Use of a refractive index-coupled diffuser to both generate and measure high-numerical aperture illumination for light microscopy.

While numerical aperture of transillumination at or above 1.25 can be achieved with a substage oiled Abbe condenser, such immersion-capable condensers can be expensive limiting their use in resource poor settings. Also the measurement of numerical aperture generated by illuminators has received relatively little attention in the literature compared to methods for measuring the numerical aperture of acceptance by objectives. In this article, I show how an inexpensive paper diffuser with refractive-index coupling to the sample slide can generate illumination of a numerical aperture of over 1.4 at a small fraction of the cost of oiled dioptric condensers of comparable numerical aperture. In addition, I present two ways in which a diffuser may also be used to measure the numerical aperture generated by an illuminator using either a calibrated index-coupled paper diffuser to implement an interpolative variation of the Horsfall method or a diffuser as a detector screen coupled to a self-built microscope slide-based illumination system apertometer.

PUMA - An open-source 3D-printed direct vision microscope with augmented reality and spatial light modulator functions.

3D-printed microscopes are a topical emerging field in the literature. However most microscopes presented to date are quite novel re-imaginings of the microscope's mechanical design and they are either solely dependent on, or primarily geared towards, camera-based observations rather than ergonomic direct vision screening through an ocular lens. The reliance on camera, computer and monitor for observation introduces a compromise between portability, cost and the quality of an instant wide field of view. In this report, I introduce the Portable Upgradeable Modular and Affordable (PUMA) microscope which is an open-source 3D-printed multimodality microscope that employs a traditional upright design for ease of human direct visual observations and slide screening. PUMA uses standard RMS or C-mount objectives, with a tube length 160 mm, 170 mm or infinity and wide field high eye point ocular lenses. PUMA can use simple mirror-based illumination or can be configured to a full Köhler system with Abbe condenser for high numerical aperture observations including oil immersion. PUMA also has advanced digital/optical imaging features such as a digital spatial light modulator and - unique to any 3D printed microscope to date - an augmented reality heads-up display for interactive calibrated measurements. Digital camera imaging can also be used with PUMA - in fact PUMA can take up to three separate digital cameras simultaneously. PUMA can also function as a direct vision multi-header microscope for teaching or discussion. The illumination system is also modular and includes transillumination, epi-illumination, fluorescence, polarisation, dark ground and also Schlieren-based phase contrast and other Fourier optics filtering modalities. All these advanced features are available through an on-board, battery operated, microprocessor so no mains supply, smartphone, network connection, PC or external monitor are required making PUMA a truly portable system suitable for remote field work.

Analysis of clonal expansions through the normal and premalignant human breast epithelium reveals the presence of luminal stem cells.

It is widely accepted that the cell of origin of breast cancer is the adult mammary epithelial stem cell; however, demonstrating the presence and location of tissue stem cells in the human breast has proved difficult. Furthermore, we do not know the clonal architecture of the normal and premalignant mammary epithelium or its cellular hierarchy. Here, we use deficiency in the mitochondrial enzyme cytochrome c oxidase (CCO), typically caused by somatic mutations in the mitochondrial genome, as a means to perform lineage tracing in the human mammary epithelium. PCR sequencing of laser-capture microdissected cells in combination with immunohistochemistry for markers of lineage differentiation was performed to determine the clonal nature of the mammary epithelium. We have shown that in the normal human breast, clonal expansions (defined here by areas of CCO deficiency) are typically uncommon and of limited size, but can occur at any site within the adult mammary epithelium. The presence of a stem cell population was shown by demonstrating multi-lineage differentiation within CCO-deficient areas. Interestingly, we observed infrequent CCO deficiency that was restricted to luminal cells, suggesting that niche succession, and by inference stem cell location, is located within the luminal layer. CCO-deficient areas appeared large within areas of ductal carcinoma in situ, suggesting that the rate of clonal expansion was altered in the premalignant lesion. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Use of methylation patterns to determine expansion of stem cell clones in human colon tissue.

