Investigating cartilage-related diseases by polarization-resolved second harmonic generation (P-SHG) imaging.

Establishing quantitative parameters for differentiating between healthy and diseased cartilage tissues by examining collagen fibril degradation patterns facilitates the understanding of tissue characteristics during disease progression. These findings could also complement existing clinical methods used to diagnose cartilage-related diseases. In this study, cartilage samples from normal, osteoarthritis (OA), and rheumatoid arthritis (RA) tissues were prepared and analyzed using polarization-resolved second harmonic generation (P-SHG) imaging and quantitative image texture analysis. The enhanced molecular contrast obtained from this approach is expected to aid in distinguishing between healthy and diseased cartilage tissues. P-SHG image analysis revealed distinct parameters in the cartilage samples, reflecting variations in collagen fibril arrangement and organization across different pathological states. Normal tissues exhibited distinct χ33/χ31 values compared with those of OA and RA, indicating collagen type transition and cartilage erosion with chondrocyte swelling, respectively. Compared with those of normal tissues, OA samples demonstrated a higher degree of linear polarization, suggesting increased tissue birefringence due to the deposition of type-I collagen in the extracellular matrix. The distribution of the planar orientation of collagen fibrils revealed a more directional orientation in the OA samples, associated with increased type-I collagen, while the RA samples exhibited a heterogeneous molecular orientation. This study revealed that the imaging technique, the quantitative analysis of the images, and the derived parameters presented in this study could be used as a reference for disease diagnostics, providing a clear understanding of collagen fibril degradation in cartilage.

An insight into optical beam induced current microscopy: Concepts and applications.

Laser scanning optical beam induced current (OBIC) microscopy has become a powerful and nondestructive alternative to other complicated methods like electron beam induced current (EBIC) microscopy, for high resolution defect analysis of electronic devices. OBIC is based on the generation of electron-hole pairs in the sample due to the raster scanning of a focused laser beam with energy equal or greater than the band gap energy and synchronized detection of resultant current profile with respect to the beam positions. OBIC is particularly suitable to localize defect sites caused by metal-semiconductor interdiffusion or electrostatic discharge (ESD). OBIC signals, thus, are capable of revealing the parameters/factors directly related to the reliability and efficiency of the electronic device under test (DUT). In this review, the basic principles of OBIC microscopy strategies and their notable applications in semiconductor device characterization are elucidated. An overview on the developments of OBIC microscopy is also presented. Specifically, the recent progresses on the following three OBIC measurement strategies have been reviewed, which include continuous laser based single photon OBIC, pulsed laser based single photon OBIC, and multiphoton OBIC microscopy for three-dimensional mapping of photocurrent response of electronic devices at high spatiotemporal resolution. Challenges and future prospects of OBIC in characterizing complex electronic devices are also discussed. HIGHLIGHTS: Characterization of electronic device quality is of paramount importance. Optical beam induced current (OBIC) microscopy offers spatially resolved mapping of local electronic properties. This review presents the principle and notable applications of OBIC microscopy.

Alcohol patch test with hue-saturation-value model analysis predicts ALDH2 genetic polymorphism.

BACKGROUND: The alcohol patch test (APT) can detect aldehyde dehydrogenase (ALDH) genetic polymorphisms used to diagnose cutaneous erythema. However, the subjective results can vary owing to confounding factors. The hue-saturation-value (HSV) model provides an objective means of image analysis with APT. METHODS: This study enrolled 57 participants (27.7 ± 9.0 years, 52.6% females) with ALDH2*1/*1, ALDH2*1/*2, and ALDH2*2/*2 percentages of 50.9%, 43.8%, and 5.3%, respectively. In total, 56 APT protocols were applied and analyzed employing both visual inspection and the HSV model. The value of the delta standard deviation (SD) of the hue histogram, which manifests the difference between the APT reaction and the baseline skin color, was obtained using the HSV model. The receiver operating characteristic (ROC) curve and area under the ROC curve (AUC) were used to predict the ALDH2*2 allele with the HSV model. RESULTS: Upon visual inspection, a maximal Youden index with a sensitivity of 82.1% and a specificity of 96.6% was determined for the ALDH2 genetic mutation. Using the delta SD of hue obtained in the HSV model, a maximal Youden index with 85.7% sensitivity and 96.6% specificity was determined using the ROC curve analysis (AUC = 0.948, p < 0.001). Thus, the use of the HSV model analysis with APT resulted in equal specificity, but better sensitivity, compared to those obtained upon visual inspection. CONCLUSION: The HSV model took into account the potential confounding factors, and thus, could help in the prediction of ALDH2 genetic polymorphisms.

