Second-harmonic generation (SHG) is uniquely capable of imaging collagen non-invasively with high-resolution, making it ideal to evaluate tissue organization in health and disease. For this, quantitative data analysis is essential. Different approaches have been proposed to quantify tissue organization from SHG images. Nevertheless, these methods have never been objectively evaluated or compared. In this study, we performed a comprehensive analysis on the performance of different metrics in computer-generated SHG images with increasing levels of disorganization to evaluate the advantages and limitations of each approach.
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.
Compressive single pixel imaging (C-SPI) is a novel imaging technique able to reconstruct images using only a single pixel detector and a partial measurement of the scene. This technique uses different structured illumination, generated by a spatial modulator, to illuminate the scene and measures the reflected or transmitted light intensities. This compressive stage allows for a sub-Nyquist set of measurements, which significantly increases the technique efficiency, while maintaining a good reconstruction image quality. An experimental setup was developed to study the ability of C-SPI to determine the lifetime and mean intensity of an oxygen sensitive biomarker (Pt(II) ring-fused chlorins). This biomarker phosphorescence is quenched in the presence of oxygen resulting in smaller intensity and lifetime. A structured illumination system was used to stimulate the sample with Hadamard patterns, in the range between 428 nm and 620 nm, while a photodiode collected the emitted light peaking at 756nm. The imaged scene was composed of two toluene solutions with the same biomarker concentration (1 μM), one in the presence of oxygen and the other in its absence. The images were reconstructed using total variation minimization by augmented lagrangian and alternating direction algorithms (TVAL3) with compressive ratios of 25%, 10% and 5%. The proposed method was able to spatially locate the sample contained in a deoxygenated environment in two distinct spatial locations. The deoxygenated sample lifetime and mean intensity of the reconstructed images were, respectively, approximate 20 μs and 2.1×10-3 AU, for the first location, and 20 μs and 4.0×10-3 AU for the second location. Regarding the oxygenated sample, the results were scattered in a long range for lifetime but achieved a mean intensity of approximately 4.8×10-5 AU. Consistent results were obtained for the three compression ratios without major loss in image quality. The proposed method showed that SPI has the ability to perform simultaneous phosphorescence lifetime and intensity imaging. The introduction of compressive sensing makes this technology more attractive to practical applications because it lowers the amount of time necessary to image the sample. The C-SPI is a simple and less expensive technique because it dismisses the use of a fast two dimensional detector (CCD) and the associated electronics, as well as mechanical scanning procedures. Also, multiple single pixel detectors, sensitive to different wavelengths, can make these instruments versatile and allow for simultaneous phosphorescence biomarkers analysis. The next steps in the research will be to study changes in the sample concentration and different percentages of dissolved oxygen.
Detecting corneal cells metabolic alterations may prove a valuable tool in the early diagnosis of corneal diseases. Nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are autofluorescent metabolic co-factors that allow the assessment of metabolic changes through non-invasive optical methods. These co-factors exhibit double-exponential fluorescence decays, with well-separated short and lifetime components, which are related to their protein-bound and free-states. Corneal metabolism can be assessed by measuring the relative contributions of these two components.
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..
Five dimensional microscopy with a 12-fs laser scanning microscope based on spectrally resolved two-photon autofluorescence lifetime and second-harmonic generation (SHG) imaging was used to characterize all layers of the porcine cornea. This setup allowed the simultaneous excitation of both metabolic cofactors, NAD(P)H and flavins, and their discrimination based on their spectral emission properties and fluorescence decay characteristics. Furthermore, the architecture of the stromal collagen fibrils was assessed by SHG imaging in both forward and backward directions. Information on the metabolic state and the tissue architecture of the porcine cornea were obtained with subcellular resolution, and high temporal and spectral resolutions.
Metabolic imaging can be a valuable tool in the early diagnosis of corneal diseases. Cell metabolic changes can be assessed through non-invasive optical methods due to the autofluorescence of metabolic co-factors nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). Both molecules exhibit double exponential fluorescence decays, with well-separated short and long lifetime components, which are related to their protein-bound and free states. Corneal metabolism can be monitored by measuring the relative contribution of these two components.
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.
