Multiphoton-Polymerized 3D Protein Assay.
Ontology highlight
ABSTRACT:
SUBMITTER: Wollhofen R
PROVIDER: S-EPMC5773935 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Controlled drug delivery systems have been utilized to enhance the therapeutic effects of many drugs by delivering drugs in a time-dependent and sustained manner. Here, with the aid of 3D printing technology, drug delivery devices were fabricated and tested using a model drug (fluorophore: rhodamine B). Poly(ethylene glycol) dimethacrylate (PEGDMA) devices were fabricated using a two-photon polymerization (TPP) system and rhodamine B was homogenously entrapped inside the polymer matrix during photopolymerization. These devices were printed with varying porosity and morphology using varying printing parameters such as slicing and hatching distance. The effects of these variables on drug release kinetics were determined by evaluating device fluorescence over the course of one week. These PEGDMA-based structures were then investigated for toxicity and biocompatibility in vitro, where MTS assays were performed using a range of cell types including induced pluripotent stem cells (iPSCs). Overall, tuning the hatching distance, slicing distance, and pore size of the fabricated devices modulated the rhodamine B release profile, in each case presumably due to resulting changes in the motility of the small molecule and its access to structure edges. In general, increased spacing provided higher drug release while smaller spacing resulted in some occlusion, preventing media infiltration and thus resulting in reduced fluorophore release. The devices had no cytotoxic effects on human embryonic kidney cells (HEK293), bone marrow stromal stem cells (BMSCs) or iPSCs. Thus, we have demonstrated the utility of two-photon polymerization to create biocompatible, complex miniature devices with fine details and tunable release of a model drug.
| S-EPMC6204107 | biostudies-literature
Project description:Quantifying skin aging changes and characterizing its 3D structure and function in a non-invasive way is still a challenging area of research, constantly evolving with the development of imaging methods and image analysis tools. In vivo multiphoton imaging offers means to assess skin constituents in 3D, however prior skin aging studies mostly focused on 2D analyses of dermal fibers through their signals' intensities or densities. In this work, we designed and implemented multiphoton multiparametric 3D quantification tools for in vivo human skin pigmentation and aging characterization. We first demonstrated that despite the limited field of view of the technic, investigation of 2 regions of interest (ROIs) per zone per volunteer is a good compromise in assessing 3D skin constituents in both epidermis and superficial dermis. We then characterized skin aging on different UV exposed areas-ventral and dorsal forearms, face. The three major facts of aging that are epidermal atrophy, the dermal-epidermal junction (DEJ) flattening and dermal elastosis can be non-invasively quantified and compared. Epidermal morphological changes occur late and were only objectified between extreme age groups. Melanin accumulation in suprabasal layers with age and chronic exposure on ventral and dorsal forearms is less known and appears earlier. Superficial dermal aging changes are mainly elastin density increase, with no obvious change in collagen density, reflected by SHGto2PEF ratio and SAAID index decrease and ImbrN index increase on all skin areas. Analysis of the z-dermal distribution of these parameters highlighted the 2nd 20 µm thickness normalized dermal sub-layer, that follows the DEJ shape, as exhibiting the highest aging differences. Moreover, the 3D ImbrN index allows refining the share of photoaging in global aging on face and the 3D SAAID index on forearm, which elastin or fibrillar collagens densities alone do not allow. Photoaging of the temple area evolves as a function of chronic exposure with a more pronounced increase in elastin density, also structurally modified from thin and straight elastic fibers in young volunteers to dense and compact pattern in older ones. More generally, multiphoton multiparametric 3D skin quantification offers rich spatial information of interest in assessing normal human skin condition and its pathological, external environment or product induced changes.
| S-EPMC9437074 | biostudies-literature
Project description:We report a method for creating stimuli-responsive biomaterials in which scanning nonlinear excitation is used to photocrosslink proteins at submicrometer 3D coordinates. Proteins with differing hydration properties can be combined to achieve tunable volume changes that are rapid and reversible in response to changes in chemical environment. Protein matrices having arbitrary 3D topographies and definable density gradients over micrometer dimensions provide the ability to effect rapid (<1 sec) and precise mechanical manipulations by means of changes in hydrogel size and shape, and applicability of these materials to cell biology is shown through the fabrication of responsive bacterial cages.
