Optical techniques for imaging membrane topography

In recent years three powerful optical imaging techniques have emerged that provide nanometer-scale information about the topography of membrane surfaces, whether cellular or artificial: intermembrane fluorescence resonance energy transfer (FRET), fluorescence interference contrast microscopy (FLIC), and reflection interference contrast microscopy (RICM). In intermembrane FRET, the sharp distance dependence of resonant energy transfer between fluorophores allows topographic measurements in the Angstrom to few-nanometer range. In FLIC and RICM, interference between light from a membrane (either from fluorescent probes, or reflected illumination) and light reflected by a planar substrate provide spatial sensitivity in the few to hundreds of nanometer range, with few-nanometer resolution. All of these techniques are fairly easy to implement. We discuss the physics and optics behind each of these tools, as well as practical concerns regarding their uses. We also provide examples of their application in imaging molecular-scale structures at intermembrane junctions.     

Closing the translation gap: AI applications in digital pathology

Recent advances in artificial intelligence show tremendous promise to improve the accuracy, reproducibility, and availability of medical diagnostics across a number of medical subspecialities. This is especially true in the field of digital pathology, which has recently witnessed a surge in publications describing state-of-the-art performance for machine learning models across a wide range of diagnostic applications. Nonetheless, despite this promise, there remain significant gaps in translating applications for any of these technologies into actual clinical practice. In this review, we will first give a brief overview of the recent progress in applying AI to digitized pathology images, focusing on how these tools might be applied in clinical workflows in the near term to improve the accuracy and efficiency of pathologists. Then we define and describe in detail the various factors that need to be addressed in order to successfully close the “translation gap” for AI applications in digital pathology.     

Photoactivatable fluorophores and techniques for biological imaging applications

Photoactivatable fluorophores (PAFs) are powerful imaging probes for tracking molecular and cellular dynamics with high spatiotemporal resolution in biological systems. Recent developments in biological microscopy have raised new demands for engineering new PAFs with improved properties, such as high two photon excitation efficiency, reversibility, cellular delivery and targeting. Here we review the history and some of the recent developments in this area, emphasizing our efforts in developing a new class of caged coumarins and related imaging methods for studying dynamic cell-cell communication through gap junction channels, and in extending the application of these caged coumarins to new areas including spatiotemporal control of microRNA activity in vivo.     

The importance of optical optimization in whole slide imaging (WSI) and digital pathology imaging

In the last 10 years, whole slide imaging (WSI) has seen impressive progress not only in image quality and scanning speed but also in the variety of systems available to pathologists. However, we have noticed that most systems have relatively simple optics axes and rely on software to optimize image quality and colour balance. While much can be done in software, this study examines the importance of optics, in particular optical filters, in WSI.Optical resolution is a function of the wavelength of light used and the numerical aperture of the lens system (Resolution = (f) wavelength/2 NA). When illumining light is not conditioned correctly with filters, there is a tendency for the wavelength to shift to longer values (more red) because of the characteristics of the lamps in common use. Most microscopes (but remarkably few WSI devices) correct for this with ND filter for brightness and Blue filter (depends on the light source) for colour correction.Using H&E slides research microscopes (Axiophot, Carl Zeiss MicroImaging, Inc. NY. Eclipse 50i., Nikon Inc. NY) at 20x, an attached digital camera (SPOT RT741 Slider Color, Diagnosis Instruments., MI USA), and a filter set, we examined the effect of filters and software enhancement on digital image quality. The focus value (as evaluated by focus evaluation software developed in house and SPOT imaging Software v4.6) was used as a proxy for image quality. Resolution of tissue features was best with the use of both the Blue and ND filters (in addition to software enhancement). Images without filters but with software enhancement while superficially good, lacked some details of specimen morphology and were unclear compared with the images with filters.The results indicate that the appropriate use of optical filters could measurably improve the appearance and resolution of WSI images.     

Optical imaging techniques for the study of malaria

Malarial infection needs to be imaged to reveal the mechanisms behind malaria pathophysiology and to provide insights to aid in the diagnosis of the disease. Recent advances in optical imaging methods are now being transferred from physics laboratories to the biological field, revolutionizing how we study malaria. To provide insight into how these imaging techniques can improve the study and treatment of malaria, we summarize recent progress on optical imaging techniques, ranging from in vitro visualization of the disease progression of malaria infected red blood cells (iRBCs) to in vivo imaging of malaria parasites in the liver.     

