Second Harmonic Generation in 3‐D Uniform Arrangement of Type I Collagen on Nonlinear Optics Microscopy

Second harmonic microscopic imaging and spectroscopy technology has become a powerful tool for biomedical studies, especially in fibrosis‐related diseases research. And type I collagen is the major risk factors for fibrotic diseases. In this study, model for three‐dimensional (3‐D) uniform arrangement type I collagen is set up for researching the second harmonic generation (SHG) on nonlinear optics microscopy. Based on this model, we discuss the influence of different length and size collagen in 3‐D arrangement type I collagen. Results can guide us to neatly judge the size, length, and molecules density effect on SHG. For practical application, this theoretical approach can lead us to analyze different severity of collagen diseases. SCANNING 35:12‐16, 2013. © 2012 Wiley Periodicals, Inc.     

TOC ‐ Issue Information

Knowledge of the collagen structure of an Achilles tendon is critical to comprehend the physiology, biomechanics, homeostasis and remodelling of the tissue. Despite intensive studies, there are still uncertainties regarding the microstructure. The majority of studies have examined the longitudinally arranged collagen fibrils as they are primarily attributed to the principal tensile strength of the tendon. Few studies have considered the structural integrity of the entire three‐dimensional (3D) collagen meshwork, and how the longitudinal collagen fibrils are integrated as a strong unit in a 3D domain to provide the tendons with the essential tensile properties. Using second harmonic generation imaging, a 3D imaging technique was developed and used to study the 3D collagen matrix in the midportion of Achilles tendons without tissue labelling and dehydration. Therefore, the 3D collagen structure is presented in a condition closely representative of the in vivo status. Atomic force microscopy studies have confirmed that second harmonic generation reveals the internal collagen matrix of tendons in 3D at a fibril level. Achilles tendons primarily contain longitudinal collagen fibrils that braid spatially into a dense rope‐like collagen meshwork and are encapsulated or wound tightly by the oblique collagen fibrils emanating from the epitenon region. The arrangement of the collagen fibrils provides the longitudinal fibrils with essential structural integrity and endows the tendon with the unique mechanical function for withstanding tensile stresses. A novel 3D microscopic method has been developed to examine the 3D collagen microstructure of tendons without tissue dehydrating and labelling. The study also provides new knowledge about the collagen microstructure in an Achilles tendon, which enables understanding of the function of the tissue. The knowledge may be important for applying surgical and tissue engineering techniques to tendon reconstruction.     

Comparison of three‐dimensional analysis and stereological techniques for quantifying lithium‐ion battery electrode microstructures

Lithium‐ion battery performance is intrinsically linked to electrode microstructure. Quantitative measurement of key structural parameters of lithium‐ion battery electrode microstructures will enable optimization as well as motivate systematic numerical studies for the improvement of battery performance. With the rapid development of 3‐D imaging techniques, quantitative assessment of 3‐D microstructures from 2‐D image sections by stereological methods appears outmoded; however, in spite of the proliferation of tomographic imaging techniques, it remains significantly easier to obtain two‐dimensional (2‐D) data sets. In this study, stereological prediction and three‐dimensional (3‐D) analysis techniques for quantitative assessment of key geometric parameters for characterizing battery electrode microstructures are examined and compared. Lithium‐ion battery electrodes were imaged using synchrotron‐based X‐ray tomographic microscopy. For each electrode sample investigated, stereological analysis was performed on reconstructed 2‐D image sections generated from tomographic imaging, whereas direct 3‐D analysis was performed on reconstructed image volumes. The analysis showed that geometric parameter estimation using 2‐D image sections is bound to be associated with ambiguity and that volume‐based 3‐D characterization of nonconvex, irregular and interconnected particles can be used to more accurately quantify spatially‐dependent parameters, such as tortuosity and pore‐phase connectivity.     

Three‐dimensional visualization of dermal skin structure using confocal laser scanning microscopy

The properties and performance of collagen‐based materials may be affected by the collagen fibre bundle pattern, orientation and weave. The aim of this study was to develop and apply methods to visualize the dermis using confocal laser scanning microscopy from thin tissue sections stained with haematoxylin and eosin. The data was processed to allow three‐dimensional (3‐D) visualization on a PC and using a 3‐D immersive technology system. The 3‐D visualization of the confocal microscope image stacks allowed the evaluation of the collagen macromolecular structure including the collagen fibre bundles. The methods developed provide a novel way of viewing complex organic structures with further potential applications in the medical field.     

