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.     

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.     

On‐Site Monitoring and Cartographical Mapping of Heavy Metals

Abstract

This paper presents a portable electrochemical instrument capable of real‐time in situ detection and automatic identification of heavy metals. The instrument is equipped with an embedded Geographical Position System and is capable of storing the geographical position of the sample under test. Software has been developed to combine pollutant results with geographical position, in order to produce a cartographical presentation of the pollution of an area. The electrochemical instrument provides the facilities found in a traditional lab based instrument in a portable design for on‐site measurements. The instrument is capable of detecting lead, cadmium, zinc, nickel, mercury, and copper with good sensitivity and precision. The system is reliable, easy to use, safe, and it may be used in a variety of situations to help environmental assessment and control.     

Harmonic optical microscopy and fluorescence lifetime imaging platform for multimodal imaging

In this work, we proposed and built a multimodal optical setup that extends a commercially available confocal microscope (Olympus VF300) to include nonlinear second harmonic generation (SHG) and third harmonic generation (THG) optical (NLO) microscopy and fluorescence lifetime imaging microscopy (FLIM). We explored all the flexibility offered by this commercial confocal microscope to include the nonlinear microscopy capabilities. The setup allows image acquisition with confocal, brightfield, NLO/multiphoton and FLIM imaging. Simultaneously, two‐photon excited fluorescence (TPEF) and SHG are well established in the biomedical imaging area, because one can use the same ultrafast laser and detectors set to acquire both signals simultaneously. Because the integration with FLIM requires a separated modulus, there are fewer reports of TPEF+SHG+FLIM in the literature. The lack of reports of a TPEF+SHG+THG+FLIM system is mainly due to difficulties with THG because the present NLO laser sources generate THG in an UV wavelength range incompatible with microscope optics. In this article, we report the development of an easy‐to‐operate platform capable to perform two‐photon fluorescence (TPFE), SHG, THG, and FLIM using a single 80 MHz femtosecond Ti:sapphire laser source. We described the modifications over the confocal system necessary to implement this integration and verified the presence of SHG and THG signals by several physical evidences. Finally, we demonstrated the use of this integrated system by acquiring images of vegetables and epithelial cancer biological samples. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

MRT Letter: An extended scanning probe microscopy system for macroscopic topography imaging

Enlightened by the principle of scanning probe microscopy or atomic force microscope (AFM), we proposed a novel surface topography imaging system based on the scanning of a piezoelectric unimorph cantilever. The height of sample surface can be obtained by recording the cantilever's strain using an ultra‐sensitive strain gauge and the Z‐axis movement is realized by electric bending of the cantilever. This system can be operated in the way similar to the contact mode in AFM, with the practical height detection resolution better than 100 nm. Imaging of the inner surface of a steel tube and on a transparent wing of a honey bee were conducted and the obtained results showed that this proposed system is a very promising solution for in situ topography mapping. Microsc. Res. Tech. 77:749–753, 2014. © 2014 Wiley Periodicals, Inc.     

Imaging ectopic fat deposition in caenorhabditis elegans muscles using nonlinear microscopy

The elucidation of the molecular mechanisms that lead to the development of metabolic syndrome, a complex of pathological conditions including type‐2 diabetes, hypertension, and cardiovascular diseases, is an important issue with high biological significance and requires accurate methods capable of monitoring lipid storage distribution and dynamics in vivo. In this study, the nonlinear phenomena of second and third harmonic generation (SHG, THG) have been employed simultaneously as label‐free, nondestructive diagnostic techniques, for the monitoring and the complementary three‐dimensional (3D) imaging and analysis of the muscular areas and the lipid content localization. THG microscopy was used as a quantitative tool in order to record the accumulation of lipids in nonadipose tissues in the pharyngeal muscles of 18 Caenorhabditis elegans (C. elegans) specimens, while the SHG imaging provided the detailed anatomical information about the structure of the muscles. The ectopic accumulation of fat on the pharyngeal muscles increases in wild‐type (N2) C. elegans between 1 and 9 days of adulthood. This suggests a correlation of ectopic fat accumulation with the process of aging. Our results can contribute to the unraveling of the link between the deposition of ectopic fat and aging, but mainly to the validation of SHG and THG microscopy modalities as new, noninvasive tools to localize and quantify selectively lipid formation and distribution. Microsc. Res. Tech. 78:523–528, 2015. © 2015 Wiley Periodicals, Inc.     

