Multiphoton Fluorescence Imaging Research Articles

Available medical imaging modalities for melanoma screening

The prevalence of melanoma of the skin has seen a significant rise in recent decades, constituting approximately one-third of all diagnosed cancer cases. Melanoma, the most fatal variant among cutaneous malignancies, exhibits a 4% probability of occurrence over an individual’s lifetime. The increasing incidence and mortality rates of skin cancer impose a substantial burden on healthcare resources and the economy. In recent years, several optical modalities, including dermoscopy, reflectance confocal microscopy (RCM), optical coherence tomography, multiphoton excited fluorescence imaging, and dermatofluorescence, have been extensively studied and utilized to improve the non-invasive diagnosis of skin cancer. This review article provides an analysis of the approach employed in the recently developed optical non-invasive diagnostic technologies. It explores the clinical uses of these techniques, while also examining their respective advantages and disadvantages. Furthermore, the paper explores the possibility for additional advancements in these technologies in the future.

Non-Linear Optical Activity of Chiral Bipyrimidine-Based Thin Films.

An original series of bipyrimidine-based chromophores featuring alkoxystyryl donor groups bearing short chiral (S)-2-methylbutyl chains in positions 4, 3,4 and 3,5, connected to electron-accepting 2,2-bipyrimidine rings, has been developed. Their linear and non-linear optical properties were studied using a variety of techniques, including one- and two-photon absorption spectroscopy, fluorescence measurements, as well as Hyper-Rayleigh scattering to determine the first hyperpolarizabilities. Their electronic and geometrical properties were rationalized by TD-DFT calculations. The thermal properties of the compounds were also investigated by a combination of polarized light optical microscopy, differential scanning calorimetry measurements and small-angle X-ray scattering experiments. The derivatives were found not to have mesomorphic properties, but to exhibit melting temperatures or cold crystallization behavior that enabled the isolation of well-organized thin films. The nonlinear optical properties of amorphous or crystalline thin films were studied by wide-field second harmonic generation and multiphoton fluorescence imaging, confirming that non-centrosymmetric crystal organization enables strong second and third harmonic generation. This new series confirms that our strategy of functionalizing 3D organic octupoles with short chiral chains to generate non-centrosymmetric organized thin films enables the development of highly second order nonlinear optical active materials without the use of corona-poling or tedious deposition techniques.

Three-Four Photon Transition Mn(II) Complex Monitoring Lysosome-Related ATP in Real Time via Fluorescence Lifetime Imaging.

Currently, in situ monitoring of the adenosine triphosphate (ATP) level in lysosomes is critical to understand their involvement in various biological processes, but it remains difficult due to the interferences of limited targeting and low resolution of fluorescent probes. Herein, we report a classic Mn(II) probe (FX2-MnCl2) with near-infrared (NIR) nonlinear (NLO) properties, accompanied by three-four photon transition and fivefold fluorescence enhancement in the presence of ATP. FX2-MnCl2 combines with ATP through dual recognition sites of diethoxy and manganese ions to reflect slightly fluorescence lifetime change. Through the synergy of multiphoton fluorescence imaging (MP-FI) and multiphoton fluorescence lifetime imaging microscopy (MP-FLIM), it is further demonstrated that FX2-MnCl2 displays lysosome-specific targeting behavior, which can monitor lysosome-related ATP migration under NIR laser light. This work provides a novel multiphoton transformation fluorescence complex, which might be a potential candidate as a simple and straightforward biomarker of lysosome ATP in vitro for clinical diagnosis.

In vivo deep brain multiphoton fluorescence imaging emitting at NIR-I and NIR-II and excited at NIR-IV.

Multiphoton microscopy (MPM) enables deep brain imaging. Three optical windows: NIR-I, NIR-II, and NIR-III are widely used. Recently, NIR-IV (the 2200 nm window) has been demonstrated to be the last and longest window for deep tissue MPM. However, so far MPM covers only two optical windows labeled by single fluorescent probe, one for emission and one for excitation. Here we demonstrate in vivo deep brain MPM covering three optical windows, with emission at NIR-I, NIR-II, and excitation at NIR-IV, labeled by ICG. The innovations include: (1) characterizing both 3-photon excitation and emission properties of ICG emitting at both NIR-I and NIR-II, in water, plasma, and circulating blood; (2) a home-built multiphoton microscope with simultaneous dual channel detection, with which we demonstrate deep brain MPM 950 μm (NIR-I) and 850 μm (NIR-II) into the mouse brain in vivo, verifying that multi-optical window MPM is promising for deep brain imaging.