Application of an advanced maximum likelihood estimation restoration method for enhanced‐resolution and contrast in second‐harmonic generation microscopy

Second‐harmonic generation (SHG) microscopy has gained popularity because of its ability to perform submicron, label‐free imaging of noncentrosymmetric biological structures, such as fibrillar collagen in the extracellular matrix environment of various organs with high contrast and specificity. Because SHG is a two‐photon coherent scattering process, it is difficult to define a point spread function (PSF) for this modality. Hence, compared to incoherent two‐photon processes like two‐photon fluorescence, it is challenging to apply the various PSF‐engineering methods to improve the spatial resolution to be close to the diffraction limit. Using a synthetic PSF and application of an advanced maximum likelihood estimation (AdvMLE) deconvolution algorithm, we demonstrate restoration of the spatial resolution in SHG images to that closer to the theoretical diffraction limit. The AdvMLE algorithm adaptively and iteratively develops a PSF for the supplied image and succeeds in improving the signal to noise ratio (SNR) for images where the SHG signals are derived from various sources such as collagen in tendon and myosin in heart sarcomere. Approximately 3.5 times improvement in SNR is observed for tissue images at depths of up to ～480 nm, which helps in revealing the underlying helical structures in collagen fibres with an ～26% improvement in the amplitude contrast in a fibre pitch. Our approach could be adapted to noisy and low resolution modalities such as micro‐nano CT and MRI, impacting precision of diagnosis and treatment of human diseases.     

Three‐dimensional analysis of injury conditions of single muscle fibers in small animals using phase‐contrast X‐ray imaging

Muscle damage can reduces the biological functions and lead to ultimately a disease state. For the reason, it is important to accurately check the state of an injury such as atrophy, and it is required to identify the state of fibers constituting the muscle. This study describes a novel method of analyzing single muscle fibers with injury conditions in three‐dimensions. The muscle fibers of the mice were visualized using phase‐contrast X‐ray projection the microstructure. In additions, it was possible to confirm the status by quantitatively analyzing the injury severity of muscle fibers. Significantly, the muscle conditions of multiple individuals were individually determined. This study could contributes to areas where it is very important to identify microdetailed and quantitative changes of state, such as new drug development.     

Real‐time three‐dimensional imaging of cell division by differential interference contrast microscopy

Differential interference contrast (DIC) microscopy can provide information about subcellular components and organelles inside living cells. Applicability to date, however, has been limited to 2D imaging. Unfortunately, understanding of cellular dynamics is difficult to extract from these single optical sections. We demonstrate here that 3D differential interference contrast microscopy has sub‐diffraction limit resolution both laterally and vertically, and can be used for following Madin Darby canine kidney cell division process in real time. This is made possible by optimization of the microscope optics and by incorporating computer‐controlled vertical scanning of the microscope stage.     

Non‐rigid alignment in electron tomography in materials science

Electron tomography is a key technique that enables the visualization of an object in three dimensions with a resolution of about a nanometre. High‐quality 3D reconstruction is possible thanks to the latest compressed sensing algorithms and/or better alignment and preprocessing of the 2D projections. Rigid alignment of 2D projections is routine in electron tomography. However, it cannot correct misalignments induced by (i) deformations of the sample due to radiation damage or (ii) drifting of the sample during the acquisition of an image in scanning transmission electron microscope mode. In both cases, those misalignments can give rise to artefacts in the reconstruction. We propose a simple‐to‐implement non‐rigid alignment technique to correct those artefacts. This technique is particularly suited for needle‐shaped samples in materials science. It is initiated by a rigid alignment of the projections and it is then followed by several rigid alignments of different parts of the projections. Piecewise linear deformations are applied to each projection to force them to simultaneously satisfy the rigid alignments of the different parts. The efficiency of this technique is demonstrated on three samples, an intermetallic sample with deformation misalignments due to a high electron dose typical to spectroscopic electron tomography, a porous silicon sample with an extremely thin end particularly sensitive to electron beam and another porous silicon sample that was drifting during image acquisitions.     

