Two-photon Fluorescence Microscopy Research Articles

Error Evaluation for Automated Diameter Measurements of Cerebral Capillaries Captured with Two-Photon Laser Scanning Fluorescence Microscopy.

Cerebral capillaries respond to changes in neural activity to maintain regional balances between energy demand and supply. However, the quantitative aspects of the capillary diameter responses and their contribution to oxygen supply to tissue remain incompletely understood. The purpose of the present study is to check if the diameters measured from large-scale angiographic image data of two-photon laser scanning fluorescent microscopy (2PLSM) are correctly determined with a custom-written MATLAB software and to investigate how the measurement errors can be reduced, such as at the junction areas of capillaries. As a result, nearly 17% of the measured locations appeared to be outliers of the automated diameter measurements, in particular arising from the junction areas where three capillary segments merged. We observed that about two-thirds of the outliers originated from the measured locations within 6μm from the branching point. The results indicate that the capillary locations in the junction areas cause non-negligible errors in the automated diameter measurements. Considering the common site of the outliers, the present study identified that the areas within 6μm from the branch point could be separately measured from the diameter analysis, and careful manual inspection with reference to the original images for these transition areas around the branch point is further recommended.

Substituted 9-Diethylaminobenzo[a]phenoxazin-5-ones (Nile Red Analogues): Synthesis and Photophysical Properties.

Nile Red is a benzo[a]phenoxazone dye containing a diethylamino substituent at the 9-position. In recent years, it has become a popular histological stain for cellular membranes and lipid droplets due to its unrivaled fluorescent properties in lipophilic environments. This makes it an attractive lead for chemical decoration to tweak its attributes and optimize it for more specialized microscopy techniques, e.g., fluorescence lifetime imaging or two-photon excited fluorescence microscopy, to which Nile Red has never been optimized. Herein, we present synthesis approaches to a series of monosubstituted Nile Red derivatives (9-diethylbenzo[a]phenoxazin-5-ones) starting from 1-naphthols or 1,3-naphthalenediols. The solvatochromic responsiveness of these fluorophores is reported with focus on how the substituents affect the absorption and emission spectra, luminosity, fluorescence lifetimes, and two-photon absorptivity. Several of the analogues emerge as strong candidates for reporting the polarity of their local environment. Specifically, the one- and two-photon excited fluorescence of Nile Red turns out to be very responsive to substitution, and the spectroscopic features can be finely tuned by judiciously introducing substituents of distinct electronic character at specific positions. This new toolkit of 9-diethylbenzo[a]phenoxazine-5-ones constitutes a step toward the next generation of optical molecular probes for advancing the understanding of lipid structures and cellular processes.

Self-Assembled Type I Collagen-Apatite Fibers with Varying Mineralization Extent and Luminescent Terbium Promote Osteogenic Differentiation of Mesenchymal Stem Cells.

This work explores in depth the simultaneous self-assembly and mineralization of type I collagen by a base-acid neutralization technique to prepare biomimetic collagen-apatite fibrils with varying mineralization extent and doped with luminescent bactericidal Tb3+ ions. Two variants of the method are tested: base-acid titration, a solution of Ca(OH)2 is added dropwise to a stirred solution containing type I collagen dispersed in H3 PO4 ; and direct mixing, the Ca(OH)2 solution is added by fast dripping onto the acidic solution. Only the direct mixing variant yielded an effective control of calcium phosphate polymorphism. Luminescence spectroscopy reveals the long luminescence lifetime and high relative luminescence intensity of the Tb3+ -doped materials, while two-photon confocal fluorescence microscopy shows the characteristic green fluorescence light when using excitation wavelength of 458nm, which is not harmful to bone tissue. Cytotoxicity/viability tests reveal that direct mixing samples show higher cell proliferation than titration samples. Additionally, osteogenic differentiation essays show that all mineralized fibrils promote the osteogenic differentiation, but the effect is more pronounced when using samples prepared by direct mixing, and more notably when using the Tb3+ -doped mineralized fibrils. Based on these findings it is concluded that the new nanocomposite is an ideal candidate for bone regenerative therapy.