Automatic setae segmentation from Chaetoceros microscopic images

A novel image processing model Grayscale Surface Direction Angle Model (GSDAM) is presented and the algorithm based on GSDAM is developed to segment setae from Chaetoceros microscopic images. The proposed model combines the setae characteristics of the microscopic images with the spatial analysis of image grayscale surface to detect and segment the direction thin and long setae from the low contrast background as well as noise which may make the commonly used segmentation methods invalid. The experimental results show that our algorithm based on GSDAM outperforms the boundary‐based and region‐based segmentation methods Canny edge detector, iterative threshold selection, Otsu's thresholding, minimum error thresholding, K‐means clustering, and marker‐controlled watershed on the setae segmentation more accurately and completely. Microsc. Res. Tech. 77:684–690, 2014. © 2014 Wiley Periodicals, Inc.     

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.     

A novel autofocusing method using the angle of hilbert space for microscopy

Autofocusing technology is indispensable for routine use of microscopes on a large scale in biological field. The autofocusing method using the angle of Hilbert space is brought forward to measure whether the image is focused or not. The angle of Hillbert space can be used to evaluate accurately the similarity degree of two images. The experiment results show that the autofocusing method can decrease the computational cost and get accuracy for real‐time biological and biomedical images with noise robustness. The focus curves are smooth and possess the unimodality, the monotonicity and the symmetry. Compared with other classic and optimum focus method, the Hilbert method demonstrates its robustness to noise and can improve the focus speed. The experiments showed that the proposed method can increase the overall performance of an autofocus system and has strong applicability in various autofocusing algorithms. Microsc. Res. Tech. 77:289–295, 2014. © 2014 Wiley Periodicals, Inc.     

New sensitive detection method for lectin hemagglutination using microscopy

The blood group system AB0 is determined by the composition of terminal oligosaccharides on red blood cells. Thanks to this structural feature, these groups can be recognized by saccharide‐recognizing compounds. Lectins are proteins that are able to reversibly bind saccharide structures. They generally occur as multimers and are known as hemagglutination agents. Hemagglutination is a process in which blood cells are cross‐linked via multivalent molecules. Apart from lectins, hemagglutination can also be caused by antibodies or viruses. A hemagglutination assay is commonly used for the detection of multivalent molecules that recognize blood cells, in order to search for their sugar specificity. It is traditionally performed on a microtiter plate, where the lectin solution is serially diluted and the lowest concentration of lectin causing agglutination is detected. This experimental set‐up is utilized further for testing lectin specificity via a hemagglutination inhibition assay. We have developed a new way of detecting hemagglutination using microscopy, which was tested on purified lectins as well as cell lysates. Hemagglutination was performed on a microscope slide directly and detected using a microscope. Comparison with the standard hemagglutination assay using microtiter plates revealed that microscopic approach is faster and more robust and allows fast determination of lectin activities immediately in bacterial cytosols. Microsc. Res. Tech. 77:841–849, 2014. © 2014 Wiley Periodicals, Inc.     

Smart portable noninvasive instrument for detection of internal air leakage of a valve using acoustic emission signals

Acoustic emission (AE) can be used to detect and determine the internal leakage rate through a valve in many applications. However, a general AE data acquisition system is expensive and bulky. This paper presents a novel low-cost instrument based on microcontroller and a novel theoretical model based on AE technique to predict the leakage rate. The system is an embedded system instead of a general PC-based data acquisition. AE_RMS parameter is used to infer the leakage rate, and the effects of various process variables on the model are also studied. The experimental results have shown that the instrument is capable of detecting possible valve leakage encountered in online operation. With its portability, ease of use and compactness, the proposed system provides faster and low cost valve leakage detection.     