Molecular Engineering of NIR‐II AIE Luminogen Excited at 1700 nm for Ultradeep Intravital Brain Two‐Photon Fluorescence Imaging

AbstractThe limited tissue penetration depth and spatial resolution are the major bottlenecks for deep‐brain imaging. In this study, molecular engineering by tailoring electron donors is conducted to develop for the first time an NIR‐II (second near‐infrared) emissive fluorescence probe, namely DCTBT, for effective deep‐brain two‐photon fluorescence imaging. Benefiting from its good biocompatibility, high photostability, bright NIR‐II emission as aggregates and large two‐photon fluorescence action cross section at the 1700 nm excitation window, DCTBT offers the imaging depths of 2180 and 1135 µm in mouse brain with removed and intact skull, respectively. These results are the record depths for brain imaging, compared to all kinds of fluorescent probes and all modalities of multiphoton microscopy at all demonstrated excitation wavelengths. Moreover, with DCTBT labeling, hemodynamic imaging of blood flow in mouse brain vessels down to a depth of 714 µm with the intact skull is achieved. Multiphoton fluorescence imaging with the NIR‐II probe DCTBT excited at the 1700 nm window may readily provide methodology for deep‐brain structural and hemodynamic research.

Impaired microvascular circulation in distant organs following renal ischemia.

Mortality in acute kidney injury (AKI) patients remains very high, although very important advances in understanding the pathophysiology and in diagnosis and supportive care have been made. Most commonly, adverse outcomes are related to extra-renal organ dysfunction and failure. We and others have documented inflammation in remote organs as well as microvascular dysfunction in the kidney after renal ischemia. We hypothesized that abnormal microvascular flow in AKI extends to distant organs. To test this hypothesis, we employed intravital multiphoton fluorescence imaging in a well-characterized rat model of renal ischemia/reperfusion. Marked abnormalities in microvascular flow were seen in every organ evaluated, with decreases up to 46% observed 48 hours postischemia (as compared to sham surgery, p = 0.002). Decreased microvascular plasma flow was found in areas of erythrocyte aggregation and leukocyte adherence to endothelia. Intravital microscopy allowed the characterization of the erythrocyte formations as rouleaux that flowed as one-dimensional aggregates. Observed microvascular abnormalities were associated with significantly elevated fibrinogen levels. Plasma flow within capillaries as well as microthrombi, but not adherent leukocytes, were significantly improved by treatment with the platelet aggregation inhibitor dipyridamole. These microvascular defects may, in part, explain known distant organ dysfunction associated with renal ischemia. The results of these studies are relevant to human acute kidney injury.

Study of fluorescent nanodiamonds concentrations in aqueous solutions for biological applications

Commercial fluorescent nanodiamonds constitute a new class of nanoprobes with excellent perspectives for biomedical applications. They produce a broad emission spectrum partially matching the biological window for high tissue penetration, which can be excited in the visible region (380–780 nm) and in the biological window (1000–2500 nm) using two-photon excitation processes, giving a chance for high-resolution multi-photon fluorescence imaging at a sub-cellular resolution scale. Because of their carbon-based structure, nanodiamonds are highly biocompatible and display low toxicity if adequately coated. The nanodiamonds fluorescence spectrum arises due to the so-called Nitrogen-Vacancy (NV) color centers, which are highly photostable and are formed by local charge density compensating defects because of the typical nitrogen presence impurity ions in the host nanocrystals. In this work, a study of different nanodiamonds’ concentrations in terms of their fluorescent activity was investigated in the range of 0.1–10 mg/mL by means of fluorescence spectroscopy with the purpose of determining which concentration of nanodiamonds suspended in milliQ water achieves the maximum intensity, in which it was found that the 5 mg/mL was the most intense in our experimental observations. Additionally, a non-linear fit of the experimental observations of intensity was performed to predict the fluorescence intensity following an expression of a fourth order polynomial with respect to concentration, with a figure of merit, FOM = 0.03–0.04 (3–4%). Based on this effect, the optimal nanodiamonds/aqueous media solution has been determined by means of cell viability assays, finding that concentrations ≤0.1 mg/mL are as biocompatible as milliQ water.

In Vivo Deep-Brain 3- and 4-Photon Fluorescence Imaging of Subcortical Structures Labeled by Quantum Dots Excited at the 2200 nm Window.