BACKGROUND & AIMS: It is a challenge to determine the dynamics of stem cells within human epithelial tissues such as colonic crypts. By tracking methylation patterns of nonexpressed genes, we have been able to determine how rapidly individual stem cells became dominant within a human colonic crypt. We also analyzed methylation patterns to study clonal expansion of entire crypts via crypt fission. METHODS: Colonic mucosa was obtained from 9 patients who received surgery for colorectal cancer. The methylation patterns of Cardiac-specific homeobox, Myoblast determination protein 1, and Biglycan were examined within clonal cell populations, comprising either part of, or multiple adjacent, normal human colonic crypts. Clonality was demonstrated by following cytochrome c oxidase-deficient (CCO⁻) cells that shared an identical somatic point mutation in mitochondrial DNA. RESULTS: Methylation pattern diversity among CCO⁻ clones that occupied only part of a crypt was proportional to clone size; this allowed us to determine rates of clonal expansion. Analysis indicated a slow rate of niche succession within the crypt. The 2 arms of bifurcating crypts had distinct methylation patterns, indicating that fission can disrupt epigenetic records of crypt ancestry. Adjacent clonal CCO⁻ crypts usually had methylation patterns as dissimilar to one another as methylation patterns of 2 unrelated crypts. Mathematical models indicated that stem cell dynamics and epigenetic drift could account for observed dissimilarities in methylation patterns. CONCLUSIONS: Methylation patterns can be analyzed to determine the rates of recent clonal expansion of stem cells, but determination of clonality over many decades is restricted by epigenetic drift. We developed a technique to follow changes in intestinal stem cell dynamics in human epithelial tissues that might be used to study premalignant disease.

The human urothelium consists of multiple clonal units, each maintained by a stem cell.

Little is known about the clonal architecture of human urothelium. It is likely that urothelial stem cells reside within the basal epithelial layer, yet lineage tracing from a single stem cell as a means to show the presence of a urothelial stem cell has never been performed. Here, we identify clonally related cell areas within human bladder mucosa in order to visualize epithelial fields maintained by a single founder/stem cell. Sixteen frozen cystectomy specimens were serially sectioned. Patches of cells deficient for the mitochondrially encoded enzyme cytochrome c oxidase (CCO) were identified using dual-colour enzyme histochemistry. To show that these patches represent clonal proliferations, small CCO-proficient and -deficient areas were individually laser-capture microdissected and the entire mitochondrial genome (mtDNA) in each area was PCR amplified and sequenced to identify mtDNA mutations. Immunohistochemistry was performed for the different cell layers of the urothelium and adjacent mesenchyme. CCO-deficient patches could be observed in normal urothelium of all cystectomy specimens. The two-dimensional length of these negative patches varied from 2-3 cells (about 30 µm) to 4.7 mm. Each cell area within a CCO-deficient patch contained an identical somatic mtDNA mutation, indicating that the patch was a clonal unit. Patches contained all the mature cell differentiation stages present in the urothelium, suggesting the presence of a stem cell. Our results demonstrate that the normal mucosa of human bladder contains stem cell-derived clonal units that actively replenish the urothelium during ageing. The size of the clonal unit attributable to each stem cell was broadly distributed, suggesting replacement of one stem cell clone by another.

Locating the stem cell niche and tracing hepatocyte lineages in human liver.

UNLABELLED: We have used immunohistochemical and histochemical techniques to identify patches of hepatocytes deficient in the enzyme cytochrome c oxidase, a component of the electron transport chain and encoded by mitochondrial DNA (mtDNA). These patches invariably abutted the portal tracts and expanded laterally as they spread toward the hepatic veins. Here we investigate, using mtDNA mutations as a marker of clonal expansion, the clonality of these patches. Negative hepatocytes were laser-capture microdissected and mutations identified by polymerase chain reaction sequencing of the entire mtDNA genome. Patches of cytochrome c oxidase-deficient hepatocytes were clonal, suggesting an origin from a long-lived cell, presumably a stem cell. Immunohistochemical analysis of function and proliferation suggested that these mutations in cytochrome c oxidase-deficient hepatocytes were nonpathogenic. CONCLUSION: These data show, for the first time, that clonal proliferative units exist in the human liver, an origin from a periportal niche is most likely, and that the trajectory of the units is compatible with a migration of cells from the periportal regions to the hepatic veins.

A methodological approach to tracing cell lineage in human epithelial tissues.

Methods for lineage tracing of stem cell progeny in human tissues are currently not available. We describe a technique for detecting the expansion of a single cell's progeny that contain clonal mitochondrial DNA (mtDNA) mutations affecting the expression of mtDNA-encoded cytochrome c oxidase (COX). Because such mutations take up to 40 years to become phenotypically apparent, we believe these clonal patches originate in stem cells. Dual-color enzyme histochemistry was used to identify COX-deficient cells, and mutations were confirmed by microdissection of single cells with polymerase chain reaction sequencing of the entire mtDNA genome. These techniques have been applied to human intestine, liver, pancreas, and skin. Our results suggest that the stem cell niche is located at the base of colonic crypts and above the Paneth cell region in the small intestine, in accord with dynamic cell kinetic studies in animals. In the pancreas, exocrine tissue progenitors appeared to be located in or close to interlobular ducts, and, in the liver, we propose that stem cells are located in the periportal region. In the skin, the origin of a basal cell carcinoma appeared to be from the outer root sheath of the hair follicle. We propose that this is a general method for detecting clonal cell populations from which the location of the niche can be inferred, also affording the generation of cell fate maps, all in human tissues. In addition, the technique allows analysis of the origin of human tumors from specific tissue sites.

