Two-photon Excited Fluorescence Imaging Research Articles

Adaptive Multiphoton Endomicroscope Incorporating a Polarization-Maintaining Multicore Optical Fibre

We present a laser scanning multiphoton endomicroscope with no distal optics or mechanical components that incorporates a polarization-maintaining (PM) multicore optical fibre to deliver, focus, and scan ultrashort pulsed radiation for two-photon excited fluorescence imaging. We show theoretically that the use of a PM multicore fibre in our experimental configuration enhances the fluorescence excitation intensity achieved in the focal spot compared to a non-PM optical fibre with the same geometry and confirm this by computer simulations based on numerical wavefront propagation. In our experimental system, a spatial light modulator (SLM) is utilised to program the phase of the light input to each of the cores of the endoscope fibre such that the radiation emerging from the distal end of the fibre interferes to provide the focused scanning excitation beam. We demonstrate that the SLM can enable dynamic phase correction of path-length variations across the multicore optical fibre whilst the fibre is perturbed with an update rate of 100 Hz.

Disassembly of myofibrils and potential imbalanced forces on Z-discs in cultured adult cardiomyocytes.

Myofibrils are the main protein structures that generate force in the beating heart. Myofibril disassembly is related to many physiological and pathological processes. This study investigated, in a cultured rat adult cardiomyocyte model, the effect of force imbalance on myofibril disassembly. The imbalance of forces that were exerted on Z-discs was induced by the synergistic effect of broken intercalated discs and actin-myosin interaction. Cardiomyocytes with well-preserved intercalated discs were isolated from adult rat ventricles. The ultrastructure of cardiomyocyte was observed using a customized two-photon excitation fluorescence and second harmonic generation imaging system. The contraction of cardiomyocytes was recorded with a high-speed CCD camera, and the movement of cellular components was analyzed using a contractile imaging assay technique. The cardiomyocyte dynamic remodeling process was recorded using a time-lapse imaging system. The role of actin-myosin interaction in myofibril disassembly was investigated by incubating cardiomyocytes with blebbistatin (25 μM). Results demonstrated that the hierarchical disassembly process of myofibrils was initiated from cardiomyocyte free ends where intercalated discs had broken, during which the desmin network near the free cell ends was destroyed to release single myofibrils. Analysis of force (based on a schematic model of cardiomyocytes connected at intercalated discs) suggests that breaking of intercalated discs caused force imbalance on both sides of the Z-discs adjacent to the cell ends due to actin-myosin interaction. The damaged intercalated discs and actin-myosin interaction induced force imbalance on both sides of the Z-discs, which played an important role in the hierarchical disassembly of myofibrils. © 2016 Wiley Periodicals, Inc.

Optimizing ultrafast illumination for multiphoton-excited fluorescence imaging.

We study the optimal conditions for high throughput two-photon excited fluorescence (2PEF) and three-photon excited fluorescence (3PEF) imaging using femtosecond lasers. We derive relations that allow maximization of the rate of imaging depending on the average power, pulse repetition rate, and noise characteristics of the laser, as well as on the size and structure of the sample. We perform our analysis using ~100 MHz, ~1 MHz and 1 kHz pulse rates and using both a tightly-focused illumination beam with diffraction-limited image resolution, as well loosely focused illumination with a relatively low image resolution, where the latter utilizes separate illumination and fluorescence detection beam paths. Our theoretical estimates agree with the experiments, which makes our approach especially useful for optimizing high throughput imaging of large samples with a field-of-view up to 10x10 cm2.

A series of novel dibenzothiophene-based two-photon fluorescent probes for cellular nucleus imaging

