Two‐Photon polymerization for microfabrication of three‐dimensional scaffolds for tissue engineering application

In natural tissues cells are embedded in a three‐dimensional fibrous network of biopolymers like collagen, hyaluronic acid etc. This extracellular matrix (ECM) influences the cell fate, the differentiation status, metabolic processes and provides structural integrity. For a three‐dimensional or physiological cell cultivation that are required in biomedical applications (e.g. tissue engineering, BioMEMS) scaffolds are needed. These scaffolds mimic the ECM according to their biocompatibility which comprises aspects of surface compatibility and importantly for tissue engineering applications aspects of structural compatibility. We have evaluated scaffold design parameters for the three‐dimensional cultivation of chondrocytes for the tissue engineering of artificial cartilage. Two‐photon polymerization is a powerful technique for fabrication of polymeric three‐dimensional micro‐ and submicro‐structures. The photoinitiation system for two‐photon polymerization is excited by simultaneous absorption of two photons leading to chemical polymerization reactions. Due to a tight confinement of the excitation volume around the focal point, this method can produce micrometer sized objects maintaining a high spatial resolution down to 100 nm. Two‐photon processes require very high photon densities which are provided by pulsed femtosecond lasers. The potential of this approach for microfabrication of scaffolds for tissue engineering is demonstrated by investigation of the cell response to microstructures with complex three‐dimensional geometry and feature sizes in the range of few micrometers.     

Enhancement of imaging depth in turbid media using a wide area detector

The depth of two‐photon fluorescence imaging in turbid media can be significantly enhanced by the use of the here described fluorescence detection method that allows to efficiently collect scattered fluorescence photons from a wide area of the turbid sample. By using this detector we were able to perform imaging of turbid samples, simulating brain tissue, at depths up to 3 mm, where the two‐photon induced fluorescence signal is too weak to be detected by means used in conventional two‐photon microscopy. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

A compact fiber‐optic probe‐based singlet oxygen luminescence detection system

This paper presents a novel compact fiberoptic based singlet oxygen near‐infrared luminescence probe coupled to an InGaAs/InP single photon avalanche diode (SPAD) detector. Patterned time gating of the single‐photon detector is used to limit unwanted dark counts and eliminate the strong photosensitizer luminescence background. Singlet oxygen luminescence detection at 1270 nm is confirmed through spectral filtering and lifetime fitting for Rose Bengal in water, and Photofrin in methanol as model photosensitizers. The overall performance, measured by the signal‐to‐noise ratio, improves by a factor of 50 over a previous system that used a fiberoptic‐coupled superconducting nanowire single‐photon detector. The effect of adding light scattering to the photosensitizer is also examined as a first step towards applications in tissue in vivo.

     

Fast non‐negative temporal deconvolution for laser scanning microscopy

Laser scanning microscopy (LSM) is a common technique for high resolution fluorescent imaging. Here we describe a fast algorithm for non‐negative deconvolution and apply it to readout of LSM detector photocurrents. By broadening photon impulses and deconvolving sampled photocurrent, effective quantum efficiency of the imaging system is increased. Using simulation and imaging with a custom‐built two‐photon microscope, we demonstrate improved fidelity of images acquired at short dwell times over a wide range of photon rates. Images formed show increased correlation‐to‐sample equivalent to a 25% increase in photon rate, lower noise, and reduced bleed‐through compared to conventional image generation. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Investigation of the source‐detector separation in near infrared spectroscopy for healthy and clinical applications