Quantification of crypt and stem cell evolution in the normal and neoplastic human colon.

Human intestinal stem cell and crypt dynamics remain poorly characterized because transgenic lineage-tracing methods are impractical in humans. Here, we have circumvented this problem by quantitatively using somatic mtDNA mutations to trace clonal lineages. By analyzing clonal imprints on the walls of colonic crypts, we show that human intestinal stem cells conform to one-dimensional neutral drift dynamics with a "functional" stem cell number of five to six in both normal patients and individuals with familial adenomatous polyposis (germline APC(-/+)). Furthermore, we show that, in adenomatous crypts (APC(-/-)), there is a proportionate increase in both functional stem cell number and the loss/replacement rate. Finally, by analyzing fields of mtDNA mutant crypts, we show that a normal colon crypt divides around once every 30-40 years, and the division rate is increased in adenomas by at least an order of magnitude. These data provide in vivo quantification of human intestinal stem cell and crypt dynamics.

Estimation of cell density to aid in assessment of percentage cells of a particular lineage or of cells expressing a specific antigen in bone marrow trephine biopsies.

The authors aimed to develop a tool to assess total cell numbers in a microscope's field of vision, which would provide the denominator for calculating the percentage of positive cells for a given antigen in bone marrow trephine biopsies (BMTBs) of varying cellularities. Precise estimates of cell densities were made from 179 images of BMTBs of varying cellularities using a cell-counting software. The estimates were then validated on an independent set of 20 BMTBs. Among the 179 images, there was a strong linear association between marrow cellularity and cell numbers (Pearson correlation: 0.788). Then standardised cell densities (cells/mm(2) of bone marrow) were deduced for BMTBs of varying cellularities. In the validation study, the actual and the estimated cell numbers correlated strongly (Pearson correlation: 0.990). The cell density estimates provided in this study can be adapted for any microscope and the same method can be used for calculation of the percentage-positive cells for any antigen.

Dual instrument for in vivo and ex vivo OCT imaging in an ENT department.

A dual instrument is assembled to investigate the usefulness of optical coherence tomography (OCT) imaging in an ear, nose and throat (ENT) department. Instrument 1 is dedicated to in vivo laryngeal investigation, based on an endoscope probe head assembled by compounding a miniature transversal flying spot scanning probe with a commercial fiber bundle endoscope. This dual probe head is used to implement a dual channel nasolaryngeal endoscopy-OCT system. The two probe heads are used to provide simultaneously OCT cross section images and en face fiber bundle endoscopic images. Instrument 2 is dedicated to either in vivo imaging of accessible surface skin and mucosal lesions of the scalp, face, neck and oral cavity or ex vivo imaging of the same excised tissues, based on a single OCT channel. This uses a better interface optics in a hand held probe. The two instruments share sequentially, the swept source at 1300 nm, the photo-detector unit and the imaging PC. An aiming red laser is permanently connected to the two instruments. This projects visible light collinearly with the 1300 nm beam and allows pixel correspondence between the en face endoscopy image and the cross section OCT image in Instrument 1, as well as surface guidance in Instrument 2 for the operator. The dual channel instrument was initially tested on phantom models and then on patients with suspect laryngeal lesions in a busy ENT practice. This feasibility study demonstrates the OCT potential of the dual imaging instrument as a useful tool in the testing and translation of OCT technology from the lab to the clinic. Instrument 1 is under investigation as a possible endoscopic screening tool for early laryngeal cancer. Larger size and better quality cross-section OCT images produced by Instrument 2 provide a reference base for comparison and continuing research on imaging freshly excised tissue, as well as in vivo interrogation of more superficial skin and mucosal lesions in the head and neck patient.

Adrenal pseudocyst: Diagnosis and laparoscopic management - A case report.