Stokes polarimetry-based second harmonic generation microscopy for collagen and skeletal muscle fiber characterization.

The complete polarization state of second harmonic (SH) light was measured and characterized by collagen type I and skeletal muscle fiber using a Stokes vector-based SHG microscope. The polarization states of the SH signal are analyzed in a pixel-by-pixel manner and displayed through two dimensional (2D) Stokes vector images. Various polarization parameters are reconstructed using Stokes values to quantify the polarization properties of SH light. Also, the measurements are extended for different input polarization states to investigate the molecular structure of second harmonic generation (SHG) active molecules such as collagen type I and myosin.

Label-free characterization of collagen fibers in cancerous esophagus tissues using ratiometric nonlinear optical microscopy.

IMPACT STATEMENT: The issue of classifying esophageal cancer at various developmental stages is crucial for determining the optimized treatment protocol for the patients, as well as the prognosis. Precision improvement in staging esophageal cancer keeps seeking quantitative and analytical imaging methods that could augment histopathological techniques. In this work, we used nonlinear optical microscopy for ratiometric analysis on the intrinsic signal of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) from single collagen fibers only in submucosa of esophageal squamous cell carcinoma (ESCC). The blind tests of TPEF/SHG and forward (F)/backward (B) SHG were demonstrated to compare with the histology conclusion. The discussion of sensitivity and specificity was provided via statistical comparison between the four stages of esophageal cancer. To the best of our knowledge, this is the first study of using these two ratios in combination for staging ESCC.

High temporal resolution and polarization resolved fluorescence lifetime measurements through stimulated emission.

We have implemented polarization-resolved fluorescence lifetime measurement through stimulated emission based pump-probe technique, which promises much higher temporal resolution (∼4 ps) than conventional time-correlated single-photon counting (TCSPC). The depolarization of ATTO 647N fluorescent dye is resolved through anisotropy fluorescence lifetime measurements, with variable time delay introduced between the pump and the probe beams. Importantly, the polarization anisotropy measurement and the corresponding rotational correlation time characterization of the fluorescent dye are carried out at various temperatures. We have also demonstrated the need of high temporal resolution via hetero Förster energy transfer (Hetero-FRET) through the interaction between the gold nanorods (GNRs) and the fluorescent dye ATTO 647N. Notably, our results compare highly favorably with conventional TCSPC method, which is rather limited in temporal resolution, for the above characterization. Additionally, this technique is applicable even under ambient light while being very cost-effective and robust.

Spontaneous loss versus stimulation gain in pump-probe microscopy: a proof of concept demonstration.

SIGNIFICANCE: The large background, narrow dynamic range, and detector saturation have been the common limiting factors in stimulated emission (SE)-based pump-probe microscopy, attributed to the very small signal overriding the very intense laser probe beam. To better differentiate the signal of interest from the background, lock-in detection is used to measure the fluorescence quenching, which is termed spontaneous loss (SL). The advantages are manifold. The spontaneous fluorescence signal can be well separated from both the pump and the probe beams with filters, thus eliminating the background, enlarging the dynamic range, and avoiding the saturation of the detector. AIM: We propose and demonstrate an integrated pump-probe microscopy technique based on lock-in detection for background removal and dynamic range enhancement through SL detection. APPROACH: The experimental setup is configured with a pulsed diode laser at a wavelength λpu = 635 nm, acting as a pump (excitation) and a mode-locked Ti:sapphire laser at a central wavelength λpr = 780 nm, serving as the probe beam (stimulation). Both pulse trains are temporally synchronized through high precision delay control by adjusting the length of the triggering cables. The pump and probe beams are alternatively modulated at different frequencies f1 and f2 to extract the stimulated gain (SG) and SL signal. RESULTS: SG signal shows saturation due to the irradiation of the intense probe beam onto the photodetector. However, the detector saturation does not occur at high probe beam power for SL detection. The fluorescence lifetime images are acquired with reduced background. The theoretical signal-to-noise ratios for SG and SL are also estimated by photon statistics. CONCLUSION: We have confirmed that the detection of SL allows the elimination of the large background without photodetector saturation, which commonly exists in SG configuration. This modality would allow unprecedented manipulation and investigation of fluorophores in fluorescence imaging.