Optical techniques for imaging membrane lipid microdomains in living cells

Lateral organisation of cellular membranes, particularly the plasma membrane, is of benefit to the cell as it allows complicated cellular processes to be regulated and efficient. For example, trafficking and secretion of molecules can be targeted and directed, cells polarised and signalling events modulated and propagated. The fluid mosaic model allows for significant heterogeneity on the part of the lipids themselves and of membrane associated proteins. By exploiting the tendency of complex lipid bilayers to undergo spontaneous or induced phase-separation into non-miscible domains, the cell could achieve this desired spatial organisation. While phase-separation is readily observed in simple, artificial bilayers, its occurrence in physiological membranes remains controversial. This stems mainly from our inability to image lipid microdomains directly - possibly due to their small size, short lifespan and/or morphological similarity to the bulk membrane. In this review, we seek to examine the techniques used to try to image membrane lipid microdomains, concentrating mainly on optical microscopy techniques that are applicable to live cells. We also look at novel emerging instruments and methods that promise to overcome our current technological limitations and shed new light on these important structures.     

High throughput slanted scanning whole slide imaging system for digital pathology

In whole slide imaging (WSI), normally only a one layer imaging of the slide is performed. Autofocus at multiple positions is usually required. But defocus blur still exists due to tissue folding or specimen thickness. Repeated Z-stack scan be applied here, which, however, is too time consuming. Here, a high throughput slanted scanning WSI system is reported. In this system, the slide surface was slanted 1° relative to the focal plane. Thus, the focal plane spanned multiple layers of the sample. By moving the slide, multi-layer image data of the sample can be acquired simultaneously at a time frame comparable to conventional 1-layer imaging. With image fusion, defocus blur can be avoided. High quality and fast imaging of both cytological and histological slide specimens was demonstrated without applying aberration correction. The system can be a highly efficient way for the application of WSI in digital pathology.     

Protein microarray biosensors based on imaging ellipsometry techniques and their applications

After years of development, biosensors based on imaging ellipsometry and biosensors based on total internal reflection imaging ellipsometry have been successfully implemented in various engineering systems. Their experimental setups, detection principles, and biological and clinical applications are briefly reviewed.     

Standardization efforts of digital pathology in Europe

BACKGROUND: EURO-TELEPATH is a European COST Action IC0604. It started in 2007 and will end in November 2011. Its main objectives are evaluating and validating the common technological framework and communication standards required to access, transmit, and manage digital medical records by pathologists and other medical specialties in a networked environment. BUSINESS MODELLING: Working Group 1, "Business Modelling in Pathology," has designed main pathology processes - Frozen Study, Formalin Fixed Specimen Study, Telepathology, Cytology, and Autopsy - using Business Process Modelling Notation (BPMN). INFORMATICS STANDARDS IN PATHOLOGY: Working Group 2 has been dedicated to promoting the application of informatics standards in pathology, collaborating with Integrating Healthcare Enterprise (IHE), Digital Imaging and Communications in Medicine (DICOM), Health Level Seven (HL7), and other standardization bodies. CONCLUSIONS: Health terminology standardization research has become a topic of great interest. Future research work should focus on standardizing automatic image analysis and tissue microarrays imaging.     

Human skeletal muscle architecture studied in vivo by non-invasive imaging techniques: functional significance and applications

The internal architecture plays an essential role in determining the functional features of skeletal muscle. Both length–force and force–velocity relationships depend on the spatial arrangement of muscle fibres in skeletal muscle. The degree of muscle pennation determines both the amount of contractile tissue packed along the tendons and fibre length, and is reflected by the force-generating capacity and shortening velocity of the muscle and by the elastic properties of the muscle–tendon complex. Until recently, knowledge on human muscle architecture was based on measurements performed on cadavers, whose muscle fibres were often shrunk by the preserving medium and by age. With the introduction of non-invasive imaging techniques, it has become possible to study muscle architecture in vivo at rest and the changes thereof upon contraction. This paper discusses the applications of these techniques, namely ultrasonography and nuclear magnetic resonance imaging, and their relevance in physiology and biomechanics.     