Label‐free detection of fibrillar collagen deposition associated with vascular elements in glioblastoma multiforme by using multiphoton microscopy

Glioblastoma multiforme (GBM‐WHO grade IV) is the most common and the most aggressive form of brain tumors in adults with the median survival of 10–12 months. The diagnostic detection of extracellular matrix (ECM) component in the tumour microenvironment is of prognostic value. In this paper, the fibrillar collagen deposition associated with vascular elements in GBM were investigated in the fresh specimens and unstained histological slices by using multiphoton microscopy (MPM) based on two‐photon excited fluorescence (TPEF) and second harmonic generation (SHG). Our study revealed the existence of fibrillar collagen deposition in the adventitia of remodelled large blood vessels and in glomeruloid vascular structures in GBM. The degree of fibrillar collagen deposition can be quantitatively evaluated by measuring the adventitial thickness of blood vessels or calculating the ratio of SHG pixel to the whole pixel of glomeruloid vascular structure in MPM images. These results indicated that MPM can not only be employed to perform a retrospective study in unstained histological slices but also has the potential to apply for in vivo brain imaging to understand correlations between malignancy of gliomas and fibrillar collagen deposition.     

In vivo second‐harmonic generation and ex vivo coherent anti‐stokes raman scattering microscopy to study the effect of obesity to fibroblast cell function using an Yb‐fiber laser‐based CARS extension unit

Nonlinear microscopy techniques are being increasingly used to perform in vivo studies in dermatology. These methods enable us to investigate the morphology and monitor the physiological process in the skin by the use of femtosecond lasers operating in the red, near‐infrared spectral range (680–1,300 nm). In this work we used two different techniques that require no labeling: second harmonic generation (SHG) for collagen detection and coherent anti‐Stokes Raman scattering (CARS) to assess lipid distribution in genetically obese murine skin. Obesity is one of the most serious public health problems due to its high and increasing prevalence and the associated risk of type 2 diabetes and cardiovascular diseases. Other than these diseases, nearly half of patients with diabetes mellitus suffer from dermatological complications such as delayed wound healing, foot ulcers and several other skin changes. In our experiment we investigated and followed the effects of obesity on dermal collagen alterations and adipocyte enlargement using a technique not reported in the literature so far. Our results indicate that the in vivo SHG and ex vivo CARS imaging technique might be an important tool for diagnosis of diabetes‐related skin disorders in the near future. Microsc. Res. Tech. 78:823–830, 2015. © 2015 Wiley Periodicals, Inc.     

An open‐source deconvolution software package for 3‐D quantitative fluorescence microscopy imaging

Deconvolution techniques have been widely used for restoring the 3‐D quantitative information of an unknown specimen observed using a wide‐field fluorescence microscope. Deconv , an open‐source deconvolution software package, was developed for 3‐D quantitative fluorescence microscopy imaging and was released under the GNU Public License. Deconv provides numerical routines for simulation of a 3‐D point spread function and deconvolution routines implemented three constrained iterative deconvolution algorithms: one based on a Poisson noise model and two others based on a Gaussian noise model. These algorithms are presented and evaluated using synthetic images and experimentally obtained microscope images, and the use of the library is explained. Deconv allows users to assess the utility of these deconvolution algorithms and to determine which are suited for a particular imaging application. The design of Deconv makes it easy for deconvolution capabilities to be incorporated into existing imaging applications.     

Label‐free imaging characteristics of colonic mucinous adenocarcinoma using multiphoton microscopy