In this study, a series of novel dibenzothiophene-based two-photon excited fluorescence (TPEF) probes with excellent nuclear staining ability were prepared and investigated. The one-photon photophysical properties indicated these labels possessed UV absorption wavelength of 390–393nm and quantum yields of 0.425–0.71. The two-photon photophysical performance exhibited two-photon absorption (TPA) cross-sections of 506–630GM by excitation of femtosecond laser at a wavelength of 800nm. 18–30-fold one-photon excited fluorescence (OPEF) enhancement and 300–450% TPEF increment binding with calf thymus DNA in Tris-HCl buffer solutions displayed obvious fluorescence “turn-on” effect. The co-localization studies showed mean co-localization coefficients of 0.88–0.94 by double-staining with commercial nuclear staining reagent DAPI in 3T3 cells, indicating accurate nuclear localization ability. The confocal fluorescence imaging displayed clear TPEF nuclear imaging. The staining penetration depth of 0–9μm exhibited good membrane permeability and three dimensional imaging ability. The observation time of 1800s and fluorescence decay amplitude of 8–10% within 300s under persistent irradiation were achieved in TPEF imaging for nuclear labeling. The DNA binding mechanism was discussed as well.

Nonlinear Optical Characterization of Membrane Protein Microcrystals and Nanocrystals.

Nonlinear optical methods such as second harmonic generation (SHG) and two-photon excited UV fluorescence (TPE-UVF)imaging are promising approaches to address bottlenecks in the membrane protein structure determination pipeline. The general principles of SHG and TPE-UVF are discussed here along with instrument design considerations. Comparisons to conventional methods in high throughput crystallization condition screening and crystal quality assessment prior to X-ray diffraction are also discussed.

Determination of the source of SHG verniers in zebrafish skeletal muscle.

SHG microscopy is an emerging microscopic technique for medically relevant imaging because certain endogenous proteins, such as muscle myosin lattices within muscle cells, are sufficiently spatially ordered to generate detectable SHG without the use of any fluorescent dye. Given that SHG signal is sensitive to the structural state of muscle sarcomeres, SHG functional imaging can give insight into the integrity of muscle cells in vivo. Here, we report a thorough theoretical and experimental characterization of myosin-derived SHG intensity profiles within intact zebrafish skeletal muscle. We determined that “SHG vernier” patterns, regions of bifurcated SHG intensity, are illusory when sarcomeres are staggered with respect to one another. These optical artifacts arise due to the phase coherence of SHG signal generation and the Guoy phase shift of the laser at the focus. In contrast, two-photon excited fluorescence images obtained from fluorescently labeled sarcomeric components do not contain such illusory structures, regardless of the orientation of adjacent myofibers. Based on our results, we assert that complex optical artifacts such as SHG verniers should be taken into account when applying functional SHG imaging as a diagnostic readout for pathological muscle conditions.

Two-photon excited fluorescence imaging of the pancreatic solid pseudopapillary tumor without hematoxylin and eosin stains.

Solid pseudopapillary tumor (SPT) of the pancreas is an epithelial tumor with low-grade malignant potential and present more common in females. At present, the gold standard for accurate diagnosis of pancreatic tumor was mostly depending on the pathological and/or cytological evaluation. In this work, TPEF microscopy was applied to obtain the images of human normal pancreas and SPT of the pancreas without hematoxylin and eosin (H&E) staining, for the purpose of identifying the organization structural, cell morphological, and cytoplasm changing, which were then compared to their corresponding H&E stained histopathological results. Our results showed that high-resolution TPEF imaging of the pancreatic SPT can clearly distinguish the pathological features from normal pancreas in unstained histological sections, and the results are consistent with the histological results. Moreover, we measured the nuclear-cytoplasmic ratios of the pancreatic SPT and normal pancreas to characterize their difference in the cytomorphological feature. It indicated that this technique can achieve the consistent information of pathological diagnosis, and has the potential to substantially improve the optical diagnosis and treatment of the pancreatic SPT without H&E staining in the future. SCANNING 38:245-250, 2016. © 2015 Wiley Periodicals, Inc.