Synchronized subharmonic modulation in stimulated emission microscopy.

In this work, we have demonstrated a stimulated emission (SE)-based pump-probe microscopy with subharmonic fast gate synchronization, which allows over an order of magnitude improvement in signal-to-noise ratio. Critically, the alternative way of modulation is implemented with the highest possible frequency that follows the lasers' repetition rate. Its working is based on a homemade frequency divider that divides the repetition frequency (76 MHz) of the Ti:sapphire (probe) laser to half of the repetition frequency, 38 MHz, which is used to synchronously drive the pump laser and to provide the reference signal for the ensuing lock-in detection. In this way, SE can be detected with sensitivity reaching the theoretical (shot noise) limits, with a much lower time constant (0.1 ms) for faster image acquisition.

Direct mineralogical imaging of economic ore and rock samples with multi-modal nonlinear optical microscopy.

Multi-modal nonlinear optical (NLO) microscopy, including stimulated Raman scattering (SRS) and second harmonic generation (SHG), was used to directly image mineralogical features of economic ore and rock samples. In SRS/SHG imaging, ore samples generally require minimal preparation and may be rapidly imaged, even in their wet state. 3D structural details, at submicron resolution, are revealed tens of microns deep within samples. Standard mineral imaging based on scanning electron microscopy (SEM), with elemental analysis via energy dispersive X-Ray spectroscopy, was used to independently validate the mineral composition of the samples. Spatially-resolved SRS from dominant Raman-resonant bands precisely maps the locations of specific minerals contained within the samples. SHG imaging reveals locally non-centrosymmetric structures, such as quartz grains. Competing absorption and nonlinear scattering processes, however, can reduce contrast in SRS imaging. Importantly, the correlation between standard electron microscopy and multi-modal NLO optical microscopy shows that the latter offers rapid image contrast based on the mineral content of the sample.

Polarization resolved second harmonic microscopy.

Second harmonic (SH) microscopy has proven to be a powerful imaging modality over the past years due to its intrinsic advantages as a multiphoton process with endogenous contrast specificity, which allows pinhole-less optical sectioning, non-invasive observation, deep tissue penetration, and the possibility of easier signal detection at visible wavelengths. Depending on the relative orientation between the polarization of the incoming light and the second-order susceptibility of non-centrosymmetric structures, SH microscopy provides the unique capacity to probe the absolute molecular structure of a broad variety of biological tissues without the necessity for additional labeling. In addition, SH microscopy, when working with polarimetry, provides clear and in-depth insights on the details of molecular orientation and structural symmetry. In this review, the working principles of the polarization resolving techniques and the corresponding implements of SH microscopy are elucidated, with focus on Stokes vector based polarimetry. An overview of the advancements on SH anisotropy measurements are also presented. Specifically, the recent progresses on the following three topics in polarization resolved SH microscopy will be elucidated, which include Stokes vector resolving for imaging molecular structure and orientation, 3-D structural chirality by SH circular dichroism, and correlation with fluorescence lifetime imaging (FLIM) for in vivo wound healing diagnosis. The potentials and challenges for future researches in exploring complex biological tissues are also discussed.

Adaptable interaction between aquaporin-1 and band 3 reveals a potential role of water channel in blood CO2 transport.

