Two-photon Scanning Fluorescence Microscopy Research Articles

Fiber enhancement and 3D orientation analysis in label-free two-photon fluorescence microscopy

Fluorescence microscopy can be exploited for evaluating the brain’s fiber architecture with unsurpassed spatial resolution in combination with different tissue preparation and staining protocols. Differently from state-of-the-art polarimetry-based neuroimaging modalities, the quantification of fiber tract orientations from fluorescence microscopy volume images entails the application of specific image processing techniques, such as Fourier or structure tensor analysis. These, however, may lead to unreliable outcomes as they do not isolate myelinated fibers from the surrounding tissue. In this work, we describe a novel image processing pipeline that enables the computation of accurate 3D fiber orientation maps from both grey and white matter regions, exploiting the selective multiscale enhancement of tubular structures of varying diameters provided by a 3D implementation of the Frangi filter. The developed software tool can efficiently generate orientation distribution function maps at arbitrary spatial scales which may support the histological validation of modern diffusion-weighted magnetic resonance imaging tractography. Despite being tested here on two-photon scanning fluorescence microscopy images, acquired from tissue samples treated with a label-free technique enhancing the autofluorescence of myelinated fibers, the presented pipeline was developed to be employed on all types of 3D fluorescence images and fiber staining.

Novel Design and Application of High-NA Fiber Imaging Bundles for In Vivo Brain Imaging with Two-Photon Scanning Fluorescence Microscopy.

Here, we provide experimental verification supporting the use of short-section imaging bundles for two-photon microscopy imaging of the mouse brain. The 8 mm long bundle is made of a pair of heavy-metal oxide glasses with a refractive index contrast of 0.38 to ensure a high numerical aperture NA = 1.15. The bundle is composed of 825 multimode cores, ordered in a hexagonal lattice with a pixel size of 14 μm and a total diameter of 914 μm. We demonstrate successful imaging through custom-made bundles with 14 μm resolution. As the input, we used a 910 nm Ti-sapphire laser with 140 fs pulse and a peak power of 9 × 104 W. The excitation beam and fluorescent image were transferred through the fiber imaging bundle. As test samples, we used 1 μm green fluorescent latex beads, ex vivo hippocampal neurons expressing green fluorescent protein and cortical neurons in vivo expressing the fluorescent reporter GCaMP6s or immediate early gene Fos fluorescent reporter. This system can be used for minimal-invasive in vivo imaging of the cerebral cortex, hippocampus, or deep brain areas as a part of a tabletop system or an implantable setup. It is a low-cost solution, easy to integrate and operate for high-throughput experiments.

The facilitation of corneal permeability of cyclosporine A loaded on chitosan-graft-cyclodextrin copolymers vector

Objective To investigate the corneal permeability of cyclosprin A (CsA) loaded on polymeric vector after topical application. Methods The grafted copolymer chitosan-graft-cyclodextrin (CS-g-CD) was synthesized, and the physicochemical structures of the polymer were investigated using nuclear magnetic resonance spectroscopy (NMR) and fourier transform infrared spectroscopy (FT-IR). A novel CsA eye drop was prepared using the grafted copolymer as carrier material.The physicochemical properties of eye drop, including drug-loading content, osmotic pressure and viscosity were investigated by high performance liquid chromatography-mass spectrometry (HPLC-MS), osmotic pressure gauge and viscometer, respectively.New Zealand albino rabbits were randomly divided into intact cornea CsA group, epithelium debrided CsA group and epithelium debrided control group.The corneal epithelia of the left eyes was debrided in the cornea epithelium debrided group.Cornea irritation test was performed on New Zealand albino rabbits.The aqueous humor was taken and the corneas were collected at 0.5 hour and 1 hour after instilled.The concentration of CsA was measured by HPLC-MS.Cy5 labeled vector loaded with Coumarin 6 served as model copolymers system, the penetration capabilities of the double fluorescent labeling copolymers system were monitored in vivo using two-photon scanning fluorescence microscopy on murine corneas after topical application.The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission. Results The polymer of CS-g-CD was successfully synthesized and confirmed using NMR and FT-IR.The drug loading of CsA in eye drop solution was 0.06 %; the osmotic pressure was 305 mOsmol/kg and the viscosity was 36.5 cP.The CsA drug delivery system had a reversible temperature-sensitive drug release behavior and had no obvious irritation on the eyes of New Zealand rabbits.One hour after treatment, the concentration of CsA in the cornea and aqueous humor of epithelium debrided CsA group was (5.88±1.46)μg/g and (149.19±3.93)ng/ml, respectively, which was significantly higher than (3.98±0.95)μg/g and (30.25±11.43)ng/ml in epithelium debrided control group (both at P<0.05); the concentration of CsA in the aqueous humor of intact cornea CsA group was (7.23±1.31)ng/ml, which was significantly lower than that in epithelium debrided CsA group (P<0.05). Polymer vectors were mainly retained in the corneal epithelium, and coumarin 6 gradually diffused into the deep corneal stroma with time. Conclusions The grafted copolymer can load CsA, and the eye drop can effectively overcome the corneal barrier and increase the corneal permeability of CsA. Key words: Cyclosporine A; Immunosuppressive agents; Corneal barrier; Fluorescent double labeling; Corneal tissue distribution

Positively charged micelles based on a triblock copolymer demonstrate enhanced corneal penetration.

