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The field of pathology is driven by microscopic images. Educational activities for trainees and practicing pathologists alike are conducted through exposure to images of a variety of pathologic entities in textbooks, publications, online tutorials, national and international conferences, and interdepartmental conferences. During the past century and a half, photographic technology has progressed from primitive and bulky, glass-lantern projector slides to static and/or whole slide digital-image formats that can now be transferred around the world in a matter of moments via the Internet. To provide a historic and technologic overview of the evolution of microscopic-image photographic tools and techniques. Primary historic methods of microscopic image capture were delineated through interviews conducted with senior staff members in the Emory University Department of Pathology. Searches for the historic image-capturing methods were conducted using the Google search engine. Google Scholar and PubMed databases were used to research methods of digital photography, whole slide scanning, and smart phone cameras for microscopic image capture in a pathology practice setting. Although film-based cameras dominated for much of the time, the rise of digital cameras outside of pathology generated a shift toward digital-image capturing methods, including mounted digital cameras and whole slide digital-slide scanning. Digital image capture techniques have ushered in new applications for slide sharing and second-opinion consultations of unusual or difficult cases in pathology. With their recent surge in popularity, we suspect that smart phone cameras are poised to become a widespread, cost-effective method for pathology image acquisition.

This study aimed to develop and test an unbiased and rapid methodology to estimate the length of external arbuscular mycorrhizal fungal (AMF) hyphae in soil. The traditional visual gridline intersection (VGI) method, which consists in a direct visual examination of the intersections of hyphae with gridlines on a microscope eyepiece after aqueous extraction, membrane-filtration, and staining (e.g., with trypan blue), was refined. For this, (i) images of the stained hyphae were taken by using a digital photomicrography technique to avoid the use of the microscope and the method was referred to as \"digital gridline intersection\" (DGI) method; and (ii), the images taken in (i) were processed and the hyphal length was measured by using ImageJ software, referred to as the \"photomicrography-ImageJ processing\" (PIP) method. The DGI and PIP methods were tested using known grade lengths of possum fur. Then they were applied to measure the hyphal lengths in soils with contrasting phosphorus (P) fertility status. Linear regressions were obtained between the known lengths (Lknown) of possum fur and the values determined by using either the DGI (LDGI) (LDGI = 0.37 + 0.97 × Lknown, r2 = 0.86) or PIP (LPIP) methods (LPIP = 0.33 + 1.01 × Lknown, r2 = 0.98). There were no significant (P > 0.05) differences between the LDGI and LPIP values. While both methods provided accurate estimation (slope of regression being 1.0), the PIP method was more precise, as reflected by a higher value of r2 and lower coefficients of variation. The average hyphal lengths (6.5-19.4 m g-1) obtained by the use of these methods were in the range of those typically reported in the literature (3-30 m g-1). Roots growing in P-deficient soil developed 2.5 times as many hyphae as roots growing in P-rich soil (17.4 vs 7.2 m g-1). These tests confirmed that the use of digital photomicrography in conjunction with either the grid-line intersection principle or image processing is a suitable method for the measurement of AMF hyphal lengths in soils for comparative investigations.

Parasitic castration of bivalves by trematodes is common, and may significantly reduce the reproductive capacity of ecologically important species. Understanding the intensity of infection is desirable, as it can indicate the time that has passed since infection, and influence the host's physiological and reproductive response. In addition, it is useful to know the developmental stage of the trematode, to understand trematode population trends and reproductive success. However, most existing methods (e.g. visually estimating the degree of infection) to assess intensity are approximate only and not reproducible. Here, we present a method to accurately quantify the percentage of bivalve gonad filled with digenean trematode tissue, based on small squashes of gonad tissue rapidly photographed under light microscopy. A maximum of 15 photographs is required to determine the percentage of the whole gonad occupied by trematodes with a minimum of 90% confidence, with smaller mussels requiring fewer. In addition, the stage of trematode infection can be assessed because full sporocysts, spent sporocysts and free cercariae are clearly distinguishable. Although variation exists in the distribution of trematodes in gonad tissue, and thus in the estimate of percentage of the gonad filled with trematodes, this method represents a marked improvement on current coarse assessments of infection which typically focus on binary presence/absence measures. This technique can be used to facilitate a more sophisticated understanding of host–parasite interactions in bivalves, and can inform the conservation and reproductive biology of environmentally crucial species.

Purpose To assess the effects of light from an integrated optical microscope and evaluate the safety of time-lapse observations using a built-in microscope incubator. Methods We prospectively compared the fertilization rate and embryonic morphology after intracytoplasmic sperm injection between embryos cultured with time-lapse observations every 15 min in an incubator with an integrated optical microscope and embryos with intermittent observations (once a day) in conventional incubators. Results No significant differences were observed in the fertilization rate (57.5% vs. 57.5%) or the rate of excellent-good cleavage embryos (36.0% vs. 36.0%). Conclusions These results suggest that time-lapse observations using an incubator with an integrated optical microscope may therefore be safely utilized in clinical practice.

