Categorization of collagen type I and II blend hydrogel using multipolarization SHG imaging with ResNet regression.

Previously, the discrimination of collagen types I and II was successfully achieved using peptide pitch angle and anisotropic parameter methods. However, these methods require fitting polarization second harmonic generation (SHG) pixel-wise information into generic mathematical models, revealing inconsistencies in categorizing collagen type I and II blend hydrogels. In this study, a ResNet approach based on multipolarization SHG imaging is proposed for the categorization and regression of collagen type I and II blend hydrogels at 0%, 25%, 50%, 75%, and 100% type II, without the need for prior time-consuming model fitting. A ResNet model, pretrained on 18 progressive polarization SHG images at 10° intervals for each percentage, categorizes the five blended collagen hydrogels with a mean absolute error (MAE) of 0.021, while the model pretrained on nonpolarization images exhibited 0.083 MAE. Moreover, the pretrained models can also generally regress the blend hydrogels at 20%, 40%, 60%, and 80% type II. In conclusion, the multipolarization SHG image-based ResNet analysis demonstrates the potential for an automated approach using deep learning to extract valuable information from the collagen matrix.

Tomato Fruit Detection Using Modified Yolov5m Model with Convolutional Neural Networks.

The farming industry is facing the major challenge of intensive and inefficient harvesting labors. Thus, an efficient and automated fruit harvesting system is required. In this study, three object classification models based on Yolov5m integrated with BoTNet, ShuffleNet, and GhostNet convolutional neural networks (CNNs), respectively, are proposed for the automatic detection of tomato fruit. The various models were trained using 1508 normalized images containing three classes of cherry tomatoes, namely ripe, immature, and damaged. The detection accuracy for the three classes was found to be 94%, 95%, and 96%, respectively, for the modified Yolov5m + BoTNet model. The model thus appeared to provide a promising basis for the further development of automated harvesting systems for tomato fruit.

Classification of Tomato Fruit Using Yolov5 and Convolutional Neural Network Models.

Four deep learning frameworks consisting of Yolov5m and Yolov5m combined with ResNet50, ResNet-101, and EfficientNet-B0, respectively, are proposed for classifying tomato fruit on the vine into three categories: ripe, immature, and damaged. For a training dataset consisting of 4500 images and a training process with 200 epochs, a batch size of 128, and an image size of 224 × 224 pixels, the prediction accuracy for ripe and immature tomatoes is found to be 100% when combining Yolo5m with ResNet-101. Meanwhile, the prediction accuracy for damaged tomatoes is 94% when using Yolo5m with the Efficient-B0 model. The ResNet-50, EfficientNet-B0, Yolov5m, and ResNet-101 networks have testing accuracies of 98%, 98%, 97%, and 97%, respectively. Thus, all four frameworks have the potential for tomato fruit classification in automated tomato fruit harvesting applications in agriculture.

Birefringence effect studies of collagen formed by nonenzymatic glycation using dual-retarder Mueller polarimetry.

Significance: Nonenzymatic glycation of collagen covalently attaches an addition of sugar molecules that initially were involved in a reversibly reaction with amino groups on the protein. Due to the ultimate formation of stable irreversible advanced glycation end products, the process of glycation leads to abnormal irreversible cross-linking, which ultimately accumulates with age and/or diabetes in the extracellular matrix, altering its organization. Aim: We report the use of dual-retarder Mueller polarimetry in conjunction with phase retardance to differentiate collagen cross-linking in a normal collagen gel matrix from that in tissues with nonenzymatic cross-linking. Approach: A dual-liquid crystal-based Mueller polarimetry system that involves electronic modulation of polarization state generators (PSGs) was employed to produce all types of polarization states without moving any part and enable detection of the signal directly using a Stokes polarimeter. The linear phase retardance response was obtained for the characterization of the solution and gel forms of collagen using differential Mueller matrix analysis. Results: We found that linear phase retardance measurements via differential Mueller matrix polarimetry successfully differentiated collagen gel matrices with different degrees of cross-linking formed by a nonenzymatic glycation process and demonstrated that this technology constitutes a quick and simple modality. Conclusions: This approach has high sensitivity for studying differences in fibrillar cross-linking in glycated collagen. Further, our work suggests that this method of structural analysis has potential clinical diagnostic value owing to its noninvasive and cost-efficient nature.

Characterization of collagen response to bone fracture healing using polarization-SHG.

