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Scientific Reports Dec 2022Chemical staining of biological specimens is commonly utilised to boost contrast in soft tissue structures, but unambiguous identification of staining location and...
Chemical staining of biological specimens is commonly utilised to boost contrast in soft tissue structures, but unambiguous identification of staining location and distribution is difficult without confirmation of the elemental signature, especially for chemicals of similar density contrast. Hyperspectral X-ray computed tomography (XCT) enables the non-destructive identification, segmentation and mapping of elemental composition within a sample. With the availability of hundreds of narrow, high resolution (~ 1 keV) energy channels, the technique allows the simultaneous detection of multiple contrast agents across different tissue structures. Here we describe a hyperspectral imaging routine for distinguishing multiple chemical agents, regardless of contrast similarity. Using a set of elemental calibration phantoms, we perform a first instance of direct stain concentration measurement using spectral absorption edge markers. Applied to a set of double- and triple-stained biological specimens, the study analyses the extent of stain overlap and uptake regions for commonly used contrast markers. An improved understanding of stain concentration as a function of position, and the interaction between multiple stains, would help inform future studies on multi-staining procedures, as well as enable future exploration of heavy metal uptake across medical, agricultural and ecological fields.
Topics: Coloring Agents; Tomography, X-Ray Computed; Staining and Labeling; Phantoms, Imaging; Calibration
PubMed: 36535963
DOI: 10.1038/s41598-022-23592-0 -
Journal of Biomedical Optics May 2023Quantification of elastic fiber in the tissue specimen is an important aspect of diagnosing different diseases. Though hematoxylin and eosin (H&E) staining is a...
SIGNIFICANCE
Quantification of elastic fiber in the tissue specimen is an important aspect of diagnosing different diseases. Though hematoxylin and eosin (H&E) staining is a routinely used and less expensive tissue staining technique, elastic and collagen fibers cannot be differentiated using it. So, in conventional pathology, special staining technique, such as Verhoeff's van Gieson (EVG), is applied physically for this purpose. However, the procedure of EVG staining is very expensive and time-consuming.
AIM
The goal of our study is to propose a deep-learning-based computerized method for the generation of RGB EVG stained tissue from hyperspectral H&E stained one to save the time and cost of conventional EVG staining procedure.
APPROACH
H&E stained hyperspectral image and EVG stained RGB whole slide image of human pancreatic tissue have been leveraged for this experiment. CycleGAN-based deep learning model has been proposed for digital stain conversion while images from source and target domains are of different modalities (hyperspectral and RGB) with different channel dimensions. A set of three basis functions have been introduced for calculating one of the losses of the proposed method, which retains the relevant features of EVG stained image within the reduced channel dimension of the H&E stained one.
RESULTS
The experimental results showed that a set of three basis functions including linear discriminant function and transmittance spectrum of eosin and hematoxylin better retained the essential properties of the elastic fiber to be discriminated from collagen fiber within the reduced dimension of the hyperspectral H&E stained image. Also, only a smaller number of paired training data with our proposed training method contributed significantly to the generation of more realistic EVG stained image with more precise identification of elastic fiber.
CONCLUSIONS
RGB EVG stained image is generated from hyperspectral H&E stained image for which our model has performed two types of image conversion simultaneously: hyperspectral to RGB and H&E to EVG. The experimental results show that the intentionally designed set of three basis functions contains more relevant information and prove the effectiveness of our proposed method in generating realistic RGB EVG stained image from hyperspectral H&E stained one.
Topics: Humans; Coloring Agents; Hematoxylin; Eosine Yellowish-(YS); Staining and Labeling; Collagen
PubMed: 37265876
DOI: 10.1117/1.JBO.28.5.056501 -
Current Protocols Nov 2021Correlating the location of subcellular structures with dynamic cellular behaviors is difficult when working with organisms that lack the molecular genetic tools needed...
Correlating the location of subcellular structures with dynamic cellular behaviors is difficult when working with organisms that lack the molecular genetic tools needed for expressing fluorescent protein fusions. Here, we describe a protocol for fixing, permeabilizing, and staining cells in a single step while imaging on a microscope. In contrast to traditional, multi-step fixing and staining protocols that take hours, the protocol outlined here achieves satisfactory staining within minutes. This approach takes advantage of well-characterized small molecules that stain specific subcellular structures, including nuclei, mitochondria, and actin networks. Direct visualization of the entire process allows for rapid optimization of cell fixation and staining, as well as straightforward identification of fixation artifacts. Moreover, live imaging prior to fixation reveals the dynamic history of cellular features, making it particularly useful for model systems without the capacity for expressing fluorescent protein fusions. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Fixing, permeabilizing, and staining mammalian cells in one step on the microscope.