Multiphoton microscopy (MPM) is an enabling technology for visualizing deep-brain structures at high spatial resolution in vivo. Within the low tissue absorption window, shifting to longer excitation wavelengths reduces tissue scattering and boosts penetration depth. Recently, the 2200 nm excitation window has emerged as the last and longest window suitable for deep-brain MPM. However, multiphoton fluorescence imaging at this window has not been demonstrated, due to the lack of characterization of multiphoton properties of fluorescent labels. Here we demonstrate technologies for measuring both the multiphoton excitation and emission properties of fluorescent labels at the 2200 nm window, using (1) 3-photon (ησ3) and 4-photon action cross sections (ησ4) and (2) 3-photon and 4-photon emission spectra both ex vivo and in vivo of quantum dots. Our results show that quantum dots have exceptionally large ησ3 and ησ4 for efficient generation of multiphoton fluorescence. Besides, the 3-photon and 4-photon emission spectra of quantum dots are essentially identical to those of one-photon emission, which change negligibly subject to the local environment of circulating blood. Based on these characterization results, we further demonstrate deep-brain vasculature imaging in vivo. Due to the superb multiphoton properties of quantum dots, 3-photon and 4-photon fluorescence imaging reaches a maximum brain imaging depth of 1060 and 940 μm below the surface of a mouse brain, respectively, which enables the imaging of subcortical structures. We thus fill the last gap in multiphoton fluorescence imaging in terms of wavelength selection.

Super-resolution SRS microscopy with A-PoD.

Stimulated Raman scattering (SRS) offers the ability to image metabolic dynamics with high signal-to-noise ratio. However, its spatial resolution is limited by the numerical aperture of the imaging objective and the scattering cross-section of molecules. To achieve super-resolved SRS imaging, we developed a deconvolution algorithm, adaptive moment estimation (Adam) optimization-based pointillism deconvolution (A-PoD) and demonstrated a spatial resolution of lower than 59 nm on the membrane of a single lipid droplet (LD). We applied A-PoD to spatially correlated multiphoton fluorescence imaging and deuterium oxide (D2O)-probed SRS (DO-SRS) imaging from diverse samples to compare nanoscopic distributions of proteins and lipids in cells and subcellular organelles. We successfully differentiated newly synthesized lipids in LDs using A-PoD-coupled DO-SRS. The A-PoD-enhanced DO-SRS imaging method was also applied to reveal metabolic changes in brain samples from Drosophila on different diets. This new approach allows us to quantitatively measure the nanoscopic colocalization of biomolecules and metabolic dynamics in organelles.

Second Near-Infrared (NIR-II) Window for Imaging-Navigated Modulation of Brain Structure and Function.

For a long time, optical imaging of the deep brain with high resolution has been a challenge. Recently, with the advance in second near-infrared (NIR-II) bioimaging techniques and imaging contrast agents, NIR-II window bioimaging has attracted great attention to monitoring deeper biological or pathophysiological processes with high signal-to-noise ratio (SNR) and spatiotemporal resolution. Assisted with NIR-II bioimaging, the modulation of structure and function of brain is promising to be noninvasive and more precise. Herein, in this review, first the advantage of NIR-II light in brain imaging from the interaction between NIR-II and tissue is elaborated. Then, several specific NIR-II bioimaging technologies are introduced, including NIR-II fluorescence imaging, multiphoton fluorescence imaging, and photoacoustic imaging. Furthermore, the corresponding contrast agents are summarized. Next, the application of various NIR-II bioimaging technologies in visualizing the characteristics of cerebrovascular network and monitoring the changes of the pathology signals will be presented. After that, the modulation of brain structure and function based on NIR-II bioimaging will be discussed, including treatment of glioblastoma, guidance of cell transplantation, and neuromodulation. In the end, future perspectives that would help improve the clinical translation of NIR-II light are proposed.

Science, Medicine, and the Anesthesiologist

Science, Medicine, and the Anesthesiologist

Coordination-Regulated Terpyridine-Mn(II) Complexes for Photodynamic Therapy Guided by Multiphoton Fluorescence/Magnetic Resonance Imaging.