The nucleus reuniens (RE) of the midline thalamus has been shown to strongly innervate structures of the limbic forebrain, prominently including the hippocampus (HF) and the medial prefrontal cortex (mPFC) and to exert pronounced excitatory effects on HF and mPFC. It was unknown, however, whether RE projections to, and hence actions on, the HF and mPFC originate from a common or largely separate groups of RE neurons. Using fluorescent retrograde tracing techniques, we examined the patterns of distribution of RE cells projecting to HF, to the mPFC or to both sites via axon collaterals. Specifically, injections of the retrograde tracers Fluorogold (FG) or Fluororuby (FR) were made in the mPFC and in various subfields of HF and patterns of single (FG or FR) or double labeled (FG + FR) cells in RE were determined. Pronounced numbers of (single) labeled neurons were present throughout RE with FG or FR injections, and although intermingled in RE, cells projecting to the mPFC were preferentially distributed along the midline or in the perireuniens nucleus (pRE), whereas those projecting to HF occupied a wide mediolateral cross sectional area of RE lying between cells projecting to the mPFC. Approximately, tenfold more labeled cells were present in RE with ventral compared to dorsal CA1 injections. Like single labeled neurons, double labeled cells were found throughout RE, but were most densely concentrated in areas of greatest overlap of FG+ and FR+ neurons or mainly in the lateral one-third of RE, medial to pRE. Depending on specific combinations of injections, double labeled cells ranged from approximately 3–9% of the labeled neurons. The nucleus reuniens has been shown to be a vital link in limbic subcortical–cortical communication and recent evidence indicates a direct RE involvement in hippocampal and medial prefrontal cortical-dependent behaviors. The present findings indicate that RE is critically positioned to influence the HF and mPFC, and their associated behaviors, via separate or collateral projections to these sites.

Chronic Obstructive Pulmonary Disease (COPD) is associated with an increased risk of myocardial infarction and stroke but it remains unclear how to identify microvascular changes in this population. We hypothesized that simple non-mydriatic retinal photography is feasible and can be used to assess microvascular damage in COPD. Novel Vascular Manifestations of COPD was a prospective study comparing smokers with and without COPD, matched for age. Non-mydriatic, retinal fundus photographs were assessed using semi-automated software. Retinal images from 24 COPD and 22 control participants were compared. Cases were of similar age to controls (65.2 vs. 63.1 years, p = 0.38), had significantly lower Forced Expiratory Volume in one second (FEV1) (53.4 vs 100.1% predicted; p < 0.001) and smoked more than controls (41.7 vs. 29.6 pack years; p = 0.04). COPD participants had wider mean arteriolar (155.6 ±15 uM vs. controls [142.2 ± 12 uM]; p = 0.002) and venular diameters (216.8 ±20.7 uM vs. [201.3± 19.1 uM]; p = 0.012). Differences in retinal vessel caliber were independent of confounders, odds ratios (OR) = 1.08 (95% confidence intervals [CI] = 1.02, 1.13; p = 0.007) and OR = 1.05 (CI = 1.01, 1.09; p = 0.011) per uM increase in arteriolar and venular diameter respectively. FEV1 remained significantly associated with retinal vessel dilatation r = -0.39 (p = 0.02). Non-mydriatic retinal imaging is easily facilitated. We found significant arteriole and venous dilation in COPD compared to age-matched smokers without COPD associated with lung function independent of standard cardiovascular risk factors. Retinal microvascular changes are known to be strongly associated with future vascular events and retinal photography offers potential to identify this risk. clinicaltrials.gov NCT02060292.

This month, we celebrate the beauty and power of microscopy images.

The retinal microcirculation can be viewed non-invasively to give a unique perspective of the cerebral microcirculation in vivo. Studying pathological changes of retinal blood vessels (microaneurysms, retinal haemorrhages, and retinal arteriolar narrowing) may help to understand the causes of various cerebrovascular disorders. Retinal photography provides such an opportunity. Several recent studies have shown that retinal microvascular changes are reliably documented by retinal photographs. These retinopathy changes seem to be fairly common in the general population, even in people without hypertension or diabetes. Retinopathy is related to incident clinical stroke and stroke mortality and to MRI-defined subclinical cerebral white-matter lesions and cerebral atrophy, independent of blood pressure, diabetes, and other cerebrovascular risk factors. Retinal microvascular abnormalities seem to be markers of concomitant cerebral microangiopathy, and retinal photography may be useful for the investigation of microvascular disorders of the brain in clinical and epidemiological settings. Future research should be aimed at the development of standardised photographic methods for the assessment of retinal microvascular changes, the replication of these findings in other populations and in people with other cerebrovascular disorders, and the examination of the increased accuracy of stoke-risk stratification given by retinal photography.