Topics: Animals; Coloring Agents; Microscopy, Fluorescence; Mitochondria; Staining and Labeling
PubMed: 34826344
DOI: 10.1002/cpz1.308 -
Diagnostic Pathology Feb 2024Staining tissue samples to visualise cellular detail and tissue structure is at the core of pathology diagnosis, but variations in staining can result in significantly...
BACKGROUND
Staining tissue samples to visualise cellular detail and tissue structure is at the core of pathology diagnosis, but variations in staining can result in significantly different appearances of the tissue sample. While the human visual system is adept at compensating for stain variation, with the growth of digital imaging in pathology, the impact of this variation can be more profound. Despite the ubiquity of haematoxylin and eosin staining in clinical practice worldwide, objective quantification is not yet available. We propose a method for quantitative haematoxylin and eosin stain assessment to facilitate quality assurance of histopathology staining, enabling truly quantitative quality control and improved standardisation.
METHODS
The stain quantification method comprises conventional microscope slides with a stain-responsive biopolymer film affixed to one side, called stain assessment slides. The stain assessment slides were characterised with haematoxylin and eosin, and implemented in one clinical laboratory to quantify variation levels.
RESULTS
Stain assessment slide stain uptake increased linearly with duration of haematoxylin and eosin staining (r = 0.99), and demonstrated linearly comparable staining to samples of human liver tissue (r values 0.98-0.99). Laboratory implementation of this technique quantified intra- and inter-instrument variation of staining instruments at one point in time and across a five-day period.
CONCLUSION
The proposed method has been shown to reliably quantify stain uptake, providing an effective laboratory quality control method for stain variation. This is especially important for whole slide imaging and the future development of artificial intelligence in digital pathology.
Topics: Humans; Artificial Intelligence; Eosine Yellowish-(YS); Staining and Labeling; Coloring Agents; Hematoxylin
PubMed: 38395890
DOI: 10.1186/s13000-024-01461-w -
PloS One 2023Traditional histological stains, such as hematoxylin-eosin (HE), special stains, and immunofluorescence (IF), have defined myriads of cellular phenotypes and tissue...
Traditional histological stains, such as hematoxylin-eosin (HE), special stains, and immunofluorescence (IF), have defined myriads of cellular phenotypes and tissue structures in a separate stained section. However, the precise connection of information conveyed by the various stains in the same section, which may be important for diagnosis, is absent. Here, we present a new staining modality-Flow chamber stain, which complies with the current staining workflow but possesses newly additional features non-seen in conventional stains, allowing for (1) quickly switching staining modes between destain and restain for multiplex staining in one single section from routinely histological preparation, (2) real-time inspecting and digitally capturing each specific stained phenotype, and (3) efficiently synthesizing graphs containing the tissue multiple-stained components at site-specific regions. Comparisons of its stains with those by the conventional staining fashions using the microscopic images of mouse tissues (lung, heart, liver, kidney, esophagus, and brain), involving stains of HE, Periodic acid-Schiff, Sirius red, and IF for Human IgG, and mouse CD45, hemoglobin, and CD31, showed no major discordance. Repetitive experiments testing on targeted areas of stained sections confirmed the method is reliable with accuracy and high reproducibility. Using the technique, the targets of IF were easily localized and seen structurally in HE- or special-stained sections, and the unknown or suspected components or structures in HE-stained sections were further determined in histological special stains or IF. By the technique, staining processing was videoed and made a backup for off-site pathologists, which facilitates tele-consultation or -education in current digital pathology. Mistakes, which might occur during the staining process, can be immediately found and amended accordingly. With the technique, a single section can provide much more information than the traditional stained counterpart. The staining mode bears great potential to become a common supplementary tool for traditional histopathology.
Topics: Humans; Animals; Mice; Coloring Agents; Reproducibility of Results; Staining and Labeling; Esophagus; Hematoxylin; Eosine Yellowish-(YS); Phenotype
PubMed: 37141296
DOI: 10.1371/journal.pone.0284444 -
Indian Journal of Dental Research :... 2020Fibro-osseous lesions (FOLs) of the jaws exhibit an overlapping histomorphologic spectrum with respect to nature of calcifications. Sometimes these calcifications may be...