基于神经网络算法的故障检测技术

针对复杂的机电产品内部构件状态检测这一工程难题,本文介绍了一种自动在线检测系统.该系统采用X射线对产品成像,运用数字图像处理技术对射线图像进行预处理,由神经网络算法进行故障诊断.故障识别模型采用了改进的BP神经网络算法,以正常装配状态时的多幅图像经预处理后作为学习样本训练BP神经网络.检测时一般只需拍摄两幅不同方位的图像,经预处理后输入神经网络与样本图像进行比较判断,即可识别出关键元器件的状态.该系统将数字射线成像技术和图像处理技术相结合,并在故障识别算法中采用了神经网络算法,提高了产品故障的检测速度和可靠性,在工业无损检测领域具有一定的实用性.     

An automated system for whole microscopic image acquisition and analysis

The field of anatomic pathology has experienced major changes over the last decade. Virtual microscopy (VM) systems have allowed experts in pathology and other biomedical areas to work in a safer and more collaborative way. VMs are automated systems capable of digitizing microscopic samples that were traditionally examined one by one. The possibility of having digital copies reduces the risk of damaging original samples, and also makes it easier to distribute copies among other pathologists. This article describes the development of an automated high‐resolution whole slide imaging (WSI) system tailored to the needs and problems encountered in digital imaging for pathology, from hardware control to the full digitization of samples. The system has been built with an additional digital monochromatic camera together with the color camera by default and LED transmitted illumination (RGB). Monochrome cameras are the preferred method of acquisition for fluorescence microscopy. The system is able to digitize correctly and form large high resolution microscope images for both brightfield and fluorescence. The quality of the digital images has been quantified using three metrics based on sharpness, contrast and focus. It has been proved on 150 tissue samples of brain autopsies, prostate biopsies and lung cytologies, at five magnifications: 2.5×, 10×, 20×, 40×, and 63×. The article is focused on the hardware set‐up and the acquisition software, although results of the implemented image processing techniques included in the software and applied to the different tissue samples are also presented. Microsc. Res. Tech. 77:697–713, 2014. © 2014 Wiley Periodicals, Inc.     

An effective and accurate identification system of Mycobacterium tuberculosis using convolution neural networks

Tuberculosis (TB) remains the leading cause of morbidity and mortality from infectious disease in developing countries. The sputum smear microscopy remains the primary diagnostic laboratory test. However, microscopic examination is always time‐consuming and tedious. Therefore, an effective computer‐aided image identification system is needed to provide timely assistance in diagnosis. The current identification system usually suffers from complex color variations of the images, resulting in plentiful of false object detection. To overcome the dilemma, we propose a two‐stage Mycobacterium tuberculosis identification system, consisting of candidate detection and classification using convolution neural networks (CNNs). The refined Faster region‐based CNN was used to distinguish candidates of M. tuberculosis and the actual ones were classified by utilizing CNN‐based classifier. We first compared three different CNNs, including ensemble CNN, single‐member CNN, and deep CNN. The experimental results showed that both ensemble and deep CNNs were on par with similar identification performance when analyzing more than 19,000 images. A much better recall value was achieved by using our proposed system in comparison with conventional pixel‐based support vector machine method for M. tuberculosis bacilli detection.     

In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy

In this study, we use combined two‐photon excitation fluorescence (TPEF), second‐harmonic generation (SHG) and third‐harmonic generation (THG) measurements to image cellular structures of the nematode Caenorhabditis elegans, in vivo. To our knowledge, this is the first time that a THG modality is employed to image live C. elegans specimens. Femtosecond laser pulses (1028 nm) were utilized for excitation. Detailed and specific structural and anatomical features can be visualized, by recording THG signals. Thus, the combination of three image‐contrast modes (TPEF‐SHG‐THG) in a single instrument has the potential to provide unique and complementary information about the structure and function of tissues and individual cells of live biological specimens.     

