Two-photon Confocal Laser Scanning Microscopy Research Articles

Advancing Glucose Sensing Through Auto-Fluorescent Polymer Brushes: From Surface Design to Nano-Arrays.

Designing smart (bio)interfaces with the capability to sense and react to changes in local environments offers intriguing possibilities for new surface-based sensing devices and technologies. Polymer brushes make ideal materials to design such adaptive and responsive interfaces given their large variety of functional and structural possibilities as well as their outstanding abilities to respond to physical, chemical, and biological stimuli. Herein, a practical sensory interface for glucose detection based on auto-fluorescent polymer brushes decorated with phenylboronic acid (PBA) receptors is presented. The glucose-responsive luminescent surfaces, which are capable of translating conformational transitions triggered by pH variations and binding events into fluorescent readouts without the need for fluorescent dyes, are grown from both nanopatterned and non-patterned substrates. Two-photon laser scanning confocal microscopy and atomic force microscopy (AFM) analyses reveal the relationship between the brush conformation and glucose concentration and confirm that the phenylboronic acid functionalized brushes can bind glucose over a range of physiologically relevant concentrations in a reversible manner. The combination of auto-fluorescent polymer brushes with synthetic receptors presents a promising avenue for designing innovative and robust sensing systems, which are essential for various biomedical applications, among other uses.

Halogen-modified carbazole derivatives for lipid droplet-specific bioimaging and two-photon photodynamic therapy

Lipid droplets (LDs) are dynamic multifunctional organelles that participate in the regulation of many metabolic processes, visualization of which is necessary for biological research. In this work, a series of two-photon responsive fluorescent probes (C-H, C-Br, and C-I) based on carbazole units were designed and synthesized. Thereinto, an iodine-modified carbazole derivative C-I exhibited an exciting lipid droplet targeting ability due to its excellent lipophilicity. Meanwhile, benefiting from its larger Stokes shift and two-photon absorption cross-section, C-I was employed for two-photon confocal laser scanning microscopy (CLSM) and stimulated emission depletion (STED) microscopy imaging to observe LDs more accurately. In addition, given the heavy atom effect, C-I can effectively generate reactive oxygen species (ROS) leading to cancer cell apoptosis under near-infrared light irradiation. Notably, we explained the process of cell apoptosis through in vitro simulation experiments. This study provides a promising platform for visualization of lipid droplets.

Micro-distribution of arsenic and polycyclic aromatic hydrocarbons and their interaction in Pteris vittata L.

Interactive effects of inorganic arsenic (As) species and polycyclic aromatic hydrocarbons (PAHs) on their uptake, accumulation and translocation in the hyperaccumulator Pteris vittata L. (P. vittata) were studied hydroponically. The presence of PAHs hindered As uptake and acropetal translocation by P. vittata, decreasing As concentrations by 29.8%–54.5% in pinnae, regardless of the initial As speciation. The inhibitive effect of PAHs was 1.6–8.7 times greater for arsenite [As(III)] than for arsenate [As(V)]. Similarly, inorganic As inhibited the uptake of fluorene (FLU) and benzo[a]pyrene (BaP) by P. vittata roots by 0.4%–21.7% and by 33.1%–69.7%, respectively. Interestingly, coexposure to As and PAHs slightly enhanced the translocation of PAHs by P. vittata with their concentrations increased 0.3 to 0.8 times in shoots, except for the As(III)+BaP treatment. The antagonistic interaction between As and PAHs uptake is likely caused by competitive inhibition or oxidative stress injury. By using synchrotron radiation micro X-ray fluorescence imaging, high concentrations of As were found distributed throughout the microstructures far from main vein of the pinnae when coexposed with PAHs, the opposite of what was observed with exposure to As only. PAHs could also significantly inhibit the accumulation and distribution of As in vascular bundles in rachis treated with As(III). The results of two-photon laser scanning confocal microscopy revealed that PAHs were mainly distributed in the vascular cylinder, epidermal cells, vascular bundles, epidermis and vein tissues, and this was independent of As speciation and treatment. This work offers new positive evidence for the interaction between As and PAHs in P. vittata, presents new information on the underlying mechanisms for interactions of As and PAHs affecting their uptake and translocation within P. vittata L., and provides direction for future research on the mechanisms of PAHs uptake by plants.

