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The biomechanical properties of the human skin are intrinsically correlated with changes associated with pathological conditions, aging, and hydration. Quantitative measurements can improve diagnostic tools, treatments, and cosmetic product evaluation. Using optical coherence elastography (OCE), an emerging imaging modality combining optical coherence tomography (OCT) with a localized excitation source to induce mechanical disturbances, a quantitative evaluation of tissue biomechanics can be achieved. OCE complements the structural information with elasticity data to attain a complete overview of skin status.
In this study, we employed a home-built OCE system, combining a swept-source OCT system with a piezoelectric actuator for tissue displacement, to evaluate changes to the skin biomechanical properties due to the application of an anti-aging cream. Skin elasticity was monitored for a total of five weeks. Anti-aging cream was applied daily for four weeks. OCE measurements continued for one additional week to assess the effect of cream application interruption. Three female volunteers were included in this proof-of-principle investigation. Their counter-arm was used as control. Although no statistical significance was reached, a decrease in skin Young’s modulus was observed with the cream application, indicating an increase in skin elasticity.
Two-photon microscopes have been successfully translated into clinical imaging tools to obtain high-resolution optical biopsies for
In this study, we investigate ACXL-induced changes to the cornea autofluorescence (AF) using MPT. ACXL was performed in de-epithelialized corneal donor buttons and keratoconus corneas by infusing the samples with 0.1% riboflavin solution followed by UVA irradiation using either an in-house adapted system or a commercial ACXL system. AF lifetime images of the tissue were acquired prior and after treatment using MPT. As a control, corneas without treatment were monitored at the same time points.
Higher AF lifetimes were observed in the stroma of treated corneas than in control samples. The stroma AF lifetime was higher anteriorly, corresponding to the area where ACXL was most effective. First changes were observed as soon as 2 ℎ after treatment. We demonstrate that MPT can be used to follow-up the outcome of ACXL and that ACXL-induced changes can be detected sooner than with conventional methods and non-invasively.
For that purpose, we have developed a wide-field time-gated fluorescence lifetime microscope based on structured illumination and one-photon excitation to record FAD lifetime images from corneas. NADH imaging was not considered as its UV excitation peak is regarded as not safe for in vivo measurements. The microscope relies on a pulsed blue diode laser (λ=443 nm) as excitation source, an ultra-high speed gated image intensifier coupled to a CCD camera to acquire fluorescence signals and a Digital Micromirror Device (DMD) to implement the Structured Illumination technique. The system has a lateral resolution better than 2.4 μm, a field of view of 160 per 120 μm and an optical sectioning of 6.91 +/- 0.45 μm when used with a 40x, 0.75 NA, Water Immersion Objective.
With this setup we were able to measure FAD contributions from ex-vivo chicken corneas collected from a local slaughterhouse..
Here we report on the development of a fluorescence lifetime imaging microscope for in vivo measurement of FAD fluorescence lifetimes in corneal cells. The microscope is based on one-photon fluorescence excitation, through a pulsed blue diode laser. Fluorescence lifetime imaging is achieved using the Time-Gated technique. Structured illumination is used to improve the low axial resolution of wide-field time-gated FLIM. A Digital Micromirror Device (DMD) is used to produce the sinusoidal patterns required by structural illumination. The DMD control is integrated with the acquisition software of the imaging system which is based on an ultra-high speed gated image intensifier coupled to a CCD camera.
We present preliminary results concerning optical and timing performance of the fluorescence lifetime microscope. Preliminary tests with ex-vivo bovine corneas are also described.
Samples were imaged using a laser-scanning microscope, consisting of a broadband sub-15 femtosecond (fs) near-infrared laser. Signal detection was performed using a 16-channel photomultiplier tube (PMT) detector (PML-16PMT). Therefore, spectral analysis of the fluorescence lifetime data was possible. To ensure a correct spectral analysis of the autofluorescence lifetime data, the spectra of the individual endogenous fluorophores were acquired with the 16-channel PMT and with a spectrometer. All experiments were performed within 12h of the porcine eye enucleation.
We were able to image the cornea, crystalline lens, and retina at multiple depths. Discrimination of each structure based on their autofluorescence intensity and lifetimes was possible. Furthermore, discrimination between different layers of the same structure was also possible. To the best of our knowledge, this was the first time that 2PE-FLIM was used for porcine lens imaging and layer discrimination. With this study we further demonstrated the feasibility of 2PE-FLIM to image and differentiate three of the main components of the eye and its potential as an ophthalmologic technique.
The reprogramming of somatic cells into induced pluripotent stem (iPS) cells can be evoked through the ectopic expression of defined transcription factors. Conventional approaches utilize retro/lenti-viruses to deliver genes/transcription factors as well as to facilitate the integration of transcription factors into that of the host genome. However, the use of viruses may result in insertional mutations caused by the random integration of genes and as a result, this may limit the use within clinical applications due to the risk of the formation of cancer. In this study, a new approach is demonstrated in realizing non-viral reprogramming through the use of ultrashort laser pulses, to introduce transcription factors into the cell so as to generate iPS cells.
Femtosecond laser-induced cell death is beneficial due to the reduced collateral side effects and therefore can be used to selectively destroy target cells within monolayers, as well as within 3D tissues, all the while preserving cells of interest. This is an important characteristic for the application in stem cell research and cancer treatment. Non-precise damage compromises the viability of neighboring cells by inducing side effects such as stress to the cells surrounding the target due to the changes in the microenvironment, resulting from both the laser and laser-exposed cells.
In this study, optimum laser parameters for optical cleaning by isolating single cells and cell colonies are exploited through the use of automated software control. Physiological equilibrium and cellular responses to the laser induced damages are also investigated. Cell death dependence on laser focus, determination and selectivity of intensity/dosage, controllable damage and cell recovery mechanisms are discussed.
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