Ruthenium(II)–terpyridine complexes-containing glyconanoparticles for one- and two-photon excited fluorescence imaging

The synthesis of ruthenium(II)–terpyridine complexes-containing glyconanoparticles (Ru–GNPs) for one- and two-photon excited fluorescence imaging was described. Ru–GNPs were built from diblock copolymers of pentafluorophenyl acrylate (PFA) and 2-(2′,3′,4′,6′-tetra-O-acetyl-β-D-glucosyloxy)ethyl methacryalte (AcGlcEMA), prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization. The pendant reactive pentafluorophenyl ester groups were substituted and the copolymer cross-linked by the two-photon absorption (TPA) chromophore, bis[4′-(4-aminophenyl)-2,2′:6′,2″-terpyridyl]ruthenium(II) hexafluorophosphate. After deacetylation of the acetyl-protecting groups in the AcGlcEMA block, TPA nanoparticles with cross-linked ruthenium(II)–terpyridine complexes as the core and glycopolymers as the corona were obtained. The resulting Ru–GNPs exhibit good water dispersity, one-photon absorption and emission and low cytotoxicity towards KB cell line. Ru–GNPs also undergo two-photon excitation when excited in the near-infrared (NIR, 720–880nm) in water, with TPA cross-sections in the range of 0.2–22 Göppert–Mayer (GM, 1 GM=10−50cm4s1photon−1). Ru–GNPs are thus potentially useful as effective cellular labeling agents for one- and two-photon excited fluorescence imaging.

Abstract 5118: In-vivo non-invasive mitochondrial evaluation of 3D human skin

Abstract Alterations in metabolism are considered one of the hallmarks of cancer and have been associated with changes in mitochondrial organization. The latter is typically studied in two-dimensional cell cultures, using exogenous stains, and qualitative or at best semi-quantitative approaches. We have recently developed a method that relies on the automated analysis of high resolution intrinsic NADH two-photon excited fluorescence (TPEF) images of three-dimensional specimens to assess quantitatively mitochondrial clustering levels. The purpose of this study was to assess whether this approach could be extended to the analysis of endogenous TPEF images acquired in vivo from human subjects. In-vivo depth-resolved TPEF and SHG images were acquired in vivo using a MPTflex clinical tomograph (Jenlab) at 790nm excitation. The TPEF signal was detected over 410nm-650nm, whereas SHG signal was detected in the 385nm-405nm range. In this proof of principle study a patient with a basal cell carcinoma lesion and a healthy volunteer were imaged. All in vivo measurements were conducted according to an approved institutional protocol. Image processing was performed in ImJ and Matlab. A series of analytical steps were implemented to exclude from the analysis the nuclei and interstitial space and also the collagen and melanin-associated fluorescence. After the cytoplasmic isolation, we proceeded with an automated, thresholding and digital object cloning method we have previously described. The power spectral density of the corresponding two-dimensional fourier transform of each processed image was fit to a simple inverse power law decay expression to extract a parameter indicative of the levels of mitochondrial clustering, β. Increased β in our study represents more clustered mitochondrial formations. The detailed depth-dependent analysis demonstrates distinct patterns of the clustering for the healthy and diseased tissue examined. An increase in mitochondrial clustering from the superficial to the basal layer is detected for the healthy sample, an outcome in accordance to previously results observed in fresh biopsies from healthy cervical epithelia. Since both the cervical and the skin epithelium are of similar histological type our finding is not startling but rather encouraging. The mitochondrial clustering values of the BCC lesion on the other hand, are depth-invariant, which implies an undifferentiated cellular phenotype, finding consistent with both the cancerous nature of the lesion and previously published in vitro and ex-vivo studies. In conclusion, we demonstrate that characterization of mitochondrial organization is feasible within three dimensional tissues of human subjects. This approach offers the potential to observe dynamically subtle changes in mitochondrial dynamics that occur during cancer development or in response to treatment, which would enrich our understanding of tissue metabolism and could further enhance current diagnostic approaches. Citation Format: Dimitra Pouli, Mihaela Balu, Bruce Tromberg, Irene Georgakoudi. In-vivo non-invasive mitochondrial evaluation of 3D human skin. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5118. doi:10.1158/1538-7445.AM2015-5118

Light- and pH-activated intracellular drug release from polymeric mesoporous silica nanoparticles

Surface modified mesoporous silica nanoparticles (MSNs) with reduced toxicity were prepared for light and pH dual triggerable drug delivery system. Both 413nm light and acidic environment can activate the drug release process, improving the pharmacological action. By applying rhodamine B (RhB) as a model drug, the accumulative RhB release is as high as 95% in pH 5.0 and in irradiation of 413nm light, compared to only 55% in pH 7.4 and in dark. The anti-cancer drug camptothecin (CPT) loaded nanoparticles can kill cancer cells with IC50 value of 0.02μgmL−1 in exposure of 413nm light, which is much lower than free CPT (about 0.1μgmL−1). Multimodal nonlinear optical imaging microscopy (NLOM) was employed to acquire in vitro coherent anti-Stokes Raman (CARS) and two-photon excited fluorescence (TPEF) images of live MCF-7 cells and showed that the nanoparticles can be taken up by breast tumor cell MCF-7 with high efficiency, indicating its great potential for anti-cancer drug delivery system.

