assessment of atherosclerotic plaques through nonlinear microscopy.

Coronary heart disease is the most common type of heart disease caused by atherosclerosis. In fact, an arterial wall lesion centered on the accumulation of cholesterol-rich lipids and the accompanying inflammatory response generates a plaque, whose rupture may result in a thrombus with fatal consequences. Plaque characterization for assessing the severity of atherosclerosis is generally performed through standard histopathological examination based on hematoxylin/eosin staining, which is operator-dependent and requires relatively long procedures. In this framework, nonlinear optical microscopy is a valid, label-free alternative to standard diagnostic methods. We combined second-harmonic generation (SHG), two-photon excited fluorescence (TPEF) and fluorescence lifetime imaging microscopy in a multimodal scheme for obtaining morphological and molecular information on human carotid ex vivo specimens affected by atherosclerosis. In this study, discrimination between different tissues within the atherosclerotic plaque was achieved based on both lifetime, TPEF-to-SHG ratio, and image pattern analysis. The presented methodology aims to be a starting point for future fully automated and fast characterization of atherosclerotic biopsies; moreover, it could be extended to the study of other tissues and pathologies. Combined TPEF/SHG mapping of a carotid specimen affected by atherosclerosis.