| S-EPMC2449329 | biostudies-literature
Project description:There is an increasing demand for rapid, effective methods to identify and detect protein micro- and nano-crystal suspensions for serial diffraction data collection at X-ray free-electron lasers or high-intensity micro-focus synchrotron radiation sources. Here, we demonstrate a compact multimodal, multiphoton microscope, driven by a fiber-based ultrafast laser, enabling excitation wavelengths at 775 nm and 1300 nm for nonlinear optical imaging, which simultaneously records second-harmonic generation, third-harmonic generation and three-photon excited ultraviolet fluorescence to identify and detect protein crystals with high sensitivity. The instrument serves as a valuable and important tool supporting sample scoring and sample optimization in biomolecular crystallography, which we hope will increase the capabilities and productivity of serial diffraction data collection in the future.
| S-EPMC7553921 | biostudies-literature
Project description:Optical recordings of neuronal activity at cellular resolution represent an invaluable tool to investigate brain mechanisms. Zebrafish larvae is one of the few model organisms where, using fluorescence-based reporters of the cell activity, it is possible to optically reconstruct the neuronal dynamics across the whole brain. Typically, leveraging the reduced light scattering, methods like lightsheet, structured illumination, and light-field microscopy use spatially extended excitation profiles to detect in parallel activity signals from multiple cells. Here, we present an alternative design for whole brain imaging based on sequential 3D point-scanning excitation. Our approach relies on a multiphoton microscope integrating an electrically tunable lens. We first apply our approach, adopting the GCaMP6s activity reporter, to detect functional responses from retinal ganglion cells (RGC) arborization fields at different depths within the zebrafish larva midbrain. Then, in larvae expressing a nuclear localized GCaMP6s, we recorded whole brain activity with cellular resolution. Adopting a semi-automatic cell segmentation, this allowed reconstructing the activity from up to 52,000 individual neurons across the brain. In conclusion, this design can easily retrofit existing imaging systems and represents a compact, versatile and reliable tool to investigate neuronal activity across the larva brain at high resolution.
| S-EPMC8154985 | biostudies-literature
Project description:In recent years, 3D printing of electronics have received growing attention due to their potential applications in emerging fields such as nanoelectronics and nanophotonics. Multiphoton lithography (MPL) is considered the state-of-the-art amongst the microfabrication techniques with true 3D fabrication capability owing to its excellent level of spatial and temporal control. Here, a homogenous and transparent photosensitive resin doped with an organic semiconductor material (OS), which is compatible with MPL process, is introduced to fabricate a variety of 3D OS composite microstructures (OSCMs) and microelectronic devices. Inclusion of 0.5 wt% OS in the resin enhances the electrical conductivity of the composite polymer about 10 orders of magnitude and compared to other MPL-based methods, the resultant OSCMs offer high specific electrical conductivity. As a model protein, laminin is incorporated into these OSCMs without a significant loss of activity. The OSCMs are biocompatible and support cell adhesion and growth. Glucose-oxidase-encapsulated OSCMs offer a highly sensitive glucose sensing platform with nearly tenfold higher sensitivity compared to previous glucose biosensors. In addition, this biosensor exhibits excellent specificity and high reproducibility. Overall, these results demonstrate the great potential of these novel MPL-fabricated OSCM devices for a wide range of applications from flexible bioelectronics/biosensors, to nanoelectronics and organ-on-a-chip devices.