Red-emitting DPSB-based conjugated polymer nanoparticles with high two-photon brightness for cell membrane imaging.

New red-emitting conjugated polymers have been successfully synthesized by incorporating classical two-photon absorption (TPA) units, electron-rich units, and a small amount of electron-deficient units along the polymer backbones. Water-dispersible nanoparticles (NPs) based on these polymers were also fabricated for applications in two-photon excitation fluorescence imaging of cell membrane. Through optimization of the polymer/matrix mass ratio and the initial feed concentration of the polymer solution, a high quantum yield (QY) of 24% was achieved for the red-emitting NPs in water. TPA cross section and two-photon action cross section values of these polymers at 750 nm reached up to 1000 GM and 190 GM per repeat unit in aqueous media, 2.5 × 10(5) GM and 4.7 × 10(4) GM per NP, respectively. Furthermore, these NPs displayed excellent photostability and biocompatibility. Their applications as two-photon excitation fluorescence probes for cell membrane imaging have been demonstrated in three different cell lines with excellent imaging contrast. These results demonstrated that these polymer NPs hold great potentials as excellent two-photon excitation fluorescence probes in various biological applications.

Label-free nonlinear optical imaging of mouse retina.

A nonlinear optical (NLO) microscopy system integrating stimulated Raman scattering (SRS), two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) was developed to image fresh mouse retinas. The morphological and functional details of various retinal layers were revealed by the endogenous NLO signals. Particularly, high resolution label-free imaging of retinal neurons and nerve fibers in the ganglion cell and nerve fiber layers was achieved by capturing endogenous SRS and TPEF signals. In addition, the spectral and temporal analysis of TPEF images allowed visualization of different fluorescent components in the retinal pigment epithelium (RPE). Fluorophores with short TPEF lifetime, such as A2E, can be differentiated from other long-lifetime components in the RPE. The NLO imaging method would provide important information for investigation of retinal ganglion cell degeneration and holds the potential to study the biochemical processes of visual cycle in the RPE.

Epi-detected quadruple-modal nonlinear optical microscopy for label-free imaging of the tooth

We present an epi-detected quadruple-modal nonlinear optical microscopic imaging technique (i.e., coherent anti-Stokes Raman scattering (CARS), second-harmonic generation (SHG), third-harmonic generation (THG), and two-photon excited fluorescence (TPEF)) based on a picosecond (ps) laser-pumped optical parametric oscillator system for label-free imaging of the tooth. We demonstrate that high contrast ps-CARS images covering both the fingerprint (500–1800 cm−1) and high-wavenumber (2500–3800 cm−1) regions can be acquired to uncover the distributions of mineral and organic biomaterials in the tooth, while high quality TPEF, SHG, and THG images of the tooth can also be acquired under ps laser excitation without damaging the samples. The quadruple-modal nonlinear microscopic images (CARS/SHG/THG/TPEF) acquired provide better understanding of morphological structures and biochemical/biomolecular distributions in the dentin, enamel, and the dentin-enamel junction of the tooth without labeling, facilitating optical diagnosis and characterization of the tooth in dentistry.