In this study, we extend on the three parameter analysis approach of utilizing a noninvasive dual-liquid-crystal-based polarization-resolved second harmonic generation (SHG) microscopy to facilitate the quantitative characterization of collagen types I and II in fracture healing tissues. The SHG images under various linear and circular polarization states are analyzed and quantified in terms of the peptide pitch angle (PA), SHG-circular dichroism (CD), and anisotropy parameter (AP). The results show that the collagen PA has a value of 49.26° after 2 weeks of fracture healing (collagen type II domination) and 49.05° after 4 weeks (collagen type I domination). Moreover, the SHG-CD and AP values of the different collagen types differ by 0.05. The change tendencies of the extracted PA, SHG-CD, and AP parameters over the healing time are consistent with the collagen properties of healthy nonfractured bone. Thus, the feasibility of the proposed dual-liquid-crystal-based polarization-SHG method for differentiating between collagen types I and II in bone fracture healing tissue is confirmed.

Single-Layer-Graphene-Coated and Gold-Film-Based Surface Plasmon Resonance Prism Coupler Sensor for Immunoglobulin G Detection.

A graphene-based surface plasmon resonance (SPR) prism coupler sensor is proposed for the rapid detection of immunoglobulin G (IgG) antibodies. The feasibility of the proposed sensor is demonstrated by measuring the IgG concentration in phantom mouse and human serum solutions over the range of 0-250 ng/mL. The results show that the circular dichroism and principal fast axis angle of linear birefringence increase in line with increases in IgG concentration over the considered range. Moreover, the proposed device has a resolution of 5-10 ng/mL and a response time of less than three minutes. In general, the sensor provides a promising approach for IgG detection and has significant potential for rapid infectious viral disease testing applications.

Dual-LC PSHG microscopy for imaging collagen type I and type II gels with pixel-resolution analysis.

Collagen of type I (Col I) and type II (Col II) are critical for cartilage and connective tissues in the human body, and several diseases may alter their properties. Assessing the identification and quantification of fibrillar collagen without biomarkers is a challenge. Advancements in non-invasive polarization-resolved second-harmonic generation (PSHG) microscopy have provided a method for the non-destructive investigation of collagen molecular level properties. Here we explored an alternative polarization modulated approach, dual-LC PSHG, that is based on two liquid crystal devices (Liquid crystal polarization rotators, LPRs) operating simultaneously with a laser scanning SHG microscope. We demonstrated that this more accessible technology allows the quick and accurate generation of any desired linear and circular polarization state without any mechanical parts. This study demonstrates that this method can aid in improving the ability to quantify the characteristics of both types of collagen, including pitch angle, anisotropy, and circular dichroism analysis. Using this approach, we estimated the effective pitch angle for Col I and Col II to be 49.7° and 51.6°, respectively. The effective peptide pitch angle for Col II gel was first estimated and is similar to the value obtained for Col I gel in the previous studies. Additionally, the difference of the anisotropy parameter of both collagen type gels was assessed to be 0.293, which reflects the different type molecular fibril assembly. Further, our work suggests a potential method for monitoring and differentiating different collagen types in biological tissues, especially cartilage or connective tissue.

Dual-Retarder Mueller Polarimetry System for Extraction of Optical Properties of Serum Albumin Protein Media.

A dual liquid-crystal variable retarder Mueller polarimetry system incorporating a gold-based surface plasmon resonance prism coupler was proposed for extracting the optical properties of serum albumin protein media in the reflectance configuration. The feasibility of the proposed system was demonstrated by measuring the circular dichroism and circular birefringence properties of glucose tissue phantom solutions with different albumin concentrations. The results showed that the circular dichroism increased with albumin concentration, while the optical rotation angle increased with glucose concentration. Both properties reduced over time as a result of the protein glycation effect, which led to a gradual reduction in the glucose content of the sample.

Surface plasmon resonance prism coupler for enhanced circular birefringence sensing and application to non-invasive glucose detection.

A surface plasmon resonance (SPR) prism coupler is proposed for the high-resolution non-invasive (NI) measurement of the circular birefringence (CB) properties of turbid media. The feasibility of the proposed device is demonstrated by means of numerical simulations. It is shown that the SPR sensor enables the CB properties to be detected with a resolution of up to 8.9 × 10-7 RIU (refractive index units) for refractive indices in the range of 1.3∼1.4. Moreover, for tissue phantom solutions containing 2% lipofundin, the device has a detection limit of 3.72 mg/dL. This resolution performance satisfies the detection limit of 10 mg/dL stipulated by the U.S FDA for point-of-care glucose monitoring devices. Thus, the proposed SPR sensor has significant potential for NI glucose sensing in such applications as diabetes detection and management.