| S-EPMC9339506 | biostudies-literature
Project description:Moxifloxacin and gatifloxacin are fourth-generation fluoroquinolone antibiotics used in the clinic to prevent or treat ocular infections. Their pharmacokinetics in the cornea is usually measured from extracted ocular fluids or tissues, and in vivo direct measurement is difficult. In this study multiphoton microscopy (MPM), which is a 3D optical microscopic technique based on multiphoton fluorescence, was applied to the measurement of moxifloxacin and gatifloxacin distribution in the cornea. Intrinsic multiphoton fluorescence properties of moxifloxacin and gatifloxacin were characterized, and their distributions in mouse cornea in vivo were measured by 3D MPM imaging. Both moxifloxacin and gatifloxacin had similar multiphoton spectra, while moxifloxacin had stronger fluorescence than gatifloxacin. MPM imaging of mouse cornea in vivo showed (1) moxifloxacin had good penetration through the superficial corneal epithelium, while gatifloxacin had relatively poor penetration, (2) both ophthalmic solutions had high intracellular distribution. In vivo MPM results were consistent with previous studies. This study demonstrates the feasibility of MPM as a method for in vivo direct measurement of moxifloxacin and gatifloxacin in the cornea.
| S-EPMC4853790 | biostudies-literature
Project description:Characterizing melanins in situ and determining their 3D z-epidermal distribution is paramount for understanding physiological/pathological processes of melanin neosynthesis, transfer, degradation or modulation with external UV exposure or cosmetic/pharmaceutical products. Multiphoton fluorescence intensity- and lifetime-based approaches have been shown to afford melanin detection, but how can one quantify melanin in vivo in 3D from multiphoton fluorescence lifetime (FLIM) data, especially since FLIM imaging requires long image acquisition times not compatible with 3D imaging in a clinical setup? We propose an approach combining (i) multiphoton FLIM, (ii) fast image acquisition times, and (iii) a melanin detection method called Pseudo-FLIM, based on slope analysis of autofluorescence intensity decays from temporally binned data. We compare Pseudo-FLIM to FLIM bi-exponential and phasor analyses of synthetic melanin, melanocytes/keratinocytes coculture and in vivo human skin. Using parameters of global 3D epidermal melanin density and z-epidermal distribution profile, we provide first insights into the in vivo knowledge of 3D melanin modulations with constitutive pigmentation versus ethnicity, with seasonality over 1 year and with topical application of retinoic acid or retinol on human skin. Applications of Pseudo-FLIM based melanin detection encompass physiological, pathological, or environmental factors-induced pigmentation modulations up to whitening, anti-photoaging, or photoprotection products evaluation.
| S-EPMC8803839 | biostudies-literature
Project description:There is a high demand for 3D multiphoton imaging in neuroscience and other fields but scanning in axial direction presents technical challenges. We developed a focusing technique based on a remote movable mirror that is conjugate to the specimen plane and translated by a voice coil motor. We constructed cost-effective z-scanning modules from off-the-shelf components that can be mounted onto standard multiphoton laser scanning microscopes to extend scan patterns from 2D to 3D. Systems were designed for large objectives and provide high resolution, high speed and a large z-scan range (>300 μm). We used these systems for 3D multiphoton calcium imaging in the adult zebrafish brain and measured odor-evoked activity patterns across >1500 neurons with single-neuron resolution and high signal-to-noise ratio.
| S-EPMC4871072 | biostudies-other
Project description:Rylene dyes functionalized with varying numbers of phenyl trifluorovinylether (TFVE) moieties were subjected to a thermal emulsion polymerization to yield shape-persistent, water-soluble chromophore nanoparticles. Perylene and terrylene diimide derivatives containing either two or four phenyl TFVE functional groups were synthesized and subjected to thermal emulsion polymerization in tetraglyme. Dynamic light scattering measurements indicated that particles with sizes ranging from 70 - 100 nm were obtained in tetraglyme, depending on monomer concentration. The photophysical properties of individual monomers were preserved in the nanoemulsions and emission colors could be tuned between yellow, orange, red, and deep red. The nanoparticles were found to retain their shape upon dissolution into water and the resulting water suspensions displayed moderate to high fluorescence quantum yield.
| S-EPMC3124817 | biostudies-literature