Cysts of the adrenal gland are rare and are usually discovered incidentally. Large adrenal cysts can however present with severe abdominal pain and can be complicated by haemorrhage, rupture or infection. Adrenal pseudocysts appear to result from haemorrhage within a normal adrenal gland and can expand to accommodate massive amounts of fluid.We report the case of a 39-year-old woman who presented with worsening right upper quadrant pain. An ultrasound scan of the abdomen confirmed a large 29 cm × 20 cm × 17 cm cyst that appeared to originate in the upper pole of the right kidney causing displacement of the liver and right kidney.Following complete aspiration the cyst re-accumulated and an MRI scan demonstrated a thickened and irregular cyst wall with haemorrhagic fluid. Laparoscopic right adrenalectomy was performed and the histopathological diagnosis was confirmed as an adrenal pseudocyst.

Computer-assisted screening of Ziehl-Neelsen-stained tissue for mycobacteria. Algorithm design and preliminary studies on 2,000 images.

Screening Ziehl-Neelsen (ZN)-stained sections for acid-alcohol-fast bacilli (AAFB) is laborious, and sparse bacilli are easily missed. This article presents an automatic screening algorithm using digital image analysis designed to assist human diagnosis of tissue sections. The algorithm uses multiderivative source potentiators and suppressors feeding into interconnected product nodes that result in a probability value for each image (the likelihood that it contains AAFB) and a spatial probability map showing the position of any bacillus. For the study, 3,000 images from ZN-stained tissues were captured, 1,000 were used to train the algorithm, and 2,000 were used to test it. The algorithm successfully ranked AAFB-containing images as the highest in the data sets, despite only single bacilli being present in sparse images (occupying 0.0024% of the image) and despite tissue and staining artifacts. These results suggest that this automated screening assistance method has the potential to save time and money, which is especially important in resource-poor health services.

On the concept of objectivity in digital image analysis in pathology.

AIMS: The term 'objective' connotes a method that is based on facts and not influenced by personal opinions, perception or emotion. One often reads in the biomedical literature claims of objectivity for methods that use digital image analysis applied to histology. Since objective assessment of histology would represent a huge leap forward in scientific measurement and clinical diagnosis, such claims should be substantiated by strong evidence. This paper takes a selective look at the literature on image analysis to assess the definition of objectivity in image analysis and asks whether such a claim is ever justified. METHODS: First, a brief background on the basic science of image analysis in histology details some of the controversies and opinions in the field. Then, a literature review of a subset of papers pertaining to image analysis in histology (with claims of objectivity) is conducted to determine what evidence exists for objectivity in these methods. RESULTS: It was found that image analysis may have many benefits (speed, indefatigability, standardisation, etc.). However, algorithms are devised and implemented by human beings who make subjective decisions at each stage of the algorithm design and implementation process. Thus, image analysis methods can be seen as deterministic processes which 'objectively' implement the subjective decisions of the programmer. This indicates that 'inter-observer' variation in image analysis is equivalent to 'inter-algorithm' variation (which is rarely studied) and that a single computer algorithm's repeatability is of lesser importance than the repeatability of the image analysis method as a whole (including the block, slide and field selection and the method of tissue processing). CONCLUSION: Repeatability and automaticity must not be confused with objectivity, but a lack of objectivity does not imply a lack of utility. Unless specific evidence of objectivity is provided, editors should insist that claims of objectivity in image analysis papers be either removed or justified prior to publication.

'Not just another appendicitis!' - a case report of acute abdominal pain caused by splenic rupture secondary to isolated splenic peliosis.

We present the case of a 31-year-old man admitted with acute abdominal pain who was subsequently found to have a ruptured spleen. A splenectomy was performed as an emergency and he was discharged from hospital 4 days later. Histological analysis revealed isolated splenic peliosis as the underlying condition predisposing to his splenic rupture.

The sources of parenchymal regeneration after chronic hepatocellular liver injury in mice.

After liver injury, parenchymal regeneration occurs through hepatocyte replication. However, during regenerative stress, oval cells (OCs) and small hepatocyte like progenitor cells (SHPCs) contribute to the process. We systematically studied the intra-hepatic and extra-hepatic sources of liver cell replacement in the hepatitis B surface antigen (HBsAg-tg) mouse model of chronic liver injury. Female HBsAg-tg mice received a bone marrow (BM) transplant from male HBsAg-negative mice, and half of these animals received retrorsine to block indigenous hepatocyte proliferation. Livers were examined 3 and 6 months post-BM transplantation for evidence of BM-derived hepatocytes, OCs, and SHPCs. In animals that did not receive retrorsine, parenchymal regeneration occurred through hepatocyte replication, and the BM very rarely contributed to hepatocyte regeneration. In mice receiving retrorsine, 4.8% of hepatocytes were Y chromosome positive at 3 months, but this was frequently attributable to cell fusion between indigenous hepatocytes and donor BM, and their frequency decreased to 1.6% by 6 months, as florid OC reactions and nodules of SHPCs developed. By analyzing serial sections and reconstructing a 3-dimensional map, continuous streams of OCs could be seen that surrounded and entered deep into the nodules of SHPCs, connecting directly with SHPCs, suggesting a conversion of OCs into SHPCs. In conclusion, during regenerative stress, the contribution to parenchymal regeneration from the BM is minor and frequently attributable to cell fusion. OCs and SHPCs are of intrinsic hepatic origin, and OCs can form SHPC nodules.