Two-color temporal focusing multiphoton excitation imaging with tunable-wavelength excitation.

Wavelength tunable temporal focusing multiphoton excitation microscopy (TFMPEM) is conducted to visualize optical sectioning images of multiple fluorophore­labeled specimens through the optimal two-photon excitation (TPE) of each type of fluorophore. The tunable range of excitation wavelength was determined by the groove density of the grating, the diffraction angle, the focal length of lenses, and the shifting distance of the first lens in the beam expander. Based on a consideration of the trade-off between the tunable-wavelength range and axial resolution of temporal focusing multiphoton excitation imaging, the presented system demonstrated a tunable-wavelength range from 770 to 920 nm using a diffraction grating with groove density of 830 ?? lines / mm . TPE fluorescence imaging examination of a fluorescent thin film indicated that the width of the axial confined excitation was 3.0 ± 0.7 ?? ? m and the shifting distance of the temporal focal plane was less than 0.95 ?? ? m within the presented wavelength tunable range. Fast different wavelength excitation and three-dimensionally rendered imaging of Hela cell mitochondria and cytoskeletons and mouse muscle fibers were demonstrated. Significantly, the proposed system can improve the quality of two-color TFMPEM images through different excitation wavelengths to obtain higher-quality fluorescent signals in multiple-fluorophore measurements.

Dual-color dynamic tracking of GM-CSF receptors/JAK2 kinases signaling activation using temporal focusing multiphoton fluorescence excitation and astigmatic imaging.

The dual-color dynamic particle tracking approach that uses temporal focusing multiphoton fluorescence excitation and two-channel astigmatic imaging is utilized to track molecular trajectories in three dimensions to explore molecular interactions. Images of two fluorophores were obtained to extract their positions by optical sectioning excitation using a fast temporal focusing multiphoton excitation microscope (TFMPEM) and by the simultaneous collection of data in two channels. The presented pair of cylindrical lenses, which was used to adjust the astigmatism effect with the minimum shifting of the imaging plane, was more feasible and flexible than single cylindrical lens for aligning two separate detection channels in astigmatic imaging. The lateral and axial positioning resolutions were observed to be approximately 9-13 nm and 23-30 nm respectively, for the two fluorescence channels. The dynamic movement and binding behavior of clusters of GM-CSF receptors and JAK2 kinases in HeLa cells in the presence of GM-CSF ligands were observed. Therefore, the proposed dual-color tracking strategy is useful for the dynamic study of molecular interactions in living specimens with a fast frame rate, less photobleaching, better penetration depth, and minimum optical trapping force.

Neurodegeneration in Amygdala Precedes Hippocampus in the APPswe/ PS1dE9 Mouse Model of Alzheimer's Disease.

Both the hippocampus and amygdala are early vulnerable brain regions in the development of Alzheimer's disease (AD). However, previous studies mainly focused on characterizing the hippocampus in the pathophysiology of AD, leaving the amygdala less explored. Here, we characterized the structures and functions of neurons in the hippocampus and amygdala of young (2, 3 and 4 months of age) APP/PS1 double transgenic (Tg) mice, a widely used AD mouse model. Compared to wild-type littermates (Wt ), Tg mice performed worse in amygdala-dominant memory at all three ages, while hippocampus-dominant memory remained intact until 4-month-old. Likewise, the dendritic arbors of neurons in the basolateral amygdala were reduced in Tg mice as early as 2-months-old, while the dendritic arbors of neurons in the hippocampal CA1 and CA3 regions were relatively intact. BDNF signaling pathways (e.g. AKT and PKC) were reduced in the amygdala, but not in the hippocampus, of young Tg mice. Furthermore, reduction of 5-HT and elevation of Aß levels also occurred earlier in the amygdala and were more pronounced than those in the hippocampus. Negative correlations between the levels of 5-HT and Aß were evident in the amygdala, but not in the hippocampus. Taken together, these results suggest that neurodegeneration occurs earlier in the amygdala than in the hippocampus. We suggest that amygdala function should be incorporated into the cognitive screening tool for the diagnosis of mild cognitive impairment due to AD.

Running exercise delays neurodegeneration in amygdala and hippocampus of Alzheimer's disease (APP/PS1) transgenic mice.