Development of a polarization imaging method to detect paraffin-embedded pathology tissues before applying other techniques

The present article describes the development of a technique, applied to paraffin-embedded tissues, which uses three different wavelengths of monochromatic light (λ1 = 445 nm, λ2 = 540 nm and λ3 = 660 nm) for the measures of the degree of polarization, degree of linear polarization, degree of circular polarization and birefringence, all obtained from measurements of Stokes parameters by using polarized light. The goal of this study was to detect changes in developing embryonic mouse eye when pregnant mice fed diets without folic acid for variable periods compared with a healthy control group. We present a biomedical diagnostic technique based on polarized light detection applied to paraffin-embedded tissues to visualize the structural damage to aid us in the diagnosis before applying other techniques. Through this method, we can visualize and identify which parts of the tissue were altered with respect to the control group.     

Optical imaging and tumor angiogenesis

Tumor angiogenesis is essential for tumor growth and progression. Therefore, targeting tumor blood vessels is a promising approach for cancer therapy. Angiogenesis, the formation of blood vessels, is a multistep process, and strongly influenced by the microenvironment. There are no in vitro assays that can resemble this dynamic process in vivo. For this reason, animal models and imaging technologies are critical for studying tumor angiogenesis, identifying therapeutic targets as well as validating the targets. Non-invasive molecular imaging in animal models presents an unprecedented opportunity and ability for us to perform repetitive observations and analysis of the biological processes underlying tumor angiogenesis and tumor progression in living animals in real time. As we gain a better understanding of the fundamental molecular nature of cancer, these techniques will be an important adjunct in translating the knowledge into clinical practice. This important information may elucidate how the tumor blood vessels behave and respond to certain treatments and therapies.     

Presynaptic imaging techniques

Understanding the detailed molecular events that support chemical synaptic transmission requires high-resolution methods that provide quantitative information combined with molecular specificity. In recent years, many new technological approaches, including genetically encoded fluorescent indicators, ultra-thin sectioning, and live-cell imaging have been brought to bear on understanding the cell biology and physiology of presynaptic terminals.     

Cervical lesion proportion measure using a digital gridded imaging technique to assess cervical pathology in women with genital schistosomiasis

Female genital schistosomiasis (FGS) is characterized by a pattern of lesions which manifest at the cervix and the vagina, such as homogeneous and grainy sandy patches, rubbery papules in addition to neovascularization. A tool for quantification of the lesions is needed to improve FGS research and control programs. Hitherto, no tools are available to quantify clinical pathology at the cervix in a standardized and reproducible manner. This study aimed to develop and validate a cervical lesion proportion (CLP) measure for quantification of cervical pathology in FGS. A digital imaging technique was applied in which a grid containing 424 identical squares was positioned on high resolution digital images from the cervix of 70 women with FGS. CLP was measured for each image by observers counting the total number of squares containing at least one type of FGS associated lesion. For assessment of inter- and intra-observer reliability, three different observers measured CLP independently. In addition, a rubbery papule count (RPC) was determined in a similar manner. The intraclass correlation coefficient was 0.94 (excellent) for the CLP inter-rater reliability and 0.90 (good) for intra-rater reliability and the coefficients for the RPC were 0.88 and 0.80 (good), respectively. The CLP facilitated a reliable and reproducible quantification of FGS associated lesions of the cervix. In the future, grading of cervical pathology by CLP may provide insight into the natural course of schistosome egg-induced pathology of the cervix and may have a role in assessing praziquantel treatment efficacy against FGS.Trial Registration: ClinicalTrials.gov, trial number {"type":"clinical-trial","attrs":{"text":"NCT04115072","term_id":"NCT04115072"}}NCT04115072; trial URL https://clinicaltrials.gov/ct2/show/{"type":"clinical-trial","attrs":{"text":"NCT04115072","term_id":"NCT04115072"}}NCT04115072?term=Female+genital+schistosomiasis+AND+Madagascar&draw=2&rank=1.     

SurfaceSlide: a multitouch digital pathology platform

Background

Digital pathology provides a digital environment for the management and interpretation of pathological images and associated data. It is becoming increasing popular to use modern computer based tools and applications in pathological education, tissue based research and clinical diagnosis. Uptake of this new technology is stymied by its single user orientation and its prerequisite and cumbersome combination of mouse and keyboard for navigation and annotation.