Adaptive optics enables aberration-free single-objective remote focusing for two-photon fluorescence microscopy.

Two-photon fluorescence microscopy has been widely applied to three-dimensional (3D) imaging of complex samples. Remote focusing by controlling the divergence of excitation light is a common approach to scanning the focus axially. However, microscope objectives induce distortion to the wavefront of non-collimated excitation beams, leading to degraded imaging quality away from the natural focal plane. In this paper, using a liquid-crystal spatial light modulator to control the divergence of the excitation beam through a single objective, we systematically characterized the aberrations introduced by divergence control through microscope objectives of NA 0.45, 0.8, and 1.05. We used adaptive optics to correct the divergence-induced-aberrations and maintain diffraction-limited focal quality over up to 800-µm axial range. We further demonstrated aberration-free remote focusing for in vivo imaging of neurites and synapses in the mouse brain.

Label-free optical biomarkers detect early calcific aortic valve disease in a wild-type mouse model

BackgroundCalcific aortic valve disease (CAVD) pathophysiology is a complex, multistage process, usually diagnosed at advanced stages after significant anatomical and hemodynamic changes in the valve. Early detection of disease progression is thus pivotal in the development of prevention and mitigation strategies. In this study, we developed a diet-based, non-genetically modified mouse model for early CAVD progression, and explored the utility of two-photon excited fluorescence (TPEF) microscopy for early detection of CAVD progression. TPEF imaging provides label-free, non-invasive, quantitative metrics with the potential to correlate with multiple stages of CAVD pathophysiology including calcium deposition, collagen remodeling and osteogenic differentiation.MethodsTwenty-week old C57BL/6J mice were fed either a control or pro-calcific diet for 16 weeks and monitored via echocardiography, histology, immunohistochemistry, and quantitative polarized light imaging. Additionally, TPEF imaging was used to quantify tissue autofluorescence (A) at 755 nm, 810 nm and 860 nm excitation, to calculate TPEF 755–860 ratio (A860/525/(A755/460 + A860/525)) and TPEF Collagen-Calcium ratio (A810/525/(A810/460 + A810/525)) in the murine valves. In a separate experiment, animals were fed the above diets till 28 weeks to assess for later-stage calcification.ResultsPro-calcific mice showed evidence of lipid deposition at 4 weeks and calcification at 16 weeks at the valve commissures. The valves of pro-calcific mice also showed positive expression for markers of osteogenic differentiation, myofibroblast activation, proliferation, inflammatory cytokines and collagen remodeling. Pro-calcific mice exhibited lower TPEF autofluorescence ratios, at locations coincident with calcification, that correlated with increased collagen disorganization and positive expression of osteogenic markers. Additionally, locations with lower TPEF autofluorescence ratios at 4 and 16 weeks exhibited increased calcification at later 28-week timepoints.ConclusionsThis study suggests the potential of TPEF autofluorescence metrics to serve as a label-free tool for early detection and monitoring of CAVD pathophysiology.

Two-Photon Absorbing AIEgens: Influence of Stereoconfiguration on Their Crystallinity and Spectroscopic Properties and Applications in Bioimaging.

This paper aims at designing chromophores with efficient aggregation-induced emission (AIE) properties for two-photon fluorescence microscopy (2PFM), which is one of the best-suited types of microscopy for the imaging of living organisms or thick biological tissues. Tetraphenylethylene (TPE) derivatives are common building blocks in the design of chromophores with efficient AIE properties. Therefore, in this study, extended TPE AIEgens specifically optimized for two-photon absorption (2PA) are synthesized and the resulting (E/Z) isomers are separated using chromatography on chiral supports. Comparative characterization of the AIE properties is performed on the pure (Z) and (E) isomers and the mixture, allowing us, in combination with powder X-ray diffraction and solid-state NMR, to document a profound impact of crystallinity on solid-state fluorescence properties. In particular, we show that stereopure AIEgens form aggregates of superior crystallinity, which in turn exhibit a higher fluorescence quantum yield compared to diastereoisomers mixtures. Preparation of stereopure organic nanoparticles affords very bright fluorescent contrast agents, which are then used for cellular and intravital two-photon microscopy on human breast cancer cells and on zebrafish embryos.