Alzheimer's disease (AD) is an age-related neurodegenerative disease. Post-mortem examination and brain imaging studies indicate that neurodegeneration is evident in the hippocampus and amygdala of very early stage AD patients. Exercise training is known to enhance hippocampus- and amygdala-associated neuronal function. Here, we investigated the effects of exercise (running) on the neuronal structure and function of the hippocampus and amygdala in APP/PS1 transgenic (Tg) mice. At 4-months-old, an age before amyloid deposition, the amygdala-associated, but not the hippocampus-associated, long-term memory was impaired in the Tg mice. The dendritic complexities of the amygdalar basolateral neurons, but not those in the hippocampal CA1 and CA3 neurons, were reduced. Furthermore, the levels of BDNF/TrkB signaling molecules (i.e. p-TrkB, p-Akt and p-PKC) were reduced in the amygdala, but not in the hippocampus of the 4-month-old Tg mice. The concentrations of Aß40 and Aß42 in the amygdala were higher than those in the hippocampus. Ten weeks of treadmill training (from 1.5- to 4-month-old) increased the hippocampus-associated memory and dendritic arbor of the CA1 and CA3 neurons, and also restored the amygdala-associated memory and the dendritic arbor of amygdalar basolateral neurons in the Tg mice. Similarly, exercise training also increased the levels of p-TrkB, p-AKT and p-PKC in the hippocampus and amygdala. Furthermore, exercise training reduced the levels of soluble Aß in the amygdala and hippocampus. Exercise training did not change the levels of APP or RAGE, but significantly increased the levels of LRP-1 in both brain regions of the Tg mice. In conclusion, our results suggest that tests of amygdala function should be incorporated into subject selection for early prevention trials. Long-term exercise protects neurons in the amygdala and hippocampus against AD-related degeneration, probably via enhancements of BDNF signaling pathways and Aß clearance. Physical exercise may serve as a means to delay the onset of AD.

Dynamic particle tracking via temporal focusing multiphoton microscopy with astigmatism imaging.

A three-dimensional (3D) single fluorescent particle tracking strategy based on temporal focusing multiphoton excitation microscopy (TFMPEM) combined with astigmatism imaging is proposed for delivering nanoscale-level axial information that reveals 3D trajectories of single fluorospheres in the axially-resolved multiphoton excitation volume without z-axis scanning. Whereas other scanning spatial focusing multiphoton excitation schemes induce optical trapping interference, temporal focusing multiphoton excitation produces widefield illumination with minimum optical trapping force on the fluorospheres. Currently, the lateral and axial positioning resolutions of the dynamic particle tracking approach are about 14 nm and 21 nm in standard deviation, respectively. Furthermore, the motion behavior and diffusion coefficients of fluorospheres in glycerol solutions with different concentrations are dynamically measured at a frame rate up to 100 Hz. This TFMPEM with astigmatism imaging holds great promise for exploring dynamic molecular behavior deep inside biotissues via its superior penetration, reduced trapping effect, fast frame rate, and nanoscale-level positioning.

Nonlinear structured-illumination enhanced temporal focusing multiphoton excitation microscopy with a digital micromirror device.

In this study, the light diffraction of temporal focusing multiphoton excitation microscopy (TFMPEM) and the excitation patterning of nonlinear structured-illumination microscopy (NSIM) can be simultaneously and accurately implemented via a single high-resolution digital micromirror device. The lateral and axial spatial resolutions of the TFMPEM are remarkably improved through the second-order NSIM and projected structured light, respectively. The experimental results demonstrate that the lateral and axial resolutions are enhanced from 397 nm to 168 nm (2.4-fold) and from 2.33 µm to 1.22 µm (1.9-fold), respectively, in full width at the half maximum. Furthermore, a three-dimensionally rendered image of a cytoskeleton cell featuring ~25 nm microtubules is improved, with other microtubules at a distance near the lateral resolution of 168 nm also able to be distinguished.

Temporal focusing-based multiphoton excitation microscopy via digital micromirror device.

This Letter presents an enhanced temporal focusing-based multiphoton excitation (MPE) microscope in which the conventional diffraction grating is replaced by a digital micromirror device (DMD). Experimental results from imaging a thin fluorescence film show that the 4.0 µm axial resolution of the microscope is comparable with that of a setup incorporating a 600 lines/mm grating; hence, the optical sectioning ability of the proposed setup is demonstrated. Similar to a grating, the DMD diffracts illuminating light frequencies for temporal focusing; additionally, it generates arbitrary patterns. Since the DMD is placed on the image-conjugate plane of the objective lens' focal plane, the MPE pattern can be projected on the focal plane precisely.