Experimental study on multicolor two-photon excited fluorescence microscopy

Two-photon excited fluorescence (TPEF) microscopy is a nonlinear optical microscopy technique. The advantages of TPEF microscopy include high temporal and spatial resolutions, high signal-to-noise ratio and inherent three-dimensional sectioning. In traditional TPEF microscopy, a wavelength tunable ultrashort pulsed laser is used as an excitation source. In practical applications, sample usually contains various fluorophores or unknown components. Therefore the excitation wavelength of the ultrafast laser has to be tuned to achieve optimal excitation efficiencies of various fluorophores. In order to acquire the fluorescent signals of different fluorophores simultaneously, we develop a multicolor TPEF microscope system based on a supercontinuum laser source. In experiments, TPEF images of Lily rhizome sample slide stained by two fluorescent dyes with different excitation and emission wavelengths are obtained without tuning the wavelength. Experimental results show that the high-contrast TPEF images of the sample with various fluorophores can be obtained simultaneously by using the multicolor TPEF microscope compared with by using traditional TPEF microscopy. The system is simple in structure, easy in operation, and can provide rich information about the sample, which allows it to be widely used in life and material sciences.

In Vivo Nonlinear Optical Imaging of Immune Responses: Tissue Injury and Infection

In this study, we demonstrate a noninvasive imaging approach based on multimodal nonlinear optical microscopy to in vivo image the responses of immune cells (neutrophils) to the tissue injury and bacterial infection in a zebrafish model. Specifically, the second harmonic generation from myosin thick filaments in sarcomere enabled a clear visualization of the muscle injury and infection. Two-photon excited fluorescence was used to track the behavior of the neutrophils that were transgenically labeled by red fluorescent protein. The corresponding reduced nicotinamide adenine dinucleotide (NADH) two-photon excited fluorescence images revealed a detailed morphological transformation process of individual neutrophils during muscle tissue injury and bacterial infection. The analysis of time-resolved NADH signals from the neutrophils provided important biological insights of the cellular energy metabolism during the immune responses. We found a significant increase of free/protein-bound NADH ratios in activated neutrophils in bacterial-infected tissue. In this study, we also discovered that, under 720 nm excitation, two wild-type strains (DH5α and BL21) of bacteria Escherichia coli emitted distinct endogenous fluorescence of double-peak at ∼450 and ∼520 nm, respectively. We demonstrated that the double-peak fluorescence signal could be used to differentiate the E. coli from surrounding tissues of dominant NADH signals, and to achieve label-free tracking of E. coli bacteria in vivo.

Multimodal Nonlinear Optical Microscopy for Simultaneous 3-D Label-Free and Immunofluorescence Imaging of Biological Samples

In this study, we demonstrated multimodal nonlinear optical (NLO) microscopy integrated simultaneously with two-photon excitation fluorescence (TPEF), second-harmonic generation (SHG), and coherent anti-Stokes Raman scattering (CARS) in order to obtain targeted cellular and label-free images in an immunofluorescence assay of the atherosclerotic aorta from apolipoprotein E-deficient mice. The multimodal NLO microscope used two laser systems: picosecond (ps) and femtosecond (fs) pulsed lasers. A pair of ps-pulsed lights served for CARS (817 nm and 1064 nm) and SHG (817 nm) images; light from the fs-pulsed laser with the center wavelength of 720 nm was incident into the sample to obtain autofluorescence and targeted molecular TPEF images for high efficiency of fluorescence intensity without cross-talk. For multicolor-targeted TPEF imaging, we stained smooth-muscle cells and macrophages with fluorescent dyes (Alexa Fluor 350 and Alexa Fluor 594) for an immunofluorescence assay. Each depth-sectioned image consisted of $512{\times}512$ pixels with a field of view of $250{\times}250{\mu}m^2$ , a lateral resolution of $0.4{\mu}m$ , and an axial resolution of $1.3{\mu}m$ . We obtained composite multicolor images with conventional label-free NLO images and targeted TPEF images in atherosclerotic-plaque samples. Multicolor 3-D imaging of atherosclerotic-plaque structural and functional composition will be helpful for understanding the pathogenesis of cardiovascular disease.