OBJECTIVES
Fibro-osseous lesions (FOLs) of the jaws exhibit an overlapping histomorphologic spectrum with respect to nature of calcifications. Sometimes these calcifications may be difficult to characterize as bone and cementum on routine Hematoxylin and Eosin (H&E) staining. This causes difficulty in assessing the origin and diagnosis of these lesions. Thus the study aimed to characterize bone, cementum, and hard tissue components in FOLs using special stains.
METHOD
The study included a histochemical evaluation of 20 samples of bone and cementum and 12 cases each of fibrous dysplasia (FD) and ossifying fibroma (OF). Three consecutive sections of each tissue were stained with H and E, modified Gallego's iron fuschin stain and Van Gieson stain. H and E and modified Gallego's iron fuschin stained sections were analyzed under light microscope whereas Van Gieson stained section was analyzed under polarizing microscope.
RESULTS
It was found that cementum stained red and bone stained greenish-yellow in color. The calcifications seen in fibrous dysplasia stained greenish-yellow in color. Three cases of OF showed greenish-yellow calcifications and nine cases showed reddish calcifications. Polarization study of bone showed lamellar pattern and tooth cementum showed quilt pattern. Four cases of FD showed lamellar pattern and eight cases showed haphazard pattern. In OF, three cases showed lamellar pattern and nine cases quilt pattern.
CONCLUSION
Calcifications having lamellar pattern and greenish-yellow color suggest their osteogenic origin thus having aggressive nature and requiring aggressive treatment. Calcifications having quilt pattern and reddish color suggest periodontal ligament origin thus having less aggressive behavior and less extensive treatment.
Topics: Coloring Agents; Dental Cementum; Fibroma, Ossifying; Humans; Iron; Staining and Labeling
PubMed: 33753659
DOI: 10.4103/ijdr.IJDR_889_18 -
Reproduction in Domestic Animals =... Jun 2022Detailed and direct analysis of semen, including sperm morphology, enables a diagnosis of male fertility. This study aimed to describe an economical and verified...
Detailed and direct analysis of semen, including sperm morphology, enables a diagnosis of male fertility. This study aimed to describe an economical and verified protocol for canine spermiograms and compare the effectiveness of Sperm Stain and Sperm Blue (Microptic, Spain) in veterinary practice. Sperm assessment was conducted manually, using a standard optical microscope, and via computerized semen analysis using the SCA CASA (Sperm Class Analyzer CASA System-MICROPTIC, Spain). This study showed that Sperm Blue is a better solution for computerized sperm quality analysis of healthy dogs. At the same time, Sperm Stain turned out to be more helpful in identifying specific morphological defects of sperm. Automated canine sperm morphology analysis worked better with Sperm Blue stain, but Sperm Stain simplified manual evaluation of various organelles' defects. Standard, manual examination is more error-prone for an inexperienced andrology technician, but it seems to be still a gold standard technique for canine sperm assessment.
Topics: Animals; Cell Count; Coloring Agents; Dogs; Male; Semen Analysis; Sperm Count; Sperm Motility; Spermatozoa; Staining and Labeling
PubMed: 35212033
DOI: 10.1111/rda.14100 -
Veterinary Medicine and Science Aug 2020Staining, as a valuable method for sperm morphological assessment, has been used to determine sperm abnormalities, fertilization capability and sperm suitability during...
Staining, as a valuable method for sperm morphological assessment, has been used to determine sperm abnormalities, fertilization capability and sperm suitability during freezing-thawing process. Synthetic dyes have been used for sperm viability and morphological evaluation. However, most of them have been made from chemical substances and have a perilous effect on the environment. In the current study, we evaluated three different natural dyes as the natural sources of dye for sperm staining. Bull frozen semen was used and prepared on slides for staining. Aqueous extract dye of black mulberry (BM), henna (HA), safflower (SA) and eosin-nigrosine (control group) were used for sperm staining. Additionally, the effect of staining dyes on viability and some morphological parameters (head area: HR, head abnormality: HB and tail abnormality: TA) were evaluated. Although none of the natural dyes could detect viability of the sperm cells, safflower stain (HR: 26.55 µm, HB: 0% TA: 28%) and black mulberry stain (HR: 25.07 µm, HB: 2% TA: 3%) compared to control group (HR: 34.29 µm, HB: 4%, TA: 4%) provoked a strong reaction in the sperm cells, so that the sperms were observed yellow and red respectively. The reaction of sperm cells to the henna dye was very poor and it did not stