Photon Excitation Microscopy Research Articles

The biophysical effects of localized electrochemical therapy on porcine skin

BackgroundMinimally-invasive methods to treat scars address a common pathway of altering collagen structure, leading to collagen remodeling. ObjectiveIn this study, we employed in situ redox chemistry to create focal pH gradients in skin, altering dermal collagen, in a process we refer to as electrochemical therapy (ECT). The effects of ECT to induce biochemical and structural changes in ex vivo porcine skin were examined. MethodsDuring ECT, two platinum electrodes were inserted into fresh porcine skin, and following saline injection, an electrical potential was applied. pH mapping, high frequency ultrasonography, and two photon excitation microscopy and second harmonic generation (SHG) microscopy were used to evaluate treatment effects. Findings were correlated with histology. ResultsFollowing ECT, pH mapping depicted acid and base production at anode and cathode sites respectively, with increasing voltage and application time. Gas formation during ECT was observed with ultrasonography. Anode sites showed significant loss of SHG signal, while cathode sites showed disorganized collagen structure with fewer fibrils emitting an attainable signal. Histologically, collagen denaturation at both sites was confirmed. ConclusionWe demonstrated the production of in situ acid and base in skin occurring via ECT. The effects chemically and precisely alter collagen structure through denaturation, giving insight on the potential of ECT as a simple, low-cost, and minimally-invasive means to remodel skin and treat scars.

P030 Intravital observation system of the colon by two photon excitation microscopy

P030 Intravital observation system of the colon by two photon excitation microscopy

LB972 Characterization of collagen in normal and diseased skin using second harmonic generation and two photon excitation microscopy

LB972 Characterization of collagen in normal and diseased skin using second harmonic generation and two photon excitation microscopy

In Vivo Evaluation of Cervical Stiffness Evolution during Induced Ripening Using Shear Wave Elastography, Histology and 2 Photon Excitation Microscopy: Insight from an Animal Model.

Prematurity affects 11% of the births and is the main cause of infant mortality. On the opposite case, the failure of induction of parturition in the case of delayed spontaneous birth is associated with fetal suffering. Both conditions are associated with precocious and/or delayed cervical ripening. Quantitative and objective information about the temporal evolution of the cervical ripening may provide a complementary method to identify cases at risk of preterm delivery and to assess the likelihood of successful induction of labour. In this study, the cervical stiffness was measured in vivo in pregnant sheep by using Shear Wave Elastography (SWE). This technique assesses the stiffness of tissue through the measurement of shear waves speed (SWS). In the present study, 9 pregnant ewes were used. Cervical ripening was induced at 127 days of pregnancy (term: 145 days) by dexamethasone injection in 5 animals, while 4 animals were used as control. Elastographic images of the cervix were obtained by two independent operators every 4 hours during 24 hours after injection to monitor the cervical maturation induced by the dexamethasone. Based on the measurements of SWS during vaginal ultrasound examination, the stiffness in the second ring of the cervix was quantified over a circular region of interest of 5 mm diameter. SWS was found to decrease significantly in the first 4–8 hours after dexamethasone compared to controls, which was associated with cervical ripening induced by dexamethasone (from 1.779 m/s ± 0.548 m/s, p < 0.0005, to 1.291 m/s ± 0.516 m/s, p < 0.000). Consequently a drop in the cervical elasticity was quantified too (from 9.5 kPa ± 0.9 kPa, p < 0.0005, to 5.0 kPa ± 0.8 kPa, p < 0.000). Moreover, SWE measurements were highly reproducible between both operators at all times. Cervical ripening induced by dexamethasone was confirmed by the significant increase in maternal plasma Prostaglandin E2 (PGE2), as evidenced by the assay of its metabolite PGEM. Histological analyses and two-photon excitation microscopy, combining both Second Harmonic Generation (SHG) and Two-photon Fluorescence microscopy (2PF) contrasts, were used to investigate, at the microscopic scale, the structure of cervical tissue. Results show that both collagen and 2PF-active fibrillar structures could be closely related to the mechanical properties of cervical tissue that are perceptible in elastography. In conclusion, SWE may be a valuable method to objectively quantify the cervical stiffness and as a complementary diagnostic tool for preterm birth and for labour induction success.

Experimenting Liver Fibrosis Diagnostic by Two Photon Excitation Microscopy and Bag-of-Features Image Classification

The accurate staging of liver fibrosis is of paramount importance to determine the state of disease progression, therapy responses, and to optimize disease treatment strategies. Non-linear optical microscopy techniques such as two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) can image the endogenous signals of tissue structures and can be used for fibrosis assessment on non-stained tissue samples. While image analysis of collagen in SHG images was consistently addressed until now, cellular and tissue information included in TPEF images, such as inflammatory and hepatic cell damage, equally important as collagen deposition imaged by SHG, remain poorly exploited to date. We address this situation by experimenting liver fibrosis quantification and scoring using a combined approach based on TPEF liver surface imaging on a Thioacetamide-induced rat model and a gradient based Bag-of-Features (BoF) image classification strategy. We report the assessed performance results and discuss the influence of specific BoF parameters to the performance of the fibrosis scoring framework.

Dual photon excitation microscopy and image threshold segmentation in live cell imaging during compression testing

Morphological studies of live connective tissue cells are imperative to helping understand cellular responses to mechanical stimuli. However, photobleaching is a constant problem to accurate and reliable live cell fluorescent imaging, and various image thresholding methods have been adopted to account for photobleaching effects. Previous studies showed that dual photon excitation (DPE) techniques are superior over conventional one photon excitation (OPE) confocal techniques in minimizing photobleaching. In this study, we investigated the effects of photobleaching resulting from OPE and DPE on morphology of in situ articular cartilage chondrocytes across repeat laser exposures. Additionally, we compared the effectiveness of three commonly-used image thresholding methods in accounting for photobleaching effects, with and without tissue loading through compression. In general, photobleaching leads to an apparent volume reduction for subsequent image scans. Performing seven consecutive scans of chondrocytes in unloaded cartilage, we found that the apparent cell volume loss caused by DPE microscopy is much smaller than that observed using OPE microscopy. Applying scan-specific image thresholds did not prevent the photobleaching-induced volume loss, and volume reductions were non-uniform over the seven repeat scans. During cartilage loading through compression, cell fluorescence increased and, depending on the thresholding method used, led to different volume changes. Therefore, different conclusions on cell volume changes may be drawn during tissue compression, depending on the image thresholding methods used. In conclusion, our findings confirm that photobleaching directly affects cell morphology measurements, and that DPE causes less photobleaching artifacts than OPE for uncompressed cells. When cells are compressed during tissue loading, a complicated interplay between photobleaching effects and compression-induced fluorescence increase may lead to interpretations in cell responses to mechanical stimuli that depend on the microscopic approach and the thresholding methods used and may result in contradictory interpretations.

Forest Filter Effect: Role of leaves in capturing/releasing air particulate matter and its associated PAHs

Plants play a key role in removing particulate matter and their associated Semi-volatile Organic Compounds (SVOCs) from the atmosphere. Understanding the processes involved in particle capture by vegetation is essential to understand the interactions between SVOCs, particles and plants. In the present study Two Photon Excitation Microscopy (TPEM) was used to visualise particle matter uptake and encapsulation, together with its distribution on leaf/needle surface of different broadleaf (cornel and maple) and conifer species (stone pine). Phenanthrene accumulation, the number of particles associated with this compound and its migration from particles into the leaf cuticle was also identified and quantified. Species-specific deposition velocities were estimated to model temporal PM10 leaf/needle accumulation and to investigate the role of Planet Boundary Layer (PBL) height variation in influencing PM10 flux to plants. Particles at the leaf/needle surface were visualised to range in size from 0.2 to 70.4 μm, but cuticular encapsulation was negligible for particles larger than 10.6 μm, which were removed by a washing procedure. Phenanthrene concentration varied between ≈5 and ≈10 ng g−1 dw according to plant species and between ≈10 and ≈200 ng g−1 dw depending on needle age; this compound was visualized to migrate from particles into the adjacent leaf cuticle. Species-specific deposition velocity range between 0.57 and 1.28 m h−1 and preliminary simulations showed that the diel variability of PBL structure influenced the temporal PM10 flux and leaf/needle concentration, e.g. during daytime hours characterized by high PBL height, PM10 accumulated on cornel leaves was about 65% lower than the amount accumulated during night time. The capability of vegetation to capture particles from the atmosphere, retain, encapsulate them into the cuticle and release them to soil and/or lower biomass, highlighted the value of vegetation in removing pollutants from the atmosphere and influencing their environmental fate.

Differential Dynamic and Structural Behavior of Lipid-Cholesterol Domains in Model Membranes

Changes in the cholesterol (Chol) content of biological membranes are known to alter the physicochemical properties of the lipid lamella and consequently the function of membrane-associated enzymes. To characterize these changes, we used steady-state and time resolved fluorescence spectroscopy and two photon-excitation microscopy techniques. The membrane systems were chosen according to the techniques that were used: large unilamellar vesicles (LUVs) for cuvette and giant unilamellar vesicles (GUVs) for microscopy measurements; they were prepared from dipalmitoyl phosphatidylcholine (DPPC) and dioctadecyl phosphatidylcholine (DOPC) in mixtures that are well known to form lipid domains. Two fluorescent probes, which insert into different regions of the bilayer, were selected: 1,6-diphenyl-1,3,5-hexatriene (DPH) was located at the deep hydrophobic core of the acyl chain regions and 2-dimethylamino-6-lauroylnaphthalene (Laurdan) at the hydrophilic-hydrophobic membrane interface. Our spectroscopy results show that (i) the changes induced by cholesterol in the deep hydrophobic phospholipid acyl chain domain are different from the ones observed in the superficial region of the hydrophilic-hydrophobic interface, and these changes depend on the state of the lamella and (ii) the incorporation of cholesterol into the lamella induces an increase in the orientation dynamics in the deep region of the phospholipid acyl chains with a corresponding decrease in the orientation at the region close to the polar lipid headgroups. The microscopy data from DOPC/DPPC/Chol GUVs using Laurdan generalized polarization (Laurdan GP) suggest that a high cholesterol content in the bilayer weakens the stability of the water hydrogen bond network and hence the stability of the liquid-ordered phase (Lo).

Capillaries are Embedded in the Sarcolemma of Murine Slow Twitch Skeletal Muscle Fibers

The purpose of this study was to evaluate the in vivo, 3D spatial relationship between capillaries (CAP), mitochondria (MITO), and three fiber types within murine Tibialis anterior (TA) muscle. Two photon excitation microscopy was utilized for in vivo imaging of MITO (NADH autofluorescence), vasculature, and interstitial space using injected dyes. A custom program was used to trace fibers and determine contact and volume ratios between CAP and 127 muscle fibers. Fiber type (slow (ST), intermediate (IT), fast‐twitch (FT)) was determined from relative NADH intensity and fiber size. Microscopy revealed that half of ST fiber CAP (53.2 ± 4.0%) had at least 50% of their circumference embedded in a groove in the sarcolemma. Embedded CAP were tightly associated with dense MITO populations lateral to the CAP grooves but nearly absent below the groove where fibrils dominated. CAP embedding was significantly lower in IT (29.5 ± 4.2%) and FT (14.2 ± 1.8%). Electron microscopy confirmed ST embedded CAP and MITO localization. A specialized sarcolemma/CAP groove is present in murine ST fibers resulting in CAP embedding in a MITO rich region. This structure optimizes CAP O2 delivery to highly aerobic ST fibers and selectively restricts O2 delivery to FT fibers. Localized contact of CAP with ST plasma membranes suggests receptors or transporters for blood borne signaling or substrate molecules may also be concentrated in these regions.

3D Imaging by Multiphoton Selective Plane Illumination

Light-sheet based techniques, such as single plane illumination microscopy (SPIM) [1] and digital scanned laser microscopy (DSLM) [2], have been found particularly useful in developmental biology applications since they provide the capability to perform fast imaging of living samples reducing photobleaching effects. In particular, single plane illumination provides high signal to noise ratio and optical sectioning capability due to the planar excitation, representing a useful tool for biological investigations of thick biological samples.On the other side, two photon excitation microscopy (2PE) has been demonstrated to be a suitable tool for improving penetration depth since it allows for reduction of the scattering effects and light-sample interactions. To improve image quality and penetration depth of light sheet illumination microscopy, two photon excitation can be coupled with planar illumination [3] thus reducing the scattering effects due to light-sample interactions.We characterized the two photon excitation process within the light sheet and we applied this technique to image large biological samples [4]. 3D imaging of mammary cell spheroids in depth has been performed using the resulting hybrid architecture.Keywords: Two photon excitation, Single plane illumination microscopy.(1) Huisken, J., et al. Science 305, 1007-1009 (2004).(2) Keller, P. J., Schmidt, A. D., Wittbrodt, J., and Stelzer, E.H.K. Science, 322 (2008).(3) Truong et al. Nature Methods 8,757-760, (2011)(4) Cella Zanacchi et al. Proc. of SPIE, vol.7903 “Multiphoton Microscopy in the Biomedical Sciences XI” (2011)

Multiphoton and STED Imaging Nanoscopy

The coupling of multi photon excitation microscopy with optical nanoscopy allows envisaging developments towards bioimaging of thick samples at nanoscale resolution. Fluorescence depletion applied to multi photon excitation allows overcoming one of the main drawbacks related to multi photon imaging, i.e. poorer resolution with respect to confocal microscopy. In order to have a potentially better control of distortions when imaging thick highly scattering specimens we focused on using the very same wavelength for excitation and depletion. To this end, we developed a new class of 2-photon excitation - stimulated emission depletion microscope (2PE- STED) using the very same wavelength for excitation and depletion. We show the opportunity to perform super-resolved fluorescence imaging, exciting and stimulating the emission of a fluorophore by exploiting the very same laser source properly split. The broadening of the cross section in the 2PE regime allows the excitation of several dyes in the visible window without changing wavelength. We show that a red-emitting fluorophore widely used for STED applications, ATTO647n, could be 2-photon excited at a wavelength that sits on its emission spectrum. This fact opens the possibility to perform super-resolved imaging at a resolution 5-6 times better than conventional 2PE microscopy.

Testate Amoebae Examined by Confocal and Two-Photon Microscopy: Implications for Taxonomy and Ecophysiology

Testate amoebae (TA) are a group of free-living protozoa, important in ecology and paleoecology. Testate amoebae taxonomy is mainly based on the morphological features of the shell, as examined by means of light microscopy or (environmental) scanning electron microscopy (SEM/ESEM). We explored the potential applications of confocal laser scanning microscopy (CLSM), two photon excitation microscopy (TPEM), phase contrast, differential interference contrast (DIC Nomarski), and polarization microscopy to visualize TA shells and inner structures of living cells, which is not possible by SEM or environmental SEM. Images captured by CLSM and TPEM were utilized to create three-dimensional (3D) visualizations and to evaluate biovolume inside the shell by stereological methods, to assess the function of TA in ecosystems. This approach broadens the understanding of TA cell and shell morphology, and inner structures including organelles and endosymbionts, with potential implications in taxonomy and ecophysiology.

Confocal Laser Scanning Microscopy and Two Photon Excitation Microscopy as Tools to Study Testate Amoebae

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

Electric field allowed molecular transitions for one and two photon excitation microscopy

We propose an excitation technique for observing single and two photon excitation in those molecules for which such transitions are forbidden by the selection rules. This is possible by the application of an external electric field that perturbs the molecular orbitals, thereby resulting in a significant shift of energy levels. Such a shift of energy levels may bring those levels in resonance with the radiation field which is normally forbidden by selection rules. Further, parity of the these states may significantly improve the emission process. The external electric field results in the mixing of excited (short lifetime) and metastable states (long lifetime), thus reducing the lifetime of metastable (or near metastable) states. This may provide an effective channel for allowing transition from the metastable states. An application of electric field may result in the excitation of poorly excitable biomolecules. This excitation technique may find applications in single- and multi-photon fluorescence microscopy, bioimaging and optical devices.