High power NIR fiber-optic femtosecond Cherenkov radiation and its application on nonlinear light microscopy.

We reported a record high power (>250 mW) and compact near-infrared fiber-optic femtosecond Cherenkov radiation source and its new application on nonlinear light microscopy for the first time (to our best knowledge). The high power femtosecond Cherenkov radiation was generated by 1.03 µm femtosecond pulses from a portable diode-pumped laser and a photonic crystal fiber as a compact, flexible, and highly efficient wavelength convertor. Sectioned nonlinear light microscopy images from mouse brain blood vessel network and rat tail tendon were then performed by the demonstrated light source. Due to the advantages of its high average output power (>250 mW), high pulse energy (>4 nJ), excellent wavelength conversion efficiency (>40%), compactness, simplicity in configuration, and turn-key operation, the demonstrated femtosecond Cherenkov radiation source could thus be widely applicable as an alternative excitation source to mode-locked Ti:Sapphire lasers for future clinical nonlinear microscopy or other applications requiring synchronized multi-wavelength light sources.

Differentiation of Col I and Col III isoforms in stromal models of ovarian cancer by analysis of second harmonic generation polarization and emission directionality.

A profound remodeling of the extracellular matrix occurs in many epithelial cancers. In ovarian cancer, the minor collagen isoform of Col III becomes upregulated in invasive disease. Here we use second harmonic generation (SHG) imaging microscopy to probe structural differences in fibrillar models of the ovarian stroma comprised of mixtures of Col I and III. The SHG intensity and forward-backward ratios decrease with increasing Col III content, consistent with decreased phasematching due to more randomized structures. We further probe the net collagen α-helix pitch angle within the gel mixtures using what is believed to be a new pixel-based polarization-resolved approach that combines and extends previous analyses. The extracted pitch angles are consistent with those of peptide models and the method has sufficient sensitivity to differentiate Col I from the Col I/Col III mixtures. We further developed the pixel-based approach to extract the SHG signal polarization anisotropy from the same polarization-resolved image matrix. Using this approach, we found that increased Col III results in decreased alignment of the dipole moments within the focal volume. Collectively, the SHG measurements and analysis all indicate that incorporation of Col III results in decreased organization across several levels of collagen organization. Furthermore, the findings suggest that the collagen isoforms comingle within the same fibrils, in good agreement with ultrastructural data. The pixel-based polarization analyses (both excitation and emission) afford determination of structural properties without the previous requirement of having well-aligned fibers, and the approaches should be generally applicable in tissue.

Image-inspired 3D multiphoton excited fabrication of extracellular matrix structures by modulated raster scanning.

Multiphoton excited photochemistry is a powerful 3D fabrication tool that produces sub-micron feature sizes. Here we exploit the freeform nature of the process to create models of the extracellular matrix (ECM) of several tissues, where the design blueprint is derived directly from high resolution optical microscopy images (e.g. fluorescence and Second Harmonic Generation). To achieve this goal, we implemented a new form of instrument control, termed modulated raster scanning, where rapid laser shuttering (10 MHz) is used to directly map the greyscale image data to the resulting protein concentration in the fabricated scaffold. Fidelity in terms of area coverage and relative concentration relative to the image data is ~95%. We compare the results to an STL approach, and find the new scheme provides significantly improved performance. We suggest the method will enable a variety of cell-matrix studies in cancer biology and also provide insight into generating scaffolds for tissue engineering.

Precise, motion-free polarization control in Second Harmonic Generation microscopy using a liquid crystal modulator in the infinity space.

Second Harmonic Generation (SHG) microscopy coupled with polarization analysis has great potential for use in tissue characterization, as molecular and supramolecular structural details can be extracted. Such measurements are difficult to perform quickly and accurately. Here we present a new method that uses a liquid crystal modulator (LCM) located in the infinity space of a SHG laser scanning microscope that allows the generation of any desired linear or circular polarization state. As the device contains no moving parts, polarization can be rotated accurately and faster than by manual or motorized control. The performance in terms of polarization purity was validated using Stokes vector polarimetry, and found to have minimal residual polarization ellipticity. SHG polarization imaging characteristics were validated against well-characterized specimens having cylindrical and/or linear symmetries. The LCM has a small footprint and can be implemented easily in any standard microscope and is cost effective relative to other technologies.