Corneal function can be drastically affected by several degenerations and dystrophies, leading to blindness. Early diagnosis of corneal disease is of major importance and it may be accomplished by monitoring changes of the metabolic state and structural organization, the first detectable pathological signs, by two-photon excitation autofluorescence lifetime and second-harmonic generation imaging. In this study, we propose to use these imaging techniques to differentiate between healthy and pathological corneas. Images were acquired using a laser-scanning microscope with a broadband sub-15 femtosecond near-infrared pulsed laser and a 16-channel photomultiplier tube detector for signal collection. This setup allows the simultaneous excitation of metabolic co-factors and to identify them based on their fluorescence spectra. We were able to discriminate between healthy and pathological corneas using two-photon excitation autofluorescence lifetime and second-harmonic generation imaging from corneal epithelium and stroma. Furthermore, differences between different pathologies were observed. Alterations in the metabolic state of corneal epithelial cells were observed using the autofluorescence lifetime of the metabolic co-factors. In the corneal stroma, we observed not only alterations in the collagen fibril structural organization but also alterations in the autofluorescence lifetime. Further tests are required as the number of pathological samples must be increased. In the future, we intend to establish a correlation between the metabolic and structural changes and the disease stage. This can be a step forward in achieving early diagnosis.
Multiphoton microscopy is a non-invasive imaging technique with ideal characteristics for biological applications. In this study, we propose to characterize three major structures of the porcine eye, the cornea, crystalline lens, and retina using two-photon excitation fluorescence lifetime imaging microscopy (2PE-FLIM).
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.
We evaluated the effect of different irradiation parameters in low-level laser therapy (LLLT) for treating inflammation induced in the gastrocnemius muscle of rats through cytokines concentration in systemic blood and analysis of muscle tissue. We used continuous (830 and 980 nm) and pulsed illuminations (830 nm). Animals were divided into five groups per wavelength (10, 20, 30, 40, and 50 mW), and a control group. LLLT was applied during 5 days with a constant irradiation time and area. TNF-α, IL-1β, IL-2, and IL-6 cytokines were quantified by ELISA. Inflammatory cells were counted using microscopy. Identical methodology was used with pulsed illumination. Average power (40 mW) and duty cycle were kept constant (80%) at five frequencies (5, 25, 50, 100, and 200 Hz). For continuous irradiation, treatment effects occurred for all doses, with a reduction of TNF-α, IL-1β, and IL-6 cytokines and inflammatory cells. Continuous irradiation at 830 nm was more effective, a result explained by the action spectrum of cytochrome c oxidase (CCO). Best results were obtained for 40 mW, with data suggesting a biphasic dose response. Pulsed wave irradiation was only effective for higher frequencies, a result that might be related to the rate constants of the CCO internal electron transfer process.
Alterations to the corneal cell metabolism or to the structural organization of collagen fibrils occur in several corneal and
systemic pathologies. In this work we resort to multiphoton microscopy corneal imaging to achieve a characterization of
the corneal state. Using fluorescence lifetime imaging microscopy (FLIM) the assessment of the metabolic state of
corneal cells is possible, whereas second harmonic generation (SHG) imaging can be used to assess corneal structural
alterations. A sub-15 fs near-infrared laser source with a broad excitation spectrum was used for SHG imaging and
FLIM. The broad spectrum allows simultaneous excitation of both metabolic co-factors. The signals were collected by a
photomultiplier tubes (PMT) detector with 16 simultaneous recording channels, which allowed the separation of
fluorophores autofluorescence based on their emission wavelengths. We were able to successfully image ex-vivo human
and porcine cornea at multiple depths. Simultaneous NADH and flavin autofluorescence, SHG of collagen fibrils, and
stroma autofluorescence imaging was performed which may in future allow an improved characterization of the
metabolic and structural alterations of the corneal tissue due to pathophysiological conditions. This would be an
important step towards a better understanding of corneal dystrophies and systemic metabolic disorders.