Colorectal carcinoma (CRC) has high mortality and increased incidence rates. An early detection of CRC is very important. Multiphoton microscopy (MPM) with high resolution and high sensitivity is used to effectively distinguish the microstructure changes of normal and mucinous adenocarcinoma slices of ex vivo human colonic tissues. In mucinous adenocarcinoma mucosa, the glands are distorted and elongated, the gland cavity is indistinct, and the mesh collagen fibers are diminished. In the submucosa, the collagens are seriously disordered, elongated, pushed aside, and sparsely visible, the content of elastic fibers is also broken and almost disappearing. Many cancer cells, some in cavity‐like shape full of mucus surrounded by some collagen fibers, occupied the submucosa, which are comparable to hematoxylin‐eosin (HE) stained images. Second harmonic generation and two‐photon excitation fluorescence (SHG/TPEF) intensity ratio can be used further to quantitatively evaluate normality and abnormality. The fast Fourier transform (FFT) images show that the normal collagen fibrils are dense and in random order, and the cancerous collagen is certainly organized. The exploratory results show that it has potential for the development of multiphoton mini‐endoscopy in real‐time early diagnosis of CRC. SCANNING 35: 277‐282, 2013. © 2012 Wiley Periodicals, Inc.     

Optimization of Picrosirius red staining protocol to determine collagen fiber orientations in vaginal and uterine cervical tissues by Mueller polarized microscopy

Polarized microscopy provides unique information on anisotropic samples. In its most complete implementation, namely Mueller microscopy, this technique is well suited for the visualization of fibrillar proteins orientations, with collagen in the first place. However, the intrinsic optical anisotropy of unstained tissues has to be enhanced by Picrosirius Red (PR) staining to enable Mueller measurements. In this work, we compared the orientation mapping provided by Mueller and second harmonic generation (SHG) microscopies on PR stained samples of vaginal and uterine cervix tissues. SHG is a multiphoton technique that is highly specific to fibrillar collagen, and was taken as the “gold standard” for its visualization. We showed that Mueller microscopy can be safely used to determine collagen orientation in PR stained cervical tissue. In contrast, in vaginal samples, Mueller microscopy revealed orientations not only of collagen but also of other anisotropic structures. Thus PR is not fully specific to collagen, which necessitates comparison to SHG microscopy in every type of tissue. In addition to this study of PR specificity, we determined the optimal values of the staining parameters. We found that staining times of 5 min, and sample thicknesses of 5 µm were sufficient in cervical and vaginal tissues. Microsc. Res. Tech. 78:723–730, 2015. © 2015 Wiley Periodicals, Inc.     

Three‐dimensional visualization of rat retina by X‐ray differential phase contrast tomographic microscopy

The retina is one of the most tiny and sophisticated tissues of the body. Three dimensional (3D) visualization of the whole retina is valuable both in clinical and research arenas. The tissue has been predominantly assessed by time‐consuming histopathology and optical coherence tomography (OCT) in research and clinical arenas. However, none of the two methods can provide 3D imaging of the retina. The purpose of this study is to give a volumetric visualization of rat retina at submicron resolution, using an emerging imaging technique‐phase‐contrast X‐ray CT. A Sprague‐Dawley (SD) rat eye specimen was scanned with X‐ray differential phase contrast tomographic microscopy (DPC‐microCT) equipped at the Swiss Light Source synchrotron. After scanning, the specimen was subjected to routine histology procedures and severed as a reference. The morphological characteristics and signal features of the retina in the DPC‐microCT images were evaluated. The total retina and its sublayers thicknesses were measured on the DPC‐microCT images and compared with those obtained from the histological sections. The retina structures revealed by DPC‐microCT were highly consistent with the histological section. In this study, we achieved nondestructive 3D visualization of SD rat retina. In addition to detailed anatomical structures, the objective parameters provided by DPC‐microCT make it a useful tool for retinal research and disease diagnosis in the early stage.     

Quantitative biomarkers of human skin photoaging based on intrinsic second harmonic generation signal

Collagen change is a major feature in the photoaged human skin. Here, we present the use of intrinsic second harmonic generation (SHG) signal as a novel means to quantify collagen change with photoaging. We obtain the SHG images of the superficial dermis from ex vivo the cheek skin and the abdomen skin of eight patients aged 55–60 years. The results show that SHG signal can quantitatively reveal collagen change between normal and photoaged human skin in three dimensions. By comparing normal with photoaged dermis, there are significant differences in the collagen content and fine structure, providing substantial potential to be applied in vivo for the clinical diagnosis of human skin photoaging. SCANNING 35: 273‐276, 2013. © 2012 Wiley Periodicals, Inc.     