High-NA two-photon single cell imaging with remote focusing using a diffractive tunable lens.

Fast, volumetric structural and functional imaging of cellular and sub-cellular dynamics inside the living brain is one of the most desired capabilities in the neurosciences, but still faces serious challenges. Specifically, while few solutions for rapid 3D scanning exist, it is generally much easier to facilitate fast in-plane scanning than it is to scan axially at high speeds. Remote focusing in which the imaging plane is shifted along the optical axis by a tunable lens while maintaining the position of the sample and objective is a promising approach to increase the axial scan speed, but existing techniques often introduce severe optical aberrations in high-NA imaging systems, eliminating the possibility of diffraction-limited single-cell imaging. Here, we demonstrate near diffraction-limited, volumetric two-photon fluorescence microscopy in which we resolve the deep sub-micron structures of single microglia cells with axial scanning performed using a novel high-NA remote focusing method. Image contrast is maintained to within 7% compared to mechanical sample stepping and the focal volume remains nearly diffraction-limited over an axial range greater than 86 µm.

Validation of red blood cell flux and velocity estimations based on optical coherence tomography intensity fluctuations

We present a validation of red blood cell flux and speed measurements based on the passage of erythrocytes through the OCT’s focal volume. We compare the performance of the so-called RBC-passage OCT technique to co-localized and simultaneously acquired two-photon excitation fluorescence microscopy (TPEF) measurements. Using concurrent multi-modal imaging, we show that fluctuations in the OCT signal display highly similar features to TPEF time traces. Furthermore, we demonstrate an overall difference in RBC flux and speed of 2.5 ± 3.27 RBC/s and 0.12 ± 0.67 mm/s (mean ± S.D.), compared to TPEF. The analysis also revealed that the OCT RBC flux estimation is most accurate between 20 RBC/s to 60 RBC/s, and is severely underestimated at fluxes beyond 80 RBC/s. Lastly, our analysis shows that the RBC speed estimations increase in accuracy as the speed decreases, reaching a difference of 0.16 ± 0.25 mm/s within the 0–0.5 mm/s speed range.

An Index Combining Lost and Remaining Nerve Fibers Correlates with Pain Hypersensitivity in Mice.

Multiple peripheral nerves are known to degenerate after nerve compression injury but the correlation between the extent of nerve alteration and pain severity remains unclear. Here, we used intravital two-photon fluorescence microscopy to longitudinally observe changes in cutaneous fibers in the hind paw of Nav1.8-Cre-tdTomato mice after chronic constriction injury (CCI). Results showed that the CCI led to variable loss of the skin nerve plexus and intraepidermal nerve fibers. The timing of Nav1.8 nerve fiber loss correlated with the development of mechanical hypersensitivity. We compared a scoring approach that assessed whole-paw nerve degeneration with an index that quantified changes in the nerve plexus and terminals in multiple small regions of interest (ROI) from intravital images of the third and fifth toe tips. We found that the number of surviving nerve fibers was not linearly correlated with mechanical hypersensitivity. On the contrary, at 14 days after CCI, the moderately injured mice showed greater mechanical hypersensitivity than the mildly or severely injured mice. This indicates that both surviving and injured nerves are required for evoked neuropathic pain. In addition, these two methods may have the estimative effect as diagnostic and prognostic biomarkers for the assessment of neuropathic pain.