Understanding near infrared light propagation in tissue is vital for designing next generation optical brain imaging devices. Monte Carlo (MC) simulations provide a controlled mechanism to characterize and evaluate contributions of diverse near infrared spectroscopy (NIRS) sensor configurations and parameters. In this study, we developed a multilayer adult digital head model under both healthy and clinical settings and assessed light‐tissue interaction through MC simulations in terms of partial differential pathlength, mean total optical pathlength, diffuse reflectance, detector light intensity and spatial sensitivity profile of optical measurements. The model incorporated four layers: scalp, skull, cerebrospinal‐fluid and cerebral cortex with and without a customizable lesion for modeling hematoma of different sizes and depths. The effect of source‐detector separation (SDS) on optical measurements' sensitivity to brain tissue was investigated. Results from 1330 separate simulations [(4 lesion volumes × 4 lesion depths for clinical +3 healthy settings) × 7 SDS × 10 simulation = 1330)] each with 100 million photons indicated that selection of SDS is critical to acquire optimal measurements from the brain and recommended SDS to be 25 to 35 mm depending on the wavelengths to obtain optical monitoring of the adult brain function. The findings here can guide the design of future NIRS probes for functional neuroimaging and clinical diagnostic systems.      

Two‐photon microscopy of healthy,infarcted and stem‐cell treated regenerating heart

Two‐photon excitation autofluorescence (produced in myocytes) and second‐harmonic generation (produced mainly by collagen) allow label‐free visualization of these two important components of myocardium. Because of their different emission wavelengths, these two signals can be separated spectrally. Here, we examine two‐photon microscopy images of healthy, infarcted and stem‐cell treated rat hearts. We find that in infarcted heart, regions distant from the site of infarct are similar to healthy tissue in composition (mostly myocytes, very little collagen) and organization (densely packed myocytes), but infarct regions are characterized by sparse myocytes and high collagen content indicative of scar tissue formation. Stem cell treated hearts, in contrast, show regions of intertwined myocytes and collagen throughout the infarct, suggesting reduced tissue damage. Finally, these results offer interesting insights into our ongoing polarized light studies of cardiac tissue anisotropy, and reveal that both tissue composition and tissue micro‐organization are reflected in polarization‐measured linear retardance values. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Effect of intrinsic and extrinsic factors on the simulated D‐band length of type I collagen

A signature feature of collagen is its axial periodicity visible in TEM as alternating dark and light bands. In mature, type I collagen, this repeating unit, D, is 67 nm long. This periodicity reflects an underlying packing of constituent triple‐helix polypeptide monomers wherein the dark bands represent gaps between axially adjacent monomers. This organization is visible distinctly in the microfibrillar model of collagen obtained from fiber diffraction. However, to date, no atomistic simulations of this diffraction model under zero‐stress conditions have reported a preservation of this structural feature. Such a demonstration is important as it provides the baseline to infer response functions of physiological stimuli. In contrast, simulations predict a considerable shrinkage of the D‐band (11–19%). Here we evaluate systemically the effect of several factors on D‐band shrinkage. Using force fields employed in previous studies we find that irrespective of the temperature/pressure coupling algorithms, assumed salt concentration or hydration level, and whether or not the monomers are cross‐linked, the D‐band shrinks considerably. This shrinkage is associated with the bending and widening of individual monomers, but employing a force field whose backbone dihedral energy landscape matches more closely with our computed CCSD(T) values produces a small D‐band shrinkage of < 3%. Since this force field also performs better against other experimental data, it appears that the large shrinkage observed in earlier simulations is a force‐field artifact. The residual shrinkage could be due to the absence of certain atomic‐level details, such as glycosylation sites, for which we do not yet have suitable data. Proteins 2015; 83:1800–1812. © 2015 Wiley Periodicals, Inc.     

Characterization and application of porous gold nanoparticles as 2‐photon luminescence imaging agents: 20‐fold brighter than gold nanorods

Two‐photon nonlinear microscopy with the aid of plasmonic contrast agents is an attractive bioimaging technique capable of generating high‐resolution images in 3 dimensions and facilitating targeted imaging with deep tissue penetration. In this work, physically synthesized gold nanoparticles containing multiple nanopores are used as 2‐photon contrast agents and are reported to emit a 20‐fold brighter 2‐photon luminescence as compared to typical contrast agents, that is, gold nanorods. A successful application of our porous gold nanoparticles is experimentally demonstrated by in vitro nonlinear optical imaging of adipocytes at subcellular level.      