The synergy of multiphoton fluorescence imaging (MP-FI) and magnetic resonance imaging (MRI) provides an imaging platform with high resolution and unlimited penetration depth for early disease detection. Herein, two kinds of terpyridine-Mn(II) complexes (FD-Mn-O2NO and FD-Mn-FD) possessing seven and six coordination modes, respectively, were designed rationally for photodynamic therapy (PDT) guided by MP-FI/MRI. The complexes obtain different multiphoton fluorescence/magnetic resonance properties by adjusting the number of terpyridine ligands. Among them, FD-Mn-FD exhibits the following superiorities: (1) The optimal three-photon excitation wavelength of FD-Mn-FD falls at 1450 nm (NIR-II), which brings high sensitivity and deep tissue penetration in MP-FI. (2) FD-Mn-FD has effective longitudinal relaxation efficiency (r1 = 2.6 m M-1 s-1), which can be used for T1-weighted MRI, overcoming the problems of limited tissue penetration depth and low spatial resolution. (3) FD-Mn-FD generates endogenous 1O2 under irradiation by 808 nm light, thereby enhancing the PDT effect in vitro and in vivo. To the best of our knowledge, the complex FD-Mn-FD is the first complex to guide PDT through MP-FI/MRI, providing a blueprint for accurate and effective early detection and timely treatment of the complex in the early stages of cancer.

Visualization of Macrophase Separation and Transformation in Immiscible Polymer Blends

Visualization of Macrophase Separation and Transformation in Immiscible Polymer Blends

Spatiotemporal beam self-cleaning for high-resolution nonlinear fluorescence imaging with multimode fiber

Beam self-cleaning (BSC) in graded-index (GRIN) multimode fibers (MMFs) has been recently reported by different research groups. Driven by the interplay between Kerr effect and beam self-imaging, BSC counteracts random mode coupling, and forces laser beams to recover a quasi-single mode profile at the output of GRIN fibers. Here we show that the associated self-induced spatiotemporal reshaping allows for improving the performances of nonlinear fluorescence (NF) microscopy and endoscopy using multimode optical fibers. We experimentally demonstrate that the beam brightness increase, induced by self-cleaning, enables two and three-photon imaging of biological samples with high spatial resolution. Temporal pulse shortening accompanying spatial beam clean-up enhances the output peak power, hence the efficiency of nonlinear imaging. We also show that spatiotemporal supercontinuum (SC) generation is well-suited for large-band NF imaging in visible and infrared domains. We substantiated our findings by multiphoton fluorescence imaging in both microscopy and endoscopy configurations.

Non-invasive optical methods for melanoma diagnosis.

Cutaneous melanoma is one of the most common malignancies with increased incidence in the past few decades, making it a significant public health problem. The early diagnosis of melanoma is a major factor in improving patient's survival. The traditional pathway to melanoma diagnosis starts with a visual diagnosis, followed by subsequent biopsy and histopathologic evaluation. Recently, multiple innovative optical technology-based methods, including dermoscopy, reflectance confocal microscopy, optical coherence tomography, multiphoton excited fluorescence imaging and stepwise two-photon excited fluorescence (dermatofluoroscopy), have been developed to increase the diagnostic accuracy for the non-invasive melanoma diagnosis. Some of them have already been applied to real-life clinical settings, others require more research and development. These technologies show promise in facilitating the diagnosis of melanoma since they are non-invasive, sensitive, objective and easy to apply. Diagnostic accuracy, detection time, portability and the cost-effectiveness of the device are all aspects that need to be improved. This article reviews the method of these emerging optical non-invasive diagnostic technologies, their clinical application, their benefits and limitations, as well as their possible future development.

Enhanced collection of scattered photons in nonlinear fluorescence microscopy by extended epi-detection with a silicon photomultiplier array

To maximize signal collection in nonlinear optical microscopy, non-descanned epi-detection is generally adopted for in vivo imaging. However, because of severe scattering in biological samples, most of the emitted fluorescence photons go beyond the collection angles of objectives and thus cannot be detected. Here, we propose an extended detection scheme to enhance the collection of scattered photons in nonlinear fluorescence microscopy using a silicon photomultiplier array ahead of the front apertures of objectives. We perform numerical simulations to demonstrate the enhanced fluorescence collection via extended epi-detection in the multi-photon fluorescence imaging of human skin and mouse brain through craniotomy windows and intact skulls. For example, with red fluorescence emission at a depth of 600 µm in human skin, the increased collection can be as much as about 150% with a 10×, 0.6-NA objective. We show that extended epi-detection is a generally applicable, feasible technique for use in nonlinear fluorescence microscopy to enhance signal detection.

Functional Platinum(II) Complexes with Four-Photon Absorption Activity, Lysosome Specificity, and Precise Cancer Therapy.