Spatiotemporal patterning of photoresponsive DNA-based hydrogels to tune local cell responses

Understanding the spatiotemporal effects of surface topographies and modulated stiffness and anisotropic stresses of hydrogels on cell growth remains a biophysical challenge. Here we introduce the photolithographic patterning or two-photon laser scanning confocal microscopy patterning of a series of o-nitrobenzylphosphate ester nucleic acid-based polyacrylamide hydrogel films generating periodically-spaced circular patterned domains surrounded by continuous hydrogel matrices. The patterning processes lead to guided modulated stiffness differences between the patterned domains and the surrounding hydrogel matrices, and to the selective functionalization of sub-regions of the films with nucleic acid anchoring tethers. HeLa cells are deposited on the circularly-shaped domains functionalized with the MUC-1 aptamers. Initiation of the hybridization chain reaction by nucleic acid tethers associated with the continuous hydrogel matrix results in stress-induced ordered orthogonal shape-changes on the patterned domains, leading to ordered shapes of cell aggregates bound to the patterns.

Different rates of endocytic activity and vesicle transport from the apical and synaptic poles of the outer hair cell.

BackgroundIntense endocytic activity at the apex of outer hair cells (OHCs)—the electromechanical cells of the cochlea—has been demonstrated using the vital plasma-membrane marker FM1-43 and confocal laser-scanning microscopy. Vesicular traffic toward the cell nucleus to distinct locations of the endoplasmic reticulum has also been shown.ObjectiveThe current study characterizes the dynamics of endocytic activity, as well as apicobasal and basoapical trafficking, using a local perfusion technique that we recently developed and published to visualize bidirectional trafficking in isolated bipolar cells.Materials and methodsThe fluorescent plasma-membrane markers FM1-43 (10 µM) and FM4-64 (10 µM), together with a fluid-phase marker, Lucifer yellow (50 µM), were used to label endocytosed vesicles in isolated OHCs of the guinea pig cochlea. Targets of endocytosed vesicles were examined with a fluorescent marker of subsurface cisternae, DiOC6 (0.87 µM). Single- and two-photon confocal laser-scanning microscopy was used to visualize labeled vesicles.ResultsThe plasma-membrane markers presented more intense vesicle internalization at the synaptic pole than at the apical pole of the OHC. Intracellular basoapical vesicle trafficking was faster than apicobasal trafficking. Vesicles endocytosed at the synaptic pole were transcytosed to the endoplasmic reticulum system. An intracellular Lucifer yellow signal was not detected.ConclusionThe larger endocytic fluorescent signals in the synaptic pole and the faster basoapical trafficking imply that membrane internalization and vesicle trafficking are more efficient at the synaptic pole than at the apical pole of the OHC.

Interactive effects of PAHs with different rings and As on their uptake, transportation, and localization in As hyperaccumulator.

In order to illuminate the mechanism of the interaction of polycyclic aromatic hydrocarbon (PAH) with different benzene rings and arsenic (As) in As hyperaccumulator, Pteris vittata L., the uptakes of PAHs were investigated using hydroponics simulation and localizations of PAHs in the plant were determined using two-photon laser scanning confocal microscopy (TPLSCM). The results showed that the total As concentration in different parts of P. vittata decreased in the presence of PAHs with increased numbers of benzene rings: 38.0-47.4% for benzo(a)pyrene (BaP, five rings), 20.5-35.9% for pyrene (PYR, four rings), and 13.7-16.6% for fluorine (FLU, three rings). BaP and PYR concentrations increased, while FLU concentration decreased in the presence of As. The results of TPLSCM revealed that PAHs distributed in epidermal cells of roots, xylem, and endothelial cells of rachis, epidermis, and stomatal cells of pinnae; however, the fluorescence intensity of BaP and PYR were higher than FLU significantly in plant. This study provided important basis to further research on interactive effects of PAHs and As in the P. vittata. These findings were important to understand the mechanisms of PAH and As translocation and distribution by P. vittata.

A series of terpyridine containing flexible amino diethylacetate derivatives with large two-photon action cross-sections for effective mitochondrial imaging in living liver cancerous cells

Small molecules possess large two-photon action cross sections (Φσ) are highly demanded for biological purpose. Herein, three novel terpyridine containing flexible amino diethylacetate organic small molecules (A1, A2 and A3) were rationally designed and their photophysical properties were investigated both experimentally and theoretically. The results revealed that the three chromophores possess large Φσ and remarkable Stokes' shift in high polar solvents, which are particularly benefit for further biological imaging application. One chromophore (A1) displayed an effective intracellular uptake against lung cancerous living cells A549. Colocalization studies suggested the internalized subcellular compartment was mitochondria. Consequently, chromophore A1 provides a promising platform to directly monitor mitochondria in living cells under two-photon confocal laser scanning microscopy.

Fluorescent probes with dual-mode for rapid detection of SO2 derivatives in living cells: Ratiometric and two-photon fluorescent sensors

The development of rapid, convenient, and sensitive methods for selective detection SO2 derivatives is of considerable significance in biomedical research and has become a topical objective. Herein four probes (DS1, DS2, DB1 and DB2) based on π-conjugated indolium/benzoindolium systems were designed for detection bisulfite/sulfite. The results indicated that probes DB1 and DB2 showed rapid response, high selectivity and sensitivity for bisulfite/sulfite anions with significant ratiometric fluorescent signal changes. Moreover, DB1 and DB2 were successfully applied for bioimaging SO2 derivatives in living cells under one- and two-photon confocal laser scanning microscopy. This paradigm provided a promising methodology for the design of ratiometric and two-photon bisulfite/sulfite sensors.

Transportation and localization of phenanthrene and its interaction with different species of arsenic in Pteris vittata L.

The interaction between arsenic (As) and phenanthrene (PHE) in Pteris vittata L. was investigated in this study. The migration and occurrence of PHE in P. vittata were determined by two-photon laser scanning confocal microscopy. Data indicated that PHE supplementation lowers the As concentration in P. vittata, decreasing As levels by 16.8–39.9% in the pinnae, 30.0–49.0% in the rachis, and 45–51.5% in the roots, respectively. Different arsenic species inhibited P. vittata PHE absorption. The most significant effect was observed using dimethylarsenic acid (DMA), which decreased PHE accumulation by 20.73%. With the exception of elevated As(V) concentrations in As(III)-treated plants, PHE treatment significantly reduced inorganic As concentrations in P. vittata. However, PHE elevated root DMA concentrations by 9%. According to in situ visualization, PHE is primarily found in the upper and lower epidermis and stomatal cells, particularly the stomata guard cells.

Label-Free Imaging of Umbilical Cord Tissue Morphology and Explant-Derived Cells.

In situ detection of MSCs remains difficult and warrants additional methods to aid with their characterization in vivo. Two-photon confocal laser scanning microscopy (TPM) and second harmonic generation (SHG) could fill this gap. Both techniques enable the detection of cells and extracellular structures, based on intrinsic properties of the specific tissue and intracellular molecules under optical irradiation. TPM imaging and SHG imaging have been used for label-free monitoring of stem cells differentiation, assessment of their behavior in biocompatible scaffolds, and even cell tracking in vivo. In this study, we show that TPM and SHG can accurately depict the umbilical cord architecture and visualize individual cells both in situ and during culture initiation, without the use of exogenously applied labels. In combination with nuclear DNA staining, we observed a variance in fluorescent intensity in the vessel walls. In addition, antibody staining showed differences in Oct4, αSMA, vimentin, and ALDH1A1 expression in situ, indicating functional differences among the umbilical cord cell populations. In future research, marker-free imaging can be of great added value to the current antigen-based staining methods for describing tissue structures and for the identification of progenitor cells in their tissue of origin.

Multimodal optical microscopy methods reveal polyp tissue morphology and structure in Caribbean reef building corals.

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues comprising the Caribbean reef building corals Montastraeaannularis and M. faveolata. These approaches include fluorescence microscopy (FM), serial block face imaging (SBFI), and two-photon confocal laser scanning microscopy (TPLSM). SBFI provides deep tissue imaging after physical sectioning; it details the tissue surface texture and 3D visualization to tissue depths of more than 2 mm. Complementary FM and TPLSM yield ultra-high resolution images of tissue cellular structure. Results have: (1) identified previously unreported lobate tissue morphologies on the outer wall of individual coral polypsand (2) created the first surface maps of the 3D distribution and tissue density of chromatophores and algae-like dinoflagellate zooxanthellae endosymbionts. Spectral absorption peaks of 500 nm and 675 nm, respectively, suggest that M. annularis and M. faveolata contain similar types of chlorophyll and chromatophores. However, M. annularis and M. faveolata exhibit significant differences in the tissue density and 3D distribution of these key cellular components. This study focusing on imaging methods indicates that SBFI is extremely useful for analysis of large mm-scale samples of decalcified coral tissues. Complimentary FM and TPLSM reveal subtle submillimeterscale changes in cellular distribution and density in nondecalcified coral tissue samples. The TPLSM technique affords: (1) minimally invasive sample preparation,(2) superior optical sectioning ability,and (3) minimal light absorption and scattering, while still permitting deep tissue imaging.

Multimodal Optical Microscopy Methods Reveal Polyp Tissue Morphology and Structure in Caribbean Reef Building Corals

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues comprising the Caribbean reef building corals Montastraeaannularis and M. faveolata. These approaches include fluorescence microscopy (FM), serial block face imaging (SBFI), and two-photon confocal laser scanning microscopy (TPLSM). SBFI provides deep tissue imaging after physical sectioning; it details the tissue surface texture and 3D visualization to tissue depths of more than 2 mm. Complementary FM and TPLSM yield ultra-high resolution images of tissue cellular structure. Results have: (1) identified previously unreported lobate tissue morphologies on the outer wall of individual coral polyps and (2) created the first surface maps of the 3D distribution and tissue density of chromatophores and algae-like dinoflagellate zooxanthellae endosymbionts. Spectral absorption peaks of 500 nm and 675 nm, respectively, suggest that M. annularis and M. faveolata contain similar types of chlorophyll and chromatophores. However, M. annularis and M. faveolata exhibit significant differences in the tissue density and 3D distribution of these key cellular components. This study focusing on imaging methods indicates that SBFI is extremely useful for analysis of large mm-scale samples of decalcified coral tissues. Complimentary FM and TPLSM reveal subtle submillimeter scale changes in cellular distribution and density in nondecalcified coral tissue samples. The TPLSM technique affords: (1) minimally invasive sample preparation, (2) superior optical sectioning ability, and (3) minimal light absorption and scattering, while still permitting deep tissue imaging.

An Explanation for the Difference in the Percutaneous Penetration Behavior of Tamsulosin Induced by Two Different O-Acylmenthol Derivatives

Using tamsulosin (TAL) as a model drug, the aim of this study was to investigate and compare the percutaneous permeation behavior of two menthol derivatives, 2-isopropyl-5-methylcyclohexyl heptanoate (M-HEP) and 2-isopropyl-5-methylcyclohexyl decanoate (M-DEC). In vitro transdermal permeation study was carried out using porcine skin. The residual amount of enhancers in the skin after permeation experiment was determined by gas chromatographic (GC) method. The penetration depths of fluorescein were visualized by two-photon confocal laser scanning microscopy (2P-LSM) after the skin being treated with different enhancers. Furthermore, changes in the stretching frequency of functional group of ceramide were investigated by using attenuated total reflectance Fourier transform infrared (ATR-FTIR) technique. After M-HEP addition, the cumulative amount of TAL permeated in 8 h (Q8) reached 20.57±0.54 μg/cm2 and the depth of fluorescein was 40 μm; the CH2 of ceramide symmetric stretching frequency was 4 cm−1 blue shifted. However, M-DEC has an opposite effect on TAL permeation compared with that of M-HEP. TAL is a crucial factor affecting permeation procedure, and microenvironment of lipid region determines promotion capability of the enhancers.

Intracellular oxygen: Similar results from two methods of measurement using phosphorescent nanoparticles

The ability to resolve the spatio-temporal complexity of intracellular O2distribution is the "Holy Grail" of cellular physiology. In an effort to obtain a minimally invasive approach to the mapping of intracellular O2tensions, two methods of phosphorescent lifetime imaging microscopy were compared in the current study and gave similar results. These were two-photon confocal laser scanning microscopy with pinhole shifting, and picosecond time-resolved epi-phosphorescence microscopy using a single 0.5 μm focused spot. Both methods utilized Ru coordination complex embedded nanoparticles (45 nm diameter) as the phosphorescent probe, excited using pulsed outputs of a titanium–sapphire Tsunami lasers (710–1050 nm).

Use of the fluorescent probe LAURDAN to label and measure inner membrane fluidity of endospores of Clostridium spp.

A method for measuring the fluidity of inner membranes of populations of endospores of Clostridium spp. with a fluorescent dye was developed. Cells of Clostridium beijerinckii ATCC 8260 and Clostridium sporogenes ATCC 7955 were allowed to sporulate in the presence of 6-dodecanoyl-2-dimethylaminonaphthalene (LAURDAN) on a soil-based media. Labeling of endospores with LAURDAN did not affect endospore viability. Removal of the outer membranes of endospores was done using a chemical treatment and confirmed using transmission electron microscopy (TEM). Two-photon confocal laser scanning microscopy (CLSM), and generalized polarization (GP) measurements were used to assess fluorescence of endospores. Lipid composition analysis of cells and endospores was done to determine whether differences in GP values are attributable to differences in membrane composition. Removal of the outer membranes of endospores did not significantly impact GP values. Decoated, labeled endospores of C. sporogenes ATCC 7955 and C. beijerinckii ATCC 8260 exhibited GP values of 0.77±0.031 and 0.74±0.027 respectively. Differences in ratios of fatty acids between cells and endospores are unlikely to be responsible for high GP values observed in endospores. These GP values indicate high levels of lipid order and the exclusion of water from within inner membranes of endospores.

Visualisation of the copepod female reproductive system using confocal laser scanning microscopy and two-photon microscopy

We examined mating behaviour in the harpacticoid copepod Tisbe battagliai Volkmann-Rocco, 1972, in particular the process of delivering spermatophore seminal contents to the female urosome. Labelling using 4′6′ diamidino-2-phenylindole (DAPI) coupled with two-photon confocal laser scanning microscopy successfully visualised the spermatophore and female internal reproductive system. Sections of the female urosome were imaged to examine seminal fluid stores. The female tissues were found to auto-fluoresce as red emission under green excitation, requiring no additional tissue labelling. DAPI-labelled seminal fluid stores were identified within the female reproductive system. The details observed agreed with previous descriptions of copepod reproductive anatomy and of spermatophores. Specimens cultured under pH 8.10 and a simulated ocean acidification scenario (pH 7.67) were compared for changes in reproductive anatomy and spermatophore size and site attachment. No differences were observed in spermatophore attachment or the female reproductive system but spermatophore size was reduced significantly at pH 7.67 compared with pH 8.10. This size reduction was, however, in proportion to an overall reduction in female body size at reduced pH. Confocal microscopy is shown here to be a valuable tool to investigate detailed reproductive processes in copepods.

Evaluation of two novel methods for assessing intracellular oxygen

The ability to resolve the spatio-temporal complexity of intracellular O2 distribution is the ‘Holy Grail’ of cellular physiology. In an effort to obtain a non-invasive approach of mapping intracellular O2 tensions, two methods of phosphorescent lifetime imaging microscopy were examined in the current study. These were picosecond time-resolved epiphosphorescence microscopy (single 0.5 µm focused spot) and two-photon confocal laser scanning microscopy with pinhole shifting. Both methods utilized nanoparticle-embedded Ru complex (45 nm diameter) as the phosphorescent probe, excited using pulsed outputs of Ti–sapphire Tsunami lasers (710–1050 nm). The former method used a 1 ps pulse width excitation beam with vertical polarization via a dichroic mirror (610 nm, XF43) and a 20× objective (NA 0.55, Nikon). Transmitted luminescence (1–2 × 104 counts s−1) was collected and time-correlated single photon counted decay times measured. Alternatively, an unmodified Zeiss LSM510 Confocal NLO microscope with 40× objective (NA 1.3) used successively shifted pinhole positions to collect image data from the lagging trail of the raster scan. Images obtained from two-photon excitation of a yeast (Schizosaccharomyces pombe) and a flagellate fish parasite (Spironucleus vortens), electroporated with Ru complex, indicated the intracellular location and magnitude of O2 gradients, thus confirming the feasibility of optical mapping under different external O2 concentrations. Both methods gave similar lifetimes for Ru complex phosphorescence under aerobic and anaerobic gas phases. Estimation of O2 tensions within individual fibroblasts (human dermal fibroblast (HDF)) and mammary adenocarcinoma (MCF-7) cells was possible using epiphosphorescence microscopy. MCF-7 cells showed lower intracellular O2 concentrations than HDF cells, possibly due to higher metabolic rates in the former. Future work should involve construction of higher resolution 3D maps of Ru coordinate complex lifetime distribution in cultured cell lines.