Human CO2 respiration requires rapid conversion between CO2 and HCO3- Carbonic anhydrase II facilitates this reversible reaction inside red blood cells, and band 3 [anion exchanger 1 (AE1)] provides a passage for HCO3- flux across the cell membrane. These 2 proteins are core components of the CO2 transport metabolon. Intracellular H2O is necessary for CO2/HCO3- conversion. However, abundantly expressed aquaporin 1 (AQP1) in erythrocytes is thought not to be part of band 3 complexes or the CO2 transport metabolon. To solve this conundrum, we used Förster resonance energy transfer (FRET) measured by fluorescence lifetime imaging (FLIM-FRET) and identified interaction between aquaporin-1 and band 3 at a distance of 8 nm, within the range of dipole-dipole interaction. Notably, their interaction was adaptable to membrane tonicity changes. This suggests that the function of AQP1 in tonicity response could be coupled or correlated to its function in band 3-mediated CO2/HCO3- exchange. By demonstrating AQP1 as a mobile component of the CO2 transport metabolon, our results uncover a potential role of water channel in blood CO2 transport and respiration.-Hsu, K., Lee, T.-Y., Periasamy, A., Kao, F.-J., Li, L.-T., Lin, C.-Y., Lin, H.-J., Lin, M. Adaptable interaction between aquaporin-1 and band 3 reveals a potential role of water channel in blood CO2 transport.

Investigating starch gelatinization through Stokes vector resolved second harmonic generation microscopy.

The changes of the morphology during heating and the degree of crystallinity of dry and hydrated starch granules are investigated using second harmonic generation (SHG) based Stokes polarimetry. A spatial distribution of various polarization parameters, such as the degree of polarization (DOP), the degree of linear polarization (DOLP), and the degree of circular polarization (DOCP) are extracted and compared with the two dimensional second harmonic (SH) Stokes images of starch granules. The SH signal from hydrated and dry starch on heating differed significantly in DOLP and DOCP values, indicating that hydrated starch has a greater degree of ultrastructural amylopectin disorder. The detail of denaturation and the phase transition of hydrated starch demonstrate the significant influence of thermal processing.

Three-dimensional trace measurements for fast-moving objects using binary-encoded fringe projection techniques.

A fringe projection technique to trace the shape of a fast-moving object is proposed. A binary-encoded fringe pattern is illuminated by a strobe lamp and then projected onto the moving object at a sequence of time. Phases of the projected fringes obtained from the sequent measurements are extracted by the Fourier transform method. Unwrapping is then performed with reference to the binary-encoded fringe pattern. Even though the inspected object is colorful, fringe orders can be identified. A stream of profiles is therefore retrieved from the sequent unwrapped phases. This makes it possible to analyze physical properties of the dynamic objects. Advantages of the binary-encoded fringe pattern for phase unwrapping also include (1) reliable performance for colorful objects, spatially isolated objects, and surfaces with large depth discontinuities; (2) unwrapped errors only confined in a local area; and (3) low computation cost.

Compact light-emitting diode lighting ring for video-assisted thoracic surgery.

In this work, a foldable ring-shaped light-emitting diode (LED) lighting assembly, designed to attach to a rubber wound retractor, is realized and tested through porcine animal experiments. Enabled by the small size and the high efficiency of LED chips, the lighting assembly is compact, flexible, and disposable while providing direct and high brightness lighting for more uniform background illumination in video-assisted thoracic surgery (VATS). When compared with a conventional fiber bundle coupled light source that is usually used in laparoscopy and endoscopy, the much broader solid angle of illumination enabled by the LED assembly allows greatly improved background lighting and imaging quality in VATS.

Dynamic photopatterning of cells in situ by Q-switched neodymium-doped yttrium ortho-vanadate laser.

Cellular micropattering has been increasingly adopted in quantitative biological experiments. A Q-switched pulsed neodymium-doped yttrium ortho-vanadate (Nd∶YVO4) laser directed in-situ microfabrication technique for cell patterning is presented. A platform is designed uniquely to achieve laser ablation. The platform is comprised of thin gold coating over a glass surface that functions as a thermal transducer and is over-layered by a cell repellant polymer layer. Micropatterns are engraved on the platform, subsequently exposing specific cell adhesive micro-domains by ablating the gold-polymer coating photothermally. Experimental results indicate that the proposed approach is applicable under culture conditions, viable toward cells, and has a higher engraving speed. Possible uses in arraying isolated single cells on the platform are also shown. Additionally, based on those micro-patterns, dynamic cellular morphological changes and migrational speed in response to geometrical barriers are studied to demonstrate the potential applications of the proposed approach. Our results further demonstrate that cells in narrower geometry had elongated shapes and higher migrational speed than those in wider geometry. Importantly, the proposed approach will provide a valuable reference for efforts to study single cell dynamics and cellular migration related processes for areas such as cell division, wound healing, and cancer invasion.