Multiphoton materials are in special demand in the field of photodynamic therapy and multiphoton fluorescence imaging. However, rational design methodology for these brands of materials is still nascent. This is despite transition-metal complexes favoring optimized nonlinear-optical (NLO) activity and heavy-atom-effected phosphorescent emission. Here, three four-photon absorption (4PA) platinum(II) complexes (Pt1-Pt3) are achieved by the incorporation of varied functionalized C^N^C ligands with high yields. Pt1-Pt3 exhibit triplet metal-to-ligand charge-transfer transitions at ∼460 nm, which are verified multiple times by transient absorption spectra, time-dependent density functional theory calculations, and low-temperature emission spectra. Further, Pt1-Pt3 undergo 4PA. Notably, one of the complexes, Pt2, has maximum 4PA cross-sectional values of up to 15.2 × 10-82 cm8 s3 photon-3 under excitation of a 1600 nm femtosecond laser (near-IR II window). The 4PA cross sections vary when Pt2 is binding to lecithin and when it displays its lysosome-specific targeting behavior. On the basis of the excellent 4PA property of Pt2, we believe that those 4PA platinum(II) complexes have great potential applications in cancer theranostics.

Accelerating precision anti-cancer therapy by time-lapse and label-free 3D tumor slice culture platform.

The feasibility of personalized medicine for cancer treatment is largely hampered by costly, labor-intensive and time-consuming models for drug discovery. Herein, establishing new pre-clinical models to tackle these issues for personalized medicine is urgently demanded.Methods: We established a three-dimensional tumor slice culture (3D-TSC) platform incorporating label-free techniques for time-course experiments to predict anti-cancer drug efficacy and validated the 3D-TSC model by multiphoton fluorescence microscopy, RNA sequence analysis, histochemical and histological analysis.Results: Using time-lapse imaging of the apoptotic reporter sensor C3 (C3), we performed cell-based high-throughput drug screening and shortlisted high-efficacy drugs to screen murine and human 3D-TSCs, which validate effective candidates within 7 days of surgery. Histological and RNA sequence analyses demonstrated that 3D-TSCs accurately preserved immune components of the original tumor, which enables the successful achievement of immune checkpoint blockade assays with antibodies against PD-1 and/or PD-L1. Label-free multiphoton fluorescence imaging revealed that 3D-TSCs exhibit lipofuscin autofluorescence features in the time-course monitoring of drug response and efficacy.Conclusion: This technology accelerates precision anti-cancer therapy by providing a cheap, fast, and easy platform for anti-cancer drug discovery.

Multi-photon fluorescence microscopy imaging of mitochondria in living cells excited by Yb-doped femtosecond fiber laser utilizing two- and three-photon competitive absorption

By employing an Yb-doped femtosecond fiber laser oscillating at 1030 nm, multi-photon fluorescence microscopy imaging was demonstrated, utilizing competitive two- and three-photon absorption processes for excitation of the fluorescence probes. Competitive two- and three-photon absorptions occurred for all six of the fluorescence probes employed. The mitochondria of human embryonic kidney 293 cells were stained with the probes, and multi-photon fluorescence images were obtained in all cases. The brightest multi-photon fluorescence image was obtained when employing the probe compound possessing the longest cut-off wavelength. This can be attributed to the larger contribution of two-photon absorption to the competitive excitation process. Thus, by utilizing competitive two- and three-photon absorption processes, multi-photon fluorescence images can be acquired using Yb-doped femtosecond fiber laser excitation with a variety of probes.

An excitation wavelength switching to enhance dual-color wide-field temporal-focusing multiphoton excitation fluorescence imaging

Dual-color two-photon excitation (TPE)-fluorescence imaging is used in conventional temporal-focusing multiphoton excitation microscopy (TFMPEM) to observe specimens with different fluorophore labels. However, concerns have been raised about the excitation efficiency and selectivity of the fluorophores under fixed-wavelength excitation. This study presents a wavelength-switching approach using a scanning mirror, beam expander, and diffraction grating in the TFMPEM to switch the excitation wavelengths and match the optimal absorption of the fluorophores to acquire dynamic dual-color TPE-fluorescence images. The presented TFMPEM system was demonstrated to have an axial excitation confinement of 2.3–5.0 μm for excitation wavelengths of 730–1000 nm, and was used to visualize three-dimensional images of the vasculature of a mouse brain. The TPE efficiencies of different fluorophores were evaluated through TFMPEM imaging with excitation wavelength scanning to obtain their TPE spectra. Consequently, time-lapsed dual-color TFMPEM imaging was performed on rhodamine 6G (R6G)–poly(lactic-co-glycolic acid) (PLGA) nanoparticles and enhanced-yellow-fluorescent protein (EYFP)-tagged clathrin using excitation wavelengths at the maximum TPEs of R6G and EYFP, respectively. Our results revealed the PLGA-nanoparticle uptake of live cells via long-lived clathrin-coated plaques in clathrin-mediated endocytosis.