High accuracy 4D cell tracking into explanted skin using two‐photon excitation microscopy

Two‐photon excitation microscopy (2PEM) analysis of large explanted organs is still laborious, principally because of tissue movements inducing lateral and axial drifts during extended imaging sessions. Here, we describe a two‐step approach to track motile T cells in murine dorsal explanted skin with the best accuracy. First, we compared various explanted skin mounting methods for 2PEM analysis to define the setup allowing for minimal sample drift over time. Second, we developed two algorithms with the ImageJ software (National Institute of Health, Bethesda, MD) to correct the residual drift using lateral and axial registration of the collagen network. Finally, we applied the macro we developed to track fluorescent T cells in explanted skin. We found that our newly developed macro is more efficient than freely or commercially available software for shift correction, leading to more accurate velocity calculations. Our work provides a practical guide for investigators interested to employ skin‐imaging approaches and offers a free alternative to commercial software for correcting lateral and axial drifts. Microsc. Res. Tech. 78:294–301, 2015. © 2015 Wiley Periodicals, Inc.     

Advances in three‐dimensional super‐resolution nanoscopy

Three‐dimensional optical super‐resolution imaging is capable of providing 3D visualization of cellular structures in nanoscale detail. The past decade has witnessed the blossoming of 3D super‐resolution imaging technologies. In this review, we comprehensively discuss and compare the imaging depth, resolution enhancement, and imaging speed of the existing 3D super‐resolution imaging techniques.     

Second harmonic generation imaging of the deep shade plant Selaginella erythropus using multifunctional two‐photon laser scanning microscopy

Background : Multifunctional two‐photon laser scanning microscopy provides attractive advantages over conventional two‐photon laser scanning microscopy. For the first time, simultaneous measurement of the second harmonic generation (SHG) signals in the forward and backward directions and two photon excitation fluorescence were achieved from the deep shade plant Selaginella erythropus. Results : These measurements show that the S. erythropus leaves produce high SHG signals in both directions and the SHG signals strongly depend on the laser's status of polarization and the orientation of the dipole moment in the molecules that interact with the laser light. The novelty of this work is (1) uncovering the unusual structure of S. erythropus leaves, including diverse chloroplasts, various cell types and micromophology, which are consistent with observations from general electron microscopy; and (2) using the multifunctional two‐photon laser scanning microscopy by combining three platforms of laser scanning microscopy, fluorescence microscopy, harmonic generation microscopy and polarizing microscopy for detecting the SHG signals in the forward and backward directions, as well as two photon excitation fluorescence. Conclusions : With the multifunctional two‐photon laser scanning microscopy, one can use noninvasive SHG imaging to reveal the true architecture of the sample, without photodamage or photobleaching, by utilizing the fact that the SHG is known to leave no energy deposition on the interacting matter because of the SHG virtual energy conservation characteristic.     

Usefulness of hard X‐ray microscope using synchrotron radiation for the structure analysis of insects

Three‐dimensional (3D) printing technology has the advantage of enabling specific visualization of creative ideas. Since synchrotron based images can provide high sensitivity and high resolution, they are a very useful technology to identify the 3D anatomy of microscale samples. X‐ray images using such synchrotron radiation are grafted to 3D printing technology. We can be obtained 3D images and modeling data of an ant using synchrotron radiation, and then, it were outputted with the 3D printer. A new way to identify the usefulness of the structure analysis is then found by visualizing the micro‐internal structure of diverse biomedical samples and creating an enlarged model. This study suggests methods of utilizing a 3D printed model produced through synchrotron radiation imaging in various fields such as bioengineering, medical, and education.     

3‐D PSF fitting for fluorescence microscopy: implementation and localization application

Localization microscopy relies on computationally efficient Gaussian approximations of the point spread function for the calculation of fluorophore positions. Theoretical predictions show that under specific experimental conditions, localization accuracy is significantly improved when the localization is performed using a more realistic model. Here, we show how this can be achieved by considering three‐dimensional (3‐D) point spread function models for the wide field microscope. We introduce a least‐squares point spread function fitting framework that utilizes the Gibson and Lanni model and propose a computationally efficient way for evaluating its derivative functions. We demonstrate the usefulness of the proposed approach with algorithms for particle localization and defocus estimation, both implemented as plugins for ImageJ.