Revealing molecular structure and orientation with Stokes vector resolved second harmonic generation microscopy.

We report on measurements and characterization of polarization properties of Second Harmonic (SH) signals using a four-channel photon counting based Stokes polarimeter. In this way, the critical polarization parameters can be obtained concurrently without the need of repeated image acquisition. The critical polarization parameters, including the degree of polarization (DOP), the degree of linear polarization (DOLP), and the degree of circular polarization (DOCP), are extracted from the reconstructed Stokes vector based SH images in a pixel-by-pixel manner. The measurements are further extended by varying the polarization states of the incident light and recording the resulting Stokes parameters of the SH signal. In turn this allows the molecular structure and orientation of the samples to be determined. Use of Stokes polarimetry is critical in determination of the full polarization state of light, and enables discrimination of material properties not possible with conventional crossed-polarized detection schemes. The combination of SHG microscopy and Stokes polarimeter hence makes a powerful tool to investigate the structural order of targeted specimens.

In situ laser micropatterning of proteins for dynamically arranging living cells.

This study shows the modification of the surface of polymer-layered glass substrates to form biofunctional micropatterns through femtosecond laser ablation in an aqueous solution. Domains of micrometer size on a substrate can be selectively converted from proteinphobic (resistant to protein adsorption) to proteinphilic, allowing patterning of protein features under physiological aqueous conditions. When femtosecond laser pulses (800 nm, 1 kHz, 200-500 nJ per pulse) were focused on and scanned on the substrate, which was glass covered with the proteinphobic polymer 2-methacryloyloxyethylphosphorylcholine (MPC), the surface became proteinphilic. Surface analysis by X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) reveals that the laser ablates the MPC polymer. Extracellular matrix (ECM) proteins were bound to the laser-ablated surface by physisorption. Since femtosecond laser ablation is induced under physiological aqueous conditions, this approach can form micropatterns of functional ECM proteins with minimal damage. This method was applied to pattern collagen, laminin, and gelatin on the substrate. Removal of an ECM protein from the substrate followed by replacement with another ECM protein was achieved on demand at a specific location and time by the same laser ablation method. Living cells adhered to the fabricated domains where ECM proteins were arranged. The modification of patterning during cell culture was used to control cell migration and form arrays of different cells.

Fluorescence lifetime imaging of alterations to cellular metabolism by domain 2 of the hepatitis C virus core protein.

Hepatitis C virus (HCV) co-opts hepatic lipid pathways to facilitate its pathogenesis. The virus alters cellular lipid biosynthesis and trafficking, and causes an accumulation of lipid droplets (LDs) that gives rise to hepatic steatosis. Little is known about how these changes are controlled at the molecular level, and how they are related to the underlying metabolic states of the infected cell. The HCV core protein has previously been shown to independently induce alterations in hepatic lipid homeostasis. Herein, we demonstrate, using coherent anti-Stokes Raman scattering (CARS) microscopy, that expression of domain 2 of the HCV core protein (D2) fused to GFP is sufficient to induce an accumulation of larger lipid droplets (LDs) in the perinuclear region. Additionally, we performed fluorescence lifetime imaging of endogenous reduced nicotinamide adenine dinucleotides [NAD(P)H], a key coenzyme in cellular metabolic processes, to monitor changes in the cofactor's abundance and conformational state in D2-GFP transfected cells. When expressed in Huh-7 human hepatoma cells, we observed that the D2-GFP induced accumulation of LDs correlated with an increase in total NAD(P)H fluorescence and an increase in the ratio of free to bound NAD(P)H. This is consistent with an approximate 10 fold increase in cellular NAD(P)H levels. Furthermore, the lifetimes of bound and free NAD(P)H were both significantly reduced--indicating viral protein-induced alterations in the cofactors' binding and microenvironment. Interestingly, the D2-expressing cells showed a more diffuse localization of NAD(P)H fluorescence signal, consistent with an accumulation of the co-factor outside the mitochondria. These observations suggest that HCV causes a shift of metabolic control away from the use of the coenzyme in mitochondrial electron transport and towards glycolysis, lipid biosynthesis, and building of new biomass. Overall, our findings demonstrate that HCV induced alterations in hepatic metabolism is tightly linked to alterations in NAD(P)H functional states.