A two-photon multi-emissive fluorescent probe for discrimination of Cys and Hcy/GSH via an aromatic substitution-rearrangement

Cys (Cysteine), Hcy (homocysteine), and GSH (glutathione) are three important kinds of biothiols, playing crucial roles in the variety of pathological and physiological processes. It is greater challenges to simultaneously identify different biothiols due to their similar molecular structures and chemical characteristics. In this work, we employed a multi-emissive fluorescent probe by sulfonyl benzoxadiazole (SBD) with halogen chloride unit as the interaction site based on aromatic substitution-rearrangement strategy to discriminate Cys and Hcy/GSH. The response of probe 1 to Cys would generate FRET and cause a red-shift of fluorescence emission, while Hcy/GSH only lead to different degrees of fluorescence enhancement owing to PET. The probe showed good selectivity, high sensitivity, and low detection limits to three biothiols (Cys: 0.86 μM, Hcy: 0.48 μM and GSH: 0.54 μM). Such capability of the probe could be demonstrated to successfully quantitatively detect the concentrations of Cys/Hcy/GSH in human plasmas. In addition, the probe was also successfully applied for imaging biothiols in lysosomes and real-time monitoring GSH changes in living cells through two-photon fluorescence microscopy.

Measurement of two-photon properties of indocyanine green in water and human plasma excited at the 1700-nm window.

Indocyanine green (ICG) is a human compatible dye and is ideal for deep-tissue two-photon fluorescence (2PF) microscopy excited at the 1700-nm window in vivo. However, the two-photon excitation and emission properties of this dye remain unknown. Here we demonstrate measurement of the two-photon excitation and emission properties of ICG in both water and human plasma, using home-built two-photon action cross-sectional measurement and two-photon emission spectrum measurement systems. Our results show that excited from 1600 to 1800 nm, 2PF can be generated from ICG dissolved in both water and human plasma. The measured two-photon action cross-sectional ησ2 of ICG dissolved in human plasma is an order-of-magnitude larger than that dissolved in water. The measured two-photon emission spectrum overlaps with the one-photon emission spectrum for ICG dissolved in both human plasma and water. Our results will provide key two-photon parameters for the clinical use of ICG.

Au nanostar nanoparticle as a bio-imaging agent and its detection and visualization in biosystems.

In the present work, we report the imaging of Au nanostars nanoparticles (AuNSt) and their multifunctional applications in biomedical research and theranostics applications. Their optical and spectroscopic properties are considered for the multimodal imaging purpose. The AuNSt are prepared by the seed-meditated method and characterized for use as an agent for bio-imaging. To demonstrate imaging with AuNSt, penetration and localization in different biological models such as cancer cell culture (A549 lung carcinoma cell), 3D tissue model (multicellular tumor spheroid on the base of human oral squamous carcinoma cell, SAS) and murine skin tissue are studied. AuNSt were visualized using fluorescence lifetime imaging (FLIM) at two-photon excitation with a pulse duration 140 fs, repetition rate 80 MHz and 780 nm wavelength femtosecond laser. Strong emission of AuNSt at two-photon excitation in the near infrared range and fluorescence lifetime less than 0.5 ns were observed. It allows using AuNSt as a fluorescent marker at two-photon fluorescence microscopy and lifetime imaging (FLIM). It was shown that AuNSt can be observed inside a thick sample (tissue and its model). This is the first demonstration using AuNSt as an imaging agent for FLIM at two-photon excitation in biosystems. Increased scattering of near-infrared light upon excitation of AuNSt surface plasmon oscillation was also observed and rendered using a possible contrast agent for optical coherence tomography (OCT). AuNSt detection in a biological system using FLIM is compared with OCT on the model of AuNSt penetrating into animal skin. The AuNSt application for multimodal imaging is discussed.

PEO-CYTOP Fluoropolymer Nanosheets as a Novel Open-Skull Window for Imaging of the Living Mouse Brain.

SummaryIn vivo two-photon deep imaging with a broad field of view has revealed functional connectivity among brain regions. Here, we developed a novel observation method that utilizes a polyethylene-oxide-coated CYTOP (PEO-CYTOP) nanosheet with a thickness of ∼130 nm that exhibited a water retention effect and a hydrophilized adhesive surface. PEO-CYTOP nanosheets firmly adhered to brain surfaces, which suppressed bleeding from superficial veins. By taking advantage of the excellent optical properties of PEO-CYTOP nanosheets, we performed in vivo deep imaging in mouse brains at high resolution. Moreover, PEO-CYTOP nanosheets enabled to prepare large cranial windows, achieving in vivo imaging of neural structure and Ca2+ elevation in a large field of view. Furthermore, the PEO-CYTOP nanosheets functioned as a sealing material, even after the removal of the dura. These results indicate that this method would be suitable for the investigation of neural functions that are composed of interactions among multiple regions.