Mitochondrial DNA mutations are established in human colonic stem cells, and mutated clones expand by crypt fission.

The understanding of the fixation of mutations within human tissues and their subsequent clonal expansion is a considerable problem, of which little is known. We have previously shown that nononcogenic mutations in the mitochondrial genome occur in one of a number of morphologically normal colonic crypt stem cells, the progeny of which later occupy the whole crypt. We propose that these wholly mutated crypts then clonally expand by crypt fission, where each crypt divides into two mutated daughter crypts. Here we show that (i) mutated crypts in the process of fission share the same mutated mitochondrial genotype not present in neighboring cytochrome c oxidase-positive crypts (the odds of this being a random event are >or=2.48 x 10(9):1); (ii) neighboring mutated crypts have the same genotype, which is different from adjacent cytochrome c oxidase-positive crypts; (iii) mutated crypts are clustered together throughout the colon; and (iv) patches of cytochrome c oxidase-deficient crypts increase in size with age. We thus demonstrate definitively that crypt fission is the mechanism by which mutations spread in the normal human colon. This has important implications for the biology of the normal adult human colon and possibly for the growth and spread of colorectal neoplasms.

Fluorescence lifetime imaging of unstained tissues: early results in human breast cancer.

Fluorescence lifetime imaging (FLIM) depends on the fluorescence decay differences between tissues to generate image contrast. In the present study FLIM has been applied to fixed (but unstained) breast cancer tissues to demonstrate the feasibility of this approach for histopathological assessment. As the FLIM method relies on natural autofluorescence, it may be possible to circumvent tissue processing altogether and so FLIM has the potential to be a powerful new method of in vivo tissue imaging via an endoscopic or per-operative approach in a variety of organs, as well as a research tool for in vivo animal models of disease. Unstained, alcohol-fixed tissue samples from 13 patients were stimulated by laser pulses at 415 nm. The temporal decay of the autofluorescence was imaged over a period of 2 ns after cessation of the pulse. The decay rate at each image pixel was calculated as the 'lifetime' factor tau. A tissue classification scheme was used to define regions in each image. The average lifetimes of different tissue regions were compared. A total of 167 tissue regions were measured. Within individual fields, stroma had a larger tau (slower decay) than epithelium (p < 0.001). Within individual patients (taking the mean tau of a given tissue type across all fields from each patient), there was a statistically significant difference between benign and malignancy-associated stroma (p < 0.05). Also, benign collagen had a longer tau than benign epithelium (p < 0.05). Multivariate analysis showed a significant difference between benign stroma, malignancy-associated stroma, blood vessels, and malignant epithelium (p < 0.05). Statistically significant differences between benign and malignancy-associated stroma were obtained even with small patient numbers, indicating that lifetime-based instruments can be developed for real-time diagnostic imaging with microscopic resolution.

Studying biological tissue with fluorescence lifetime imaging: microscopy, endoscopy, and complex decay profiles.

We have applied fluorescence lifetime imaging (FLIM) to the autofluorescence of different kinds of biological tissue in vitro, including animal tissue sections and knee joints as well as human teeth, obtaining two-dimensional maps with functional contrast. We find that fluorescence decay profiles of biological tissue are well described by the stretched exponential function (StrEF), which can represent the complex nature of tissue. The StrEF yields a continuous distribution of fluorescence lifetimes, which can be extracted with an inverse Laplace transformation, and additional information is provided by the width of the distribution. Our experimental results from FLIM microscopy in combination with the StrEF analysis indicate that this technique is ready for clinical deployment, including portability that is through the use of a compact picosecond diode laser as the excitation source. The results obtained with our FLIM endoscope successfully demonstrated the viability of this modality, though they need further optimization. We expect a custom-designed endoscope with optimized illumination and detection efficiencies to provide significantly improved performance.