Identifying the Relative Importance of Leaf versus Shredder Species Loss on Litter Decomposition in Streams

Understanding how species loss influences ecosystem function is a contemporary issue in ecology. However, most research has focused on species loss at one trophic‐level. We explored the relationship between functional diversity (FD) and species richness separately for trees and aquatic leaf‐shredding detritivores. For trees, we collected information on species‐specific leaf tissue chemistry and species co‐occurrences in the mid‐Atlantic region (USA). For shredders, we used a published trait database with information on communities from 38 streams in the same region. We used a clustering algorithm to estimate FD for each community and for randomly assembled communities. If FD was high, we concluded that species loss was important to change in function; if low, species were functionally redundant and insensitive to species loss. We found tree FD to be significantly different than expected, but shredders exhibited FD levels similar to patterns based on random assembly. Furthermore, there were more leaf species exclusively associated with very high or very low levels of functional diversity compared to shredders. This approach revealed greater implications for leaf than shredder species loss for litter breakdown. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Two‐photon focal modulation microscopy for high‐resolution imaging in deep tissue

Two‐photon microscopy (2PM) is one of the most widely used tools for in vivo deep tissue imaging. However, the spatial resolution and penetration depth are still limited due to the strong scattering background. Here we demonstrate a two‐photon focal modulation microscopy. By utilizing the modulation and demodulation techniques, background rejection capability is enhanced, thus spatial resolution and imaging penetration depth are improved. Compared with 2PM, the transverse resolution is increased by 70%, while the axial resolution is increased to 2‐fold. Furthermore, when applied in conventional 2PM mode, it can achieve inertial‐free scanning in either transverse or axial direction with in principle unlimited scanning speed. Finally, we applied 2PFMM in thick scattering samples to further examine the imaging performance. The results show that the signal‐to‐background ratio of 2PFMM can be improved up to five times of 2PM at the depth of 500 μm. Fluorescent imaging in the mouse brain tissue. 3D Thy1‐GFP hippocampal neurons imaged by (A) 2PM compared with (B) 2PFMM; (C‐H) xy maximum‐intensity projection imaged by 2PM compared with 2PFMM. Scale bar 50 μm.      

Hyperspectral characterization of re‐epithelialization in an in vitro wound model

In vitro wound models are useful for research on wound re‐epithelialization. Hyperspectral imaging represents a non‐destructive alternative to histology analysis for detection of re‐epithelialization. This study aims to characterize the main optical behavior of a wound model in order to enable development of detection algorithms. K‐Means clustering and agglomerative analysis were used to group spatial regions based on the spectral behavior, and an inverse photon transport model was used to explain differences in optical properties. Six samples of the wound model were prepared from human tissue and followed over 22 days. Re‐epithelialization occurred at a mean rate of 0.24 mm²/day after day 8 to 10. Suppression of wound spectral features was the main feature characterizing re‐epithelialized and intact tissue. Modeling the photon transport through a diffuse layer placed on top of wound tissue properties reproduced the spectral behavior. The missing top layer represented by wounds is thus optically detectable using hyperspectral imaging.     

Assessment of the metabolism and morphology of the porcine cornea,lens and retina by 2‐photon imaging

Two‐photon imaging is a noninvasive imaging technique with increasing importance in the biological and medical fields since it allows intratissue cell imaging with high resolution. We demonstrate the feasibility of using a single 2‐photon instrument to evaluate the cornea, the crystalline lens and the retina based on their autofluorescence (AF). Image acquisition was performed using a custom‐built 2‐photon microscope for 5‐dimensional microscopy with a near infrared broadband sub‐15 femtosecond laser centered at 800 nanometers. Signals were detected using a spectral photomultiplier tube. The spectral ranges for the analysis of each tissue/layer AF were determined based on the spectra of each tissue as well as of pure endogenous fluorophores. The cornea, lens and retina are characterized at multiple depths with subcellular resolution based on their morphology and AF lifetime. Additionally, the AF lifetime of NAD(P)H was used to assess the metabolic activity of the cornea epithelium, endothelium and keratocytes. The feasibility to evaluate the metabolic activity of lens epithelial cells was also demonstrated, which may be used to further investigate the pathogenesis of cataracts. The results illustrate the potential of multimodal multiphoton imaging as a novel ophthalmologic technique as well as its potential as a diagnostic tool.      

