Standing wave microscopy of red blood cell membrane morphology with high temporal resolution

Widefield fluorescence microscopy is an integral tool for life science imaging though the achievable resolutions are limited by the diffraction nature of light. One technique to increase the axial resolution is known as standing wave microscopy [1]. The standing wave can be generated by placing a mirror at the specimen plane which causes interference between the incoming and reflected excitation illumination. The axial resolution is reduced to λ/4n as only fluorophores which are in the location of the full width at the half maximum of the antinodes are excited [2] resulting in periodic bands of fluorescence

A computational method for two-dimensional quantitative analysis of standing wave images of red blood cells

Standing wave (SW) microscopy allows for an improvement in the axial resolution that can be obtained in optical microscopy. In SW microscopy two counter propagating waves interfere producing a SW with anti-nodal planes that are separated by λ/2n and a FWHM of λ/4n which is the axial resolution, where λ is the excitation wavelength and n is the refractive index Multi-planar SW microscopy, with the addition of a mirror below the specimen, allows for selective plane contour mapping of one concave surface of a red blood cell (RBC). We report a computational method to extract SW anti-nodal plane and boundaries positions (x,y) and extract each pixel intensity value. By doing so, we can create 2D reconstruction of SW RBC images captured at video rate. In future, by utilising the positional information for the each of the anti-nodal SW planes, we aim to create 3D and 4D reconstruction of the RBCs concave surface. Additionally, applying the extended computational method to SW RBC images we aim to study healthy and diseased erythrocytes and the changes in the concave surface morphology over time

Timing of urinary catheter removal after colorectal surgery with pelvic dissection : A systematic review and meta-analysis

Peer reviewedPublisher PD

Three-dimensional observations of an aperiodic oscillatory gliding behavior in Myxococcus xanthus using confocal interference reflection microscopy

The deltaproteobacterium Myxococcus xanthus is a model for bacterial motility and has provided unprecedented insights into bacterial swarming behaviors. Fluorescence microscopy techniques have been invaluable in defining the mechanisms that are involved in gliding motility, but these have almost entirely been limited to two-dimensional (2D) studies, and there is currently no understanding of gliding motility in a three-dimensional (3D) context. We present here the first use of confocal interference reflection microscopy (IRM) to study gliding bacteria, revealing aperiodic oscillatory behavior with changes in the position of the basal membrane relative to the substrate on the order of 90 nm in vitro. First, we use a model planoconvex lens specimen to show how topological information can be obtained from the wavelength-dependent interference pattern in IRM. We then use IRM to observe gliding M. xanthus bacteria and show that cells undergo previously unobserved changes in their adhesion profile as they glide. We compare the wild type with mutants that have reduced motility, which also exhibit the same changes in the adhesion profile during gliding. We find that the general gliding behavior is independent of the proton motive force-generating complex AglRQS and suggest that the novel behavior that we present here may be a result of recoil and force transmission along the length of the cell body following firing of the type IV pili. IMPORTANCE 3D imaging of live bacteria with optical microscopy techniques is a challenge due to the small size of bacterial cells, meaning that previous studies have been limited to observing motility behavior in 2D. We introduce the application of confocal multiwavelength interference reflection microscopy to bacteria, which enables visualization of 3D motility behaviors in a single 2D image. Using the model organism Myxococcus xanthus, we identified novel motility behaviors that are not explained by current motility models, where gliding bacteria exhibit aperiodic changes in their adhesion to an underlying solid surface. We concluded that the 3D behavior was not linked to canonical motility mechanisms and that IRM could be applied to study a range of microbiological specimens with minimal adaptation to a commercial microscope

A simple image processing pipeline to sharpen topology maps in multi-wavelength interference microscopy

Multi-wavelength standing wave (SW) microscopy and interference reflection microscopy (IRM) are powerful techniques that use optical interference to study topographical structure. However, the use of more than two wavelengths to image the complex cell surface results in complicated topographical maps and it can be difficult to resolve the three-dimensional contours. We present a simple image processing method to reduce the thickness and spacing of antinodal fringes in multiwavelength interference microscopy by up to a factor of two to produce clearer and more precise topographical maps of cellular structures. We first demonstrate this improvement using model non-biological specimens, and we subsequently demonstrate the benefit of our method for reducing the ambiguity of surface topography and revealing obscured features in live and fixed cell specimens

Optical mesoscopy, machine learning, and computational microscopy enable high information content diagnostic imaging of blood films