A compact CCD‐monitored atomic force microscope with optical vision and improved performances

A novel CCD‐monitored atomic force microscope (AFM) with optical vision and improved performances has been developed. Compact optical paths are specifically devised for both tip‐sample microscopic monitoring and cantilever's deflection detecting with minimized volume and optimal light‐amplifying ratio. The ingeniously designed AFM probe with such optical paths enables quick and safe tip‐sample approaching, convenient and effective tip‐sample positioning, and high quality image scanning. An image stitching method is also developed to build a wider‐range AFM image under monitoring. Experiments show that this AFM system can offer real‐time optical vision for tip‐sample monitoring with wide visual field and/or high lateral optical resolution by simply switching the objective; meanwhile, it has the elegant performances of nanometer resolution, high stability, and high scan speed. Furthermore, it is capable of conducting wider‐range image measurement while keeping nanometer resolution. Microsc. Res. Tech. 76:931–935, 2013. © 2013 Wiley Periodicals, Inc.     

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.     

An automated cell viability quantification method for low‐resolution confocal images of closely packed cells based on a modified gradient flow tracking algorithm

Fluorescent‐based live/dead labelling combined with fluorescent microscopy is one of the widely used and reliable methods for assessment of cell viability. This method is, however, not quantitative. Many image‐processing methods have been proposed for cell quantification in an image. Among all these methods, several of them are capable of quantifying the number of cells in high‐resolution images with closely packed cells. However, no method has addressed the quantification of the number of cells in low‐resolution images containing closely packed cells with variable sizes. This paper presents a novel method for automatic quantification of live/dead cells in 2D fluorescent low‐resolution images containing closely packed cells with variable sizes using a mean shift‐based gradient flow tracking. Accuracy and performance of the method was tested on growth plate confocal images. Experimental results show that our algorithm has a better performance in comparison to other methods used in similar detection conditions.     

Image enhancement for increased dot-counting efficiency in FISH

Background: The most commonly used molecular cytogenetic technique is fluorescence in situ hybridization (FISH). It has been widely applied in many areas of diagnosis and research, including pre‐natal and post‐natal screening of chromosomal aberrations, pre‐implantation genetic diagnosis, cancer cytogenetics, gene mapping, molecular pathology and developmental molecular biology. The analysis of FISH images consists of detecting fluorescent dots, after which the number of dots per cell can be counted or their relative positions can be measured. A major impediment in the analysis of FISH specimens is signal (dot) quality, which is influenced by the hybridization efficiency and/or the sensitivity of the camera that records the images. Method: In this paper, we present an approach to improve the efficiency of detecting fluorescent signals in FISH images by recovering the radiance map of the camera. This allows us to generate a high‐dynamic‐range image wherein an extended range of the sample radiance captured by the camera can be visualized at distinct intensity values. The resulting higher‐order numeric complexity of the transformed image is adjusted (or simplified) by examining the intensity distribution in each of the three colour channels (red, green and blue), and remapping the intensity values to generate a high‐contrast image with a lower‐order (compressed) dynamic range. The remapping is based on a criterion that optimizes the detection of the hybridized signals, allowing attenuation of saturated intensity values while amplifying low‐intensity signals. Results: A simple dot‐counting algorithm is used to automatically process 2000 FISH images. The images are taken for lymphocytes from cultured blood specimens for cytogenetic testing. Images are manually analyzed by an expert to obtain ground truth for dot counts. A quantitative analysis is performed by comparing results of automated dot detection on images before and after enhancement with the developed algorithms. In addition, common errors in dot counting due to split dots, dust, poor segmentation and overlapping signals are analyzed and the robustness of the developed approach against these errors evaluated. It is observed that dot‐detection efficiency is increased by an average of 9% across all colour channels while reducing errors in missed and false dot counts. Conclusions: Our proposed method and results demonstrate that dot‐counting specificity and sensitivity can be improved by pre‐processing and enhancing the image using the radiance curve of the camera and generating a high‐contrast, remapped high‐dynamic‐range image prior to using any algorithm for dot counting.     

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.