The feasibility of using reflective surfaces to increase the detection of SHG signals from corneal histological sections in the backward direction was attempted. Three reflective surfaces were tested: aluminum foil, a silver mirror, and a dielectric mirror with specific high reflectivity around 400 nm. To compare the SHG signal detected with and without reflective surfaces, the translation caused by the bending of the sample due to the extra weight on top of the microscope slide was determined. All reflective surfaces resulted in an increase of the detection of SHG signals. Increases of 4%, 11%, and 16% were observed for aluminum foil, dielectric mirror and silver mirror, respectively. A method for increased detection of backward SHG signals when forward detection is not possible was herein demonstrated.
Low level laser therapy (LLLT) has been used for inflammation treatment. Here, we evaluate the effect of different
doses, using continuous (830 and 980 nm) and pulsed illumination (830 nm), in the treatment of inflammation induced in
the gastrocnemius muscle of Wistar rats, through cytokines concentration in systemic blood and histological analysis of
muscle tissue. Animals were randomly divided into five groups per wavelength (5 animals per group: 10, 20, 30, 40 and
50 mW) plus a control group. LLLT was applied during five days, with constant exposure time and irradiated area (3
minutes; 0.5026 cm2). Blood was collected on days 0, 3 and 6. TNF-α, IL-1β, IL-2 and IL-6 cytokines were quantified
by ELISA. Rats were killed on day 6. Muscle inflammatory cells were counted using optical microscopy. Treatment
effects occurred for all applied doses (largest effect at 40 mW: 7.2 J, 14 J/cm2 per irradiation), with reduction of proinflammatory
TNF-α, IL-1β and IL-6 cytokines and lower number of inflammatory cells. Results were better for 830 nm.
Identical methodology was used with pulsed illumination. Average power (40 mW) and duty cycle were kept constant
(80%) at five frequencies (5, 25, 50, 100 and 200 Hz). Treatment effects were observed at higher frequencies, with no
significant differences between them. However, the treatment effect was lower than for continuous illumination. LLLT
effect on inflammation treatment can be monitored by measuring systemic blood cytokines. A larger treatment effect was
observed with continuous illumination, where results seem to be compatible with a biphasic dose response.
Several studies in human and animals show the clinical effectiveness of low level laser therapy (LLLT) in reducing some types of pain, treating inflammation and wound healing. However, more scientific evidence is required to prove the effectiveness of LLLT since many aspects of the cellular and molecular mechanisms triggered by irradiation of injured tissue with laser remain unknown. Here, we present a methodology that can be used to evaluate the effect of different LLLT irradiation parameters on the treatment of muscle inflammation on animals, through the quantification of four cytokines (TNF-α, IL-1β, IL-2 and IL-6) in systemic blood and histological analysis of muscle tissue. We have used this methodology to assess the effect of LLLT parameters (wavelength, dose, power and type of illumination) in the treatment of inflammation induced in the gastrocnemius muscle of Wistar rats. Results obtained for laser dose evaluation with continuous illumination are presented.
Although studies show the clinical effectiveness of low level laser therapy (LLLT) in facilitating the muscle healing process, scientific evidence is still required to prove the effectiveness of LLLT and to clarify the cellular and molecular mechanisms triggered by irradiation. Here we evaluate the effect of different LLLT wavelengths, using continuous coherent Laser illumination (830 nm and 980 nm) and non-coherent LED illumination (850 nm), in the treatment of inflammation induced in the gastrocnemius muscle of Wistar rats, through the quantification of cytokines in systemic blood. We verified that all applied doses of coherent radiation produce an effect on reducing the concentration of pro-inflammatory TNF-α and IL-1β cytokines, while no treatment effect was observed after irradiation with non-coherent radiation. The best results were obtained for 40 mW at 830 nm. The results may suggest an important role of coherence properties of laser in LLLT.
Although studies show the clinical effectiveness of low level laser therapy (LLLT) in facilitating the muscle healing process, scientific evidence is still required to prove the effectiveness of LLLT and to clarify the cellular and molecular mechanisms triggered by irradiation. Here we evaluate the effect of different LLLT doses, using continuous illumination (830 nm), in the treatment of inflammation induced in the gastrocnemius muscle of Wistar rats, through the quantification of cytokines in systemic blood and histological analysis of muscle tissue. We verified that all applied doses produce an effect on reducing the number of inflammatory cells and the concentration of pro-inflammatory TNF-α and IL-1β cytokines. The best results were obtained for 40 mW. The results may suggest a biphasic dose response curve.
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