Optical characterization of lesions and identification of surgical margins in pancreatic metastasis from renal cell carcinoma by using two-photon excited fluorescence microscopy

Two-photon excited fluorescence (TPEF) microscopy has become a powerful instrument for imaging unstained tissue samples in biomedical research. The purpose of this study was to determine whether TPEF imaging of histological sections without hematoxylin-eosin (H-E) stain can be used to characterize lesions and identify surgical margins in pancreatic metastasis from renal cell carcinoma (RCC). The specimens of a pancreatic metastasis from RCC, as well as a primary RCC from a patient, were examined by TPEF microscopy and compared with their corresponding H-E stained histopathological results. The results showed that high-resolution TPEF imaging of unstained histological sections of pancreatic metastasis from RCC can reveal that the typical morphology of the tissue and cells in cancer tissues is different from the normal pancreas. It also clearly presented histopathological features of the collagenous capsule, which is an important boundary symbol to identify normal and cancerous tissue and to instruct surgical operation. It indicated the feasibility of using TPEF microscopy to make an optical diagnosis of lesions and identify the surgical margins in pancreatic metastasis from RCC.

Optical diagnosis of gallbladder cancers via two-photon excited fluorescence imaging of unstained histological sections.

Two-photon excited fluorescence (TPEF) microscopy, based on signal from cells, can provide detailed information on tissue architecture and cellular morphology in unstained histological sections to generate subcellular-resolution images from tissue directly. In this paper, we used TPEF microscopy to image microstructure of human normal gallbladder and three types of differentiated carcinomas in order to investigate the morphological changes of tissue structure, cell, cytoplasm, and nucleus without hematoxylin and eosin (H&E) staining. It displayed that TPEF microscopy can well image the stratified normal gallbladder tissue, including the mucosa, the muscularis, and the serosa. The typical cancer cell, characterized by cellular and nuclear pleomorphism, enlarged nuclei, and augmented nucleolus, can be identified in histological sections without H-E staining as well. The quantitative results showed that the areas of the nucleus and the nucleolus in three types of cancerous cells were all significantly greater than those in normal gallbladder columnar epithelial cells derived from TPEF microscopic images. The studies demonstrated that TPEF microscopy has the ability to characterize tissue structures and cell morphology of gallbladder cancers differentiated from a normal gallbladder in a manner similar to traditional histological analysis. As a novel tool, it has the potential for future retrospective studies of tumor staging and migration by utilizing histological section specimens without H-E staining.

Label-free three-dimensional imaging of cell nucleus using third-harmonic generation microscopy

We report the implementation of the combined third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) microscopy for label-free three-dimensional (3-D) imaging of cell nucleus morphological changes in liver tissue. THG imaging shows regular spherical shapes of normal hepatocytes nuclei with inner chromatin structures while revealing the condensation of chromatins and nuclear fragmentations in hepatocytes of diseased liver tissue. Colocalized THG and TPEF imaging provides complementary information of cell nuclei and cytoplasm in tissue. This work suggests that 3-D THG microscopy has the potential for quantitative analysis of nuclear morphology in cells at a submicron-resolution without the need for DNA staining.

P16.20 * ANALYSIS OF GLIOBLASTOMA'S PERITUMORAL BRAIN ZONE USING BIPHOTONIC MICROSCOPY

Glial tumors are primary tumors of the central nervous system. Gross total resection (GTR) of the tumor is the first step of its therapeutic management. Several techniques of per-operative analysis of the margin of the peritumoral brain zone (PBZ) have been developed in order to ensure a GTR of the tumor. We present here a new approach to the per-operative analysis of the PBZ using biphotonic microscopy analysis, a method allowing a fast and reproducible analysis of brain sample tissues. GB tumor zone, low-grade glioma and their respective PBZ were fixed on paraffine-embedded slices. These slices were analyzed using second harmonic generation microscopy (SHG) and two-photon excitation fluorescence (TPEF) microscopy. Data were collected and interpreted using a custom-wricen program with Labview, and compared to the histopathological diagnosis made by a certified neuropathologist. Each kind of brain tumor tissue and PBZ displayed a specific pattern in SHG and TPEF imaging. Also, distinction between tumor and PBZ was easy in SHG and TPEF analysis, allowing a clear vizualisation of the brain/tumor interface. SHG and TPEF imaging are fast and efficient methods allowing per-operative analysis of brain tumor tissue. The next step of our study will be to create an atlas of SHG and TPEF profile of the different brain tumors to allow direct per-operative analysis of PBZ without waiting for the pathological diagnosis, to ensure an optimal resection's quality.