PurposeThe cornea is a main barrier to drug penetration after topical application. The aim of this study was to evaluate the abilities of micelles generated from a positively charged triblock copolymer to penetrate the cornea after topical application.MethodsThe triblock copolymer poly(ethylene glycol)-poly(ε-caprolactone)-g-polyethyleneimine was synthesized, and the physicochemical properties of the self-assembled polymeric micelles were investigated, including hydrodynamic size, zeta potential, morphology, drug-loading content, drug-loading efficiency, and in vitro drug release. Using fluorescein diacetate as a model drug, the penetration capabilities of the polymeric micelles were monitored in vivo using a two-photon scanning fluorescence microscopy on murine corneas after topical application.ResultsThe polymer was successfully synthesized and confirmed using nuclear magnetic resonance and Fourier transform infrared. The polymeric micelles had an average particle size of 28 nm, a zeta potential of approximately +12 mV, and a spherical morphology. The drug-loading efficiency and drug-loading content were 75.37% and 3.47%, respectively, which indicates that the polymeric micelles possess a high drug-loading capacity. The polymeric micelles also exhibited controlled-release behavior in vitro. Compared to the control, the positively charged polymeric micelles significantly penetrated through the cornea.ConclusionPositively charged micelles generated from a triblock copolymer are a promising vehicle for the topical delivery of hydrophobic agents in ocular applications.

Grating imager systems for fluorescence optical-sectioning microscopy.

In fluorescence microscopy, optical sectioning is defined as the attenuation or removal of out-of-focus features from an image, and it is a prerequisite for quantitative analysis of three-dimensional structure or function within the specimen. Optical sectioning is most commonly performed by confocal scanning fluorescence microscopy or two-photon scanning fluorescence microscopy. However, structured illumination can be used in conventional fluorescence microscopes to obtain optical sectioning performance, and, in advanced systems, 3D superresolution. The simplest structured-illumination system uses a Ronchi grating as a mask to project parallel stripes within the sharp depth-of-focus of the objective to encode in-focus specimen features differently from out-of-focus features. By shifting the grating, the in-focus image component can be discriminated and separated by elementary image processing operations. This implementation of structured illumination, the fluorescence grating imager, uses a conventional light source, is compatible with all high-quality fluorescence filter sets, and provides high optical-sectioning performance when used to image specimens in which (1) the out-of-focus image component is not much brighter than the in-focus features and (2) there is no significant movement in the specimen during the grating shift and image capture process.

Two-photon microscopy with wavelength switchable fiber laser excitation

Two-photon scanning fluorescence microscopy has become a powerful tool for imaging living cells and tissues. Most applications of two-photon microscopy employ a Ti:sapphire laser excitation source, which is not readily portable or rapidly tunable. This work explores the use of two-photon fiber laser excitation (TP-FLEX) as an excitation source for scanning two-photon microscopy. We have further demonstrated the use of a photonic crystal fiber (PCF) for facile tuning of the excitation wavelength over the range from 810 nm to 1100 nm. We generated two-photon fluorescence images at excitation wavelengths from 850 nm to 1100 nm detected on a scanning-stage microscope. By PCF wavelength tuning the dye BODIPY fl was selectively excited at 1000 nm whereas MitoTracker red was excited preferentially at 1100 nm. We discuss the potential for fiber laser sources coupled with PCF wavelength tuning as an attractive tunable excitation source for two-photon scanning fluorescence microscopy.

Fibre-optic two-photon scanning fluorescence microscopy.

Two geometries of a novel two-photon fluorescence microscope incorporating single-mode fibre optics for the delivery of ultrashort-pulsed illumination to a remote sample are characterized. First, a 785 nm single-mode optical fibre is implemented in a scanning microscope, which demonstrates that an improvement in axial resolution is achieved due to the non-linear response of the fibre to intense ultrashort-pulsed light. Second, a 785 nm single-mode optical fibre coupler is adapted, in which case spectral broadening and blue shifting of the ultrashort-pulsed laser beam caused by the non-linear response of the fibre to ultrashort-pulsed illumination are experimentally characterized. An investigation into the impact of temporal broadening of the ultrashort-pulsed beam on the systems is also considered. The coupling efficiency of both geometries for various illumination wavelengths is also presented. The introduction of the fibre coupler to the system has significant advantages, including an improved optical sectioning effect and a reduction in the number of bulk optical components resulting in a low-cost, compact instrument. Sets of three-dimensional images of fluorescent polymer microspheres and biological material confirm these features.

In Vitro Visualization and Quantification of Oleic Acid Induced Changes in Transdermal Transport Using Two-Photon Fluorescence Microscopy

In a novel application of two-photon scanning fluorescence microscopy, three-dimensional spatial distributions of the hydrophilic and hydrophobic fluorescent probes, sulforhodamine B and rhodamine B hexyl ester, in excised full-thickness human cadaver skin were visualized and quantified. Both sulforhodamine B and rhodamine B hexyl ester were observed to lie primarily in the lipid multilamellae region surrounding the corneocytes within the stratum corneum. From the two-photon scanning fluorescence microscopy scans, the changes in the concentration gradient and the vehicle to skin partition coefficient of each probe induced by the oleic acid enhancer action were calculated relative to the control sample (not exposed to oleic acid), and subsequently applied to theoretically derived mathematical expressions of transdermal transport to quantitatively characterize the oleic acid-induced relative changes in the skin diffusion coefficient and the skin barrier diffusion length of the permeant. For the hydrophobic probe rhodamine B hexyl ester, the permeability enhancement was primarily driven by an increase in the vehicle to skin partition coefficient, leading to an increase in the steepness of the concentration gradient across the skin. The primary oleic acid-induced changes in the transport properties of the hydrophilic probe sulforhodamine B included increases in the vehicle to skin partition coefficient and the skin diffusion coefficient. These findings utilizing the two-photon scanning fluorescence microscopy methodology and data analysis described here demonstrate that, in addition to providing three-dimensional images that clearly delineate probe distributions in the direction of increasing skin depth, the subsequent quantification of these images provides additional important insight into the mechanistic changes in transdermal transport underlying the visualized changes in probe distributions across the skin.