Automated image-based assessment of blood films has tremendous potential to support clinical haematology within overstretched healthcare systems. To achieve this, efficient and reliable digital capture of the rich diagnostic information contained within a blood film is a critical first step. However, this is often challenging, and in many cases entirely unfeasible, with the microscopes typically used in haematology due to the fundamental trade-off between magnification and spatial resolution. To address this, we investigated three state-of-the-art approaches to microscopic imaging of blood films which leverage recent advances in optical and computational imaging and analysis to increase the information capture capacity of the optical microscope: optical mesoscopy, which uses a giant microscope objective (Mesolens) to enable high-resolution imaging at low magnification; Fourier ptychographic microscopy, a computational imaging method which relies on oblique illumination with a series of LEDs to capture high-resolution information; and deep neural networks which can be trained to increase the quality of low magnification, low resolution images. We compare and contrast the performance of these techniques for blood film imaging for the exemplar case of Giemsa-stained peripheral blood smears. Using computational image analysis and shape-based object classification, we demonstrate their use for automated analysis of red blood cell morphology and visualization and detection of small blood-borne parasites such as the malarial parasite Plasmodium falciparum. Our results demonstrate that these new methods greatly increase the information capturing capacity of the light microscope, with transformative potential for haematology and more generally across digital pathology

An evaluation of multi-excitation-wavelength standing-wave fluorescence microscopy (TartanSW) to improve sampling density in studies of the cell membrane and cytoskeleton

Conventional standing-wave (SW) fluorescence microscopy uses a single wavelength to excite fluorescence from the specimen, which is normally placed in contact with a first surface reflector. The resulting excitation SW creates a pattern of illumination with anti-nodal maxima at multiple evenly-spaced planes perpendicular to the optical axis of the microscope. These maxima are approximately 90 nm thick and spaced 180 nm apart. Where the planes intersect fluorescent structures, emission occurs, but between the planes are non-illuminated regions which are not sampled for fluorescence. We evaluate a multi-excitation-wavelength SW fluorescence microscopy (which we call TartanSW) as a method for increasing the density of sampling by using SWs with different axial periodicities, to resolve more of the overall cell structure. The TartanSW method increased the sampling density from 50% to 98% over seven anti-nodal planes, with no notable change in axial or lateral resolution compared to single-excitation-wavelength SW microscopy. We demonstrate the method with images of the membrane and cytoskeleton of living and fixed cells

Millions of historical monthly rainfall observations taken in the UK and Ireland rescued by citizen scientists

Recovering additional historical weather observations from known archival sources will improve understanding of how the climate is changing and enable detailed examination of unusual events within the historical record. The UK National Meteorological Archive recently scanned more than 66,000 paper sheets containing 5.28 million hand-written monthly rainfall observations taken across the UK and Ireland between 1677 and 1960. Only a small fraction of these observations were previously digitally available for climate scientists to analyse. More than 16,000 volunteer citizen scientists completed the transcription of these sheets of observations during early 2020 using the RainfallRescue.org website, built using the Zooniverse platform. A total of 3.34 million observations from more than 6000 locations have so far been quality controlled and made openly available. This has increased the total number of monthly rainfall observations that are available for this time period and region by a factor of six. The newly rescued observations will enable longer and much improved reconstructions of past variations in rainfall across the British and Irish Isles, including for periods of significant flooding and drought. Specifically, this data should allow the official gridded monthly rainfall reconstructions for the UK to be extended back to 1836, and even earlier for some regions

Development and preliminary validation of the 'Caring for Country' questionnaire: measurement of an Indigenous Australian health determinant

<p>Abstract</p> <p>Background</p> <p>'Caring for Country' is defined as Indigenous participation in interrelated activities with the objective of promoting ecological and human health. Ecological services on Indigenous-owned lands are belatedly attracting some institutional investment. However, the health outcomes associated with Indigenous participation in 'caring for country' activities have never been investigated. The aims of this study were to pilot and validate a questionnaire measuring caring for country as an Indigenous health determinant and to relate it to an external reference, obesity.</p> <p>Methods</p> <p>Purposively sampled participants were 301 Indigenous adults aged 15 to 54 years, recruited during a cross-sectional program of preventive health checks in a remote Australian community. Questionnaire validation was undertaken with psychometric tests of internal consistency, reliability, exploratory factor analysis and confirmatory one-factor congeneric modelling. Accurate item weightings were derived from the model and used to create a single weighted composite score for caring for country. Multiple linear regression modelling was used to test associations between the caring for country score and body mass index adjusting for socio-demographic factors and health behaviours.</p> <p>Results</p> <p>The questionnaire demonstrated adequate internal consistency, test-retest validity and proxy-respondent validity. Exploratory factor analysis of the 'caring for country' items produced a single factor solution that was confirmed via one-factor congeneric modelling. A significant and substantial association between greater participation in caring for country activities and lower body mass index was demonstrated. Adjusting for socio-demographic factors and health behaviours, an inter-quartile range rise in caring for country scores was associated with 6.1 Kg and 5.3 Kg less body weight for non-pregnant women and men respectively.</p> <p>Conclusion</p> <p>This study indicates preliminary support for the validity of the caring for country concept and a questionnaire designed to measure it. This study also highlights the importance of investigating Indigenous-asserted health promotion activities. Further studies in similar populations are merited to test the generalisability of this questionnaire and to explore associations with other important Indigenous health outcomes.</p