Fluorescence excitation analysis by two-photon confocal laser scanning microscopy: a new method to identify fluorescent nanoparticles on histological tissue sections

In the present study, we make use of the ability of two-photon confocal laser scanning microscopes (CLSMs) equipped with tunable lasers to produce spectral excitation image sequences. Furthermore, unmixing, which is usually performed on emission image sequences, is performed on these excitation image sequences. We use factor analysis of medical image sequences (FAMIS), which produces factor images, to unmix spectral image sequences of stained structures in tissue sections to provide images of characterized stained cellular structures. This new approach is applied to histological tissue sections of mouse aorta containing labeled iron nanoparticles stained with Texas Red and counterstained with SYTO13, to obtain visual information about the accumulation of these nanoparticles in the arterial wall. The possible presence of Texas Red is determined using a two-photon CLSM associated with FAMIS via the excitation spectra. Texas Red and SYTO13 are thus differentiated, and corresponding factor images specify their possible presence and cellular localization. In conclusion, the designed protocol shows that sequences of images obtained by excitation in a two-photon CLSM enables characterization of Texas Red-stained nanoparticles and other markers. This methodology offers an alternative and complementary solution to the conventional use of emission spectra unmixing to localize fluorescent nanoparticles in tissue samples.

Structural integrity of collagen and elastin in SynerGraft® decellularized–cryopreserved human heart valves

SynerGraft® (SG) decellularized–cryopreserved cardiac valve allografts have been developed to provide a valve replacement option that has reduced antigenicity, retained structural integrity, and the ability to be stored long-term until needed for implantation. However, it is critical to ensure that both the SG processing and cryopreservation of these allografts do not detrimentally affect the extracellular matrix architecture within the tissue. This study evaluates the effects of SG decellularization and subsequent cryopreservation on the extracellular matrix integrity of allograft heart valves. Human aortic and pulmonary valves were trisected, with one-third of each either left fresh (no further processing after dissection), decellularized, or decellularized and cryopreserved. Two-photon laser scanning confocal microscopy was used to visualize collagen and elastin in leaflets and conduits. The optimized percent laser transmission (OPLT) required for full dynamic range imaging of each site was determined, and changes in OPLT were used to infer changes in collagen and elastin signal intensity. Collagen fiber crimp period and collagen and elastin fiber diameter were measured in leaflet tissue. Statistically significant differences in OPLT and the dimensional characteristics of collagen and elastin in study groups were determined through single factor ANOVA. The majority of donor-aggregated average OPLT observations showed no statistically significant differences among all groups, indicating no difference in collagen or elastin signal strength. Morphometric analysis of collagen and elastin fibers revealed no significant alterations in treated leaflet tissues relative to fresh tissues. Collagen and elastin structural integrity within allograft heart valves are maintained through SynerGraft® decellularization and subsequent cryopreservation.

Measurement of capillary pulsations in the rat neocortex with two-photon laser scanning confocal microscopy

Background Hydrocephalus is associated with increased pulsations in the cerebral aqueduct, as demonstrated by cine MRI, as well as increased pulse pressure, as demonstrated by invasive intracranial pressure monitoring. What has yet to be elucidated is the relationship between increased pulsations and the pathophysiology of hydrocephalus. Are increased pulsations an important component of the pathophysiology, or simply an artefact of decreased intracranial compliance? We have shown that under normal circumstances, the transmission of arterial pulsations into the cranium is minimized (the so-called Windkessel effect). In this paper, we sought to demonstrate this effect directly by measuring capillary pulsations with two-photon laser scanning confocal microscopy.