Methodology

In this study we developed SurfaceSlide, a dedicated viewing platform which enables the navigation and annotation of gigapixel digitised pathological images using fingertip touch. SurfaceSlide was developed using the Microsoft Surface, a 30 inch multitouch tabletop computing platform. SurfaceSlide users can perform direct panning and zooming operations on digitised slide images. These images are downloaded onto the Microsoft Surface platform from a remote server on-demand. Users can also draw annotations and key in texts using an on-screen virtual keyboard. We also developed a smart caching protocol which caches the surrounding regions of a field of view in multi-resolutions thus providing a smooth and vivid user experience and reducing the delay for image downloading from the internet. We compared the usability of SurfaceSlide against Aperio ImageScope and PathXL online viewer.

Conclusion

SurfaceSlide is intuitive, fast and easy to use. SurfaceSlide represents the most direct, effective and intimate human–digital slide interaction experience. It is expected that SurfaceSlide will significantly enhance digital pathology tools and applications in education and clinical practice.     

SDOCT imaging to identify macular pathology in patients diagnosed with diabetic maculopathy by a digital photographic retinal screening programme

Introduction

Diabetic macular edema (DME) is an important cause of vision loss. England has a national systematic photographic retinal screening programme to identify patients with diabetic eye disease. Grading retinal photographs according to this national protocol identifies surrogate markers for DME. We audited a care pathway using a spectral-domain optical coherence tomography (SDOCT) clinic to identify macular pathology in this subset of patients.

Methods

A prospective audit was performed of patients referred from screening with mild to moderate non-proliferative diabetic retinopathy (R1) and surrogate markers for diabetic macular edema (M1) attending an SDOCT clinic. The SDOCT images were graded by an ophthalmologist as SDOCT positive, borderline or negative. SDOCT positive patients were referred to the medical retina clinic. SDOCT negative and borderline patients were further reviewed in the SDOCT clinic in 6 months.

Results

From a registered screening population of 17 551 patients with diabetes mellitus, 311 patients met the inclusion criteria between (March 2008 and September 2009). We analyzed images from 311 patients’ SDOCT clinic episodes. There were 131 SDOCT negative and 12 borderline patients booked for revisit in the OCT clinic. Twenty-four were referred back to photographic screening for a variety of reasons. A total of 144 were referred to ophthalmology with OCT evidence of definite macular pathology requiring review by an ophthalmologist.

Discussion

This analysis shows that patients with diabetes, mild to moderate non-proliferative diabetic retinopathy (R1) and evidence of diabetic maculopathy on non-stereoscopic retinal photographs (M1) have a 42.1% chance of having no macular edema on SDOCT imaging as defined by standard OCT definitions of DME when graded by a retinal specialist. SDOCT imaging is a useful adjunct to colour fundus photography in screening for referable diabetic maculopathy in our screening population.     

Spatially resolved cytosolic calcium response to angiotensin II and potassium in rat glomerulosa cells measured by digital imaging techniques

The response of cytosolic calcium [Ca2+]i to angiotensin II (AII) and potassium (K+) in individual rat glomerulosa cells was determined using the calcium-sensitive fluorescent dye, fura-2 and digital imaging. Control (4 mM K+) cytosolic calcium levels were generally in the 80-120 nM range and increased monotonically as [K+] was increased from 4 to 12 mM. There was no delay in the onset of the response. In most cells the [Ca2+]i decreased from its peak after 3-4 min, even in the presence of superfusate containing elevated K+. The time course of the change in [Ca2+]i in response to AII stimulation, on the other hand, was more variable. It was most often characterized by an early decrease followed by a large delayed increase. The response also was observed to decline during sustained AII stimulation. The majority of the cells showed some response to one or the other secretagogue with a sizeable minority (25%) having an increase in [Ca2+]i in excess of 200%. While the majority showed a response, the cell to cell variation was substantial. Finally, the pattern of cytosolic calcium increase sometimes showed a marked dependence on the secretagogue used, with different regions of the same cell being more strongly affected by one agent or the other. A few cells (10%) responded to AII only at one pole, establishing a large concentration gradient of calcium across the cell. Because of differences in time course, pattern, and degree of responsiveness, it is likely that the mechanisms underlying the Ca2+ elevation with K+ and AII are different.