Imaging directed photothermolysis through two‐photon absorption demonstrated on mouse skin – a potential novel tool for highly targeted skin treatment

One‐photon absorption based traditional laser treatment may not necessarily be selective at the microscopic level, thus could result in un‐intended tissue damage. Our objective is to test whether two‐photon absorption (TPA) could provide highly targeted tissue alteration of specific region of interest without damaging surrounding tissues. TPA based laser treatments (785 nm, 140 fs pulse width, 90 MHz) were performed on ex vivo mouse skin using different average power levels and irradiation times. Reflectance confocal microscopy (RCM) and combined second‐harmonic‐generation (SHG) and two‐photon fluorescence (TPF) imaging channels were used to image before, during, and after each laser treatment. The skin was fixed, sectioned and H & E stained after each experiment for histological assessment of tissue alterations and for comparison with the non‐invasive imaging assessments. Localized destruction of dermal fibers was observed without discernible epidermal damage on both RCM and SHG + TPF images for all the experiments. RCM and SHG + TPF images correlated well with conventional histological examination. This work demonstrated that TPA‐based light treatment provides highly localized intradermal tissue alteration. With further studies on optimizing laser treatment parameters, this two‐photon absorption photothermolysis method could potentially be applied in clinical dermatology. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Functional identity is the main driver of diversity effects in young tree communities

Two main effects are proposed to explain biodiversity–ecosystem functioning relationships: niche complementarity and selection effects. Both can be functionally defined using the functional diversity (FD) and functional identity (FI) of the community respectively. Herein, we present results from the first tree diversity experiment that separated the effect of selection from that of complementarity by varying community composition in high‐density plots along a gradient of FD, independent of species richness and testing for the effects of FD and community weighted means of traits (a proxy for FI) on stem biomass increment (a proxy for productivity). After 4 years of growth, most mixtures did not differ in productivity from the averages of their respective monocultures, but some did overyield significantly. Those positive diversity effects resulted mostly from selection effects, primarily driven by fast‐growing deciduous species and associated traits. Net diversity effect did not increase with time over 4 years.     

Effects of force fields on the conformational and dynamic properties of amyloid β(1‐40) dimer explored by replica exchange molecular dynamics simulations

The conformational space and structural ensembles of amyloid beta (Aβ) peptides and their oligomers in solution are inherently disordered and proven to be challenging to study. Optimum force field selection for molecular dynamics (MD) simulations and the biophysical relevance of results are still unknown. We compared the conformational space of the Aβ(1‐40) dimers by 300 ns replica exchange MD simulations at physiological temperature (310 K) using: the AMBER‐ff99sb‐ILDN, AMBER‐ff99sb*‐ILDN, AMBER‐ff99sb‐NMR, and CHARMM22* force fields. Statistical comparisons of simulation results to experimental data and previously published simulations utilizing the CHARMM22* and CHARMM36 force fields were performed. All force fields yield sampled ensembles of conformations with collision cross sectional areas for the dimer that are statistically significantly larger than experimental results. All force fields, with the exception of AMBER‐ff99sb‐ILDN (8.8 ± 6.4%) and CHARMM36 (2.7 ± 4.2%), tend to overestimate the α‐helical content compared to experimental CD (5.3 ± 5.2%). Using the AMBER‐ff99sb‐NMR force field resulted in the greatest degree of variance (41.3 ± 12.9%). Except for the AMBER‐ff99sb‐NMR force field, the others tended to under estimate the expected amount of β‐sheet and over estimate the amount of turn/bend/random coil conformations. All force fields, with the exception AMBER‐ff99sb‐NMR, reproduce a theoretically expected β‐sheet‐turn‐β‐sheet conformational motif, however, only the CHARMM22* and CHARMM36 force fields yield results compatible with collapse of the central and C‐terminal hydrophobic cores from residues 17‐21 and 30‐36. Although analyses of essential subspace sampling showed only minor variations between force fields, secondary structures of lowest energy conformers are different.     