Generalized spectral phase-only time-domain ptychographic phase reconstruction applied in nonlinear microscopy

Nonlinear microscopy has evolved over the last few decades to become a powerful tool for imaging and spectroscopic applications in biological sciences. In this study, i 2 P I E , a novel spectral phase control technique, was implemented in order to compress broad-bandwidth supercontinuum light pulses generated in an all-normal-dispersion (ANDi) photonic crystal fiber (PCF). The technique, based on time-domain ptychography, is demonstrated here in a nonlinear microscopy application for the first time, to the best of our knowledge. The first real-world application of this technique for second-harmonic generation and two-photon excitation fluorescence microscopies in biological samples is presented. We further show that in our implementation, i 2 P I E leads to improved contrast and signal-to-noise ratios in the generated images, compared to conventional compression techniques used in nonlinear microscopy.

Changes in Collagen Structure and Permeability of Rat and Human Sclera After Crosslinking.

PurposeTo use second harmonic generation imaging and fluorescence recovery after photobleaching to demonstrate alterations in scleral collagen structure and permeability after crosslinking in rat and human eyes.MethodsExcised rat and human scleras were imaged ex vivo with an inverted two-photon excitation fluorescence microscope before and after photochemical crosslinking using riboflavin and 405-nm laser light. Fluorescence recovery after photobleaching was applied to measure the diffusion of fluorescein isothiocyanate–dextran across the sclera.ResultsCrosslinking caused scleral collagen fibers to become wavier and more densely packed, with surface collagen being more affected than deeper collagen fibers. Crosslinked sclera showed significantly decreased permeability in the irradiation zone and also extended as far as 250 µm outside the irradiation zone.ConclusionsPhotochemical crosslinking induced changes in scleral structure and permeability that extended to tissue even outside the irradiation zone.Translational RelevanceUltrastructural changes associated with the emerging clinical technique of photochemical scleral crosslinking have not been well characterized. We demonstrate not only changes in scleral collagen by second harmonic generation imaging but also the associated functional changes in tissue permeability by fluorescence recovery after photobleaching. We report the novel finding of reduced permeability extending well beyond the direct irradiation zone. This has implications for control in the clinical setting.

Pupil plane actuated remote focusing for rapid focal depth control.

Laser scanning is widely employed in imaging and material processing. Common laser scanners are often fast for 2D transverse scanning. Rapid focal depth control is highly desired in many applications. Although remote focusing has been developed to achieve fast focal depth control, the implementation is limited by the laser damage to the actuator near laser focus. Here, we present a new method named pupil plane actuated remote focusing, which enables sub-millisecond response time while avoiding laser damage. We demonstrate its application by implementing a dual-plane two-photon laser scanning fluorescence microscope for in vivo recording of calcium transient of neurons in mouse neocortex.

The effects of voluntary running on cerebrovascular morphology and spatial short-term memory in a mouse model of amyloidosis

Physical activity has been correlated with a reduced risk of cognitive decline, including that associated with vascular dementia, mild cognitive impairment (MCI) and Alzheimer's disease (AD); recent literature suggests this may in part result from benefits to the cerebrovascular network. Using a transgenic (Tg) mouse model of AD, we evaluated the effect of running on cortical and hippocampal vascular morphology, cerebral amyloid angiopathy, amyloid plaque load, and spatial memory. TgCRND8 mice present with progressive amyloid pathology, advancing from the cortex to the hippocampus in a time-dependent manner. We postulated that the characteristic progression of pathology could lead to differential, time-dependent effects of physical activity on vascular morphology in these brain regions at 6 months of age. We used two-photon fluorescent microscopy and 3D vessel tracking to characterize vascular and amyloid pathology in sedentary TgCRND8 mice compared those who have a history of physical activity (unlimited access to a running wheel, from 3 to 6 months of age). In sedentary TgCRND8 mice, capillary density was found to be lower in the cortex and higher in the hippocampus compared to non-transgenic (nonTg) littermates. Capillary length, vessel branching, and non-capillary vessel tortuosity were also higher in the hippocampus of sedentary TgCRND8 compared to nonTg mice. Three months of voluntary running resulted in normalizing cortical and hippocampal microvascular morphology, with no significant difference between TgCRND8 and nonTg mice. The benefits of physical activity on cortical and hippocampal vasculature in 6-month old TgCRND8 mice were not paralleled by significant changes on parenchymal and cerebral amyloid pathology. Short-term spatial memory— as evaluated by performance in the Y-maze— was significantly improved in running compared to sedentary TgCRND8 mice. These results suggest that long-term voluntary running contributes to the maintenance of vascular morphology and spatial memory in TgCRND8 mice, even in the absence of an effect on amyloid pathology.

Frequency-doubled femtosecond Er-doped fiber laser for two-photon excited fluorescence imaging.

A femtosecond frequency-doubled erbium-doped fiber laser with an adjustable pulse repetition rate is developed and applied in two-photon excited fluorescence microscopy. The all-fiber laser system provides the fundamental pulse at 1560 nm wavelength with 22 fs duration for the second harmonic generation, resulting in 1.35 nJ, 60 fs pulses at 780 nm. The repetition rate is adjusted by a pulse picker unit built-in within the amplifier chain, directly providing transform-limited pulses for any chosen repetition rate between 1 and 12 MHz. We employed the laser source to drive a scanning two-photon excited fluorescence microscope for ex vivo rat skin and other samples' imaging at various pulse repetition rates. Due to compactness, ease of operation, and suitable pulse characteristics, the laser source can be considered as an attractive alternative for Ti:Sapphire laser in biomedical imaging.

Two-Photon Fluorescence Microscopy at Extremely Low Excitation Intensity: The Power of Quantum Correlations.

Quantum entanglement has been shown to imply correlations stronger than those allowed by classical models. The possibility of performing tasks that are classically impossible has made quantum entanglement a powerful resource for the development of novel methods and applications in various fields of research such as quantum computing, quantum cryptography, and quantum metrology. There is a great need for the development of next generation instrumentation and technologies utilizing entangled quantum light. Among the many applications of nonclassical states of light, nonlinear microscopy has the potential to make an impact in broad areas of science from physics to biology. Here, the microscopic image created by the fluorescence selectively excited by the process of the entangled two-photon absorption is reported. Entangled two-photon microscopy offers nonlinear imaging capabilities at an unprecedented low excitation intensity 107, which is 6 orders of magnitude lower than the excitation level for the classical two-photon image. The nonmonotonic dependence of the image on the femtosecond delay between the components of the entangled photon pair is demonstrated. This delay dependence is a result of specific quantum interference effects associated with the entanglement and this is not observable with classical excitation light. In combination with novel spectroscopic capabilities provided by a nonclassical light excitation, this is of critical importance for sensing and biological applications.

Two-photon imaging of hydrogen polysulfides in living cells and hippocampal tissues

Hydrogen polysulfide (H2Sn) is an emerging topic of research because it plays a critical role in the pathological and physiological activities of an organism. Therefore, visual monitoring of H2Sn activity in living cells is critical. In our work, a two-photon fluorescent probe NI-H2S2 based on a naphthalimide skeleton was developed as a molecular tool for detecting hydrogen polysulfides in solution and live HeLa cells. NI-H2S2 showed rapid response and high sensitivity. Notably, hydrogen polysulfides in hippocampal tissue of mice could also be tracked using the probe under a two-photon fluorescence microscope. Therefore, this probe NI-H2S2 is expected to serve as a pragmatic tool for studying the physiological roles of hydrogen polysulfides in living cells and in vivo.