Shortening amplitude affects the incomplete force recovery after active shortening in mouse soleus muscle

Compared to isometric contraction, the force producing capacity of muscle is reduced (force depression, FD) after a work producing shortening phase. It has been suggested that FD results from an inhibition of cross-bridge binding. Because the rate constants of the exponential force (re)development are thought to be primarily determined by cross-bridge attachment/detachment rate, we aimed to investigate the components of force redevelopment (REDEV) after 0.6, 1.2 and 2.4 mm shortening, resulting in varying amounts of FD (from about 5% to about 16%), in mouse soleus muscle (n=11). Compared to isometric force development (DEV), the time to reach steady-state during REDEV was about 3 times longer (370 versus 1261 ms) increasing with increasing amplitude. Contrary to a single, a double exponential function with one component set equal to the rate constant of DEV (14.3 s^?1), accurately described REDEV (RMS<0.8%). The rate constant of the additional slow component decreased with increasing shortening amplitude and was associated with work delivered during shortening (R²=0.75) and FD (R²=0.77). We concluded that a work related slow exponential component is induced to the trajectory of incomplete force recovery after shortening, causing FD. These results suggest that after shortening, aside from cross-bridges with normal attachment/detachment rate, cross-bridges with reduced cycling rate are active.     

Two‐photon excitation and direct emission from S2 state of U.S. Food and Drug Administration approved near‐infrared dye: Application of anti‐Kasha's rule for two‐photon fluorescence imaging

In recent years, two‐photon fluorescence microscopy has gained significant interest in bioimaging. It allows the visualization of deeply buried inhomogeneities in tissues. The near‐infrared (NIR) dyes are also used for deep tissue imaging. Indocyanine green (ICG) is the only U.S. Food and Drug Administration (FDA) approved exogenous contrast agent in the NIR region for clinical applications. However, despite its potential candidature, it had never been used as a two‐photon contrast agent for biomedical imaging applications. This letter provides an insight into the scope and application of the two‐photon excitation property of ICG to the second excited singlet (S₂) state in aqueous solution. Furthermore, in this work, we demonstrate the two‐photon cellular imaging application of ICG using direct fluorescence emission from S₂ state for the first time. Our results show that two‐photon excitation to S₂ state of ICG could be achieved with approximately 790 nm wavelength of femtosecond laser, which lies in well‐known “tissue‐optical window.” This property would enable light to penetrate much deeper in the turbid medium such as biological tissues. Thus, ICG could be used as the first FDA approved NIR exogenous contrast agent for two‐photon imaging. These findings can make remarkable influence on preclinical and clinical cell imaging.      

Lightsheet fluorescence lifetime imaging microscopy with wide‐field time‐correlated single photon counting

We report on wide‐field time‐correlated single photon counting (TCSPC)‐based fluorescence lifetime imaging microscopy (FLIM) with lightsheet illumination. A pulsed diode laser is used for excitation, and a crossed delay line anode image intensifier, effectively a single‐photon sensitive camera, is used to record the position and arrival time of the photons with picosecond time resolution, combining low illumination intensity of microwatts with wide‐field data collection. We pair this detector with the lightsheet illumination technique, and apply it to 3D FLIM imaging of dye gradients in human cancer cell spheroids, and C. elegans.     

Two‐photon excited fluorescence lifetime measurements through a double‐clad photonic crystal fiber for tissue micro‐endoscopy

This paper presents an endoscopic configuration for measurements of tissue autofluorescence using two–photon excitation and time‐correlated single photon counting detection through a double‐clad photonic crystal fiber (DC‐PCF) without pre‐chirping of laser pulses. The instrument performance was evaluated by measurements of fluorescent standard dyes, biological fluorophores (collagen and elastin), and tissue specimens (muscle, cartilage, tendon). Current results demonstrate the ability of this system to accurately retrieve the fluorescence decay profile and lifetime of these samples. This simple setup, which offers larger penetration depth than one‐photon‐based techniques, may be combined with morphology‐yielding techniques such as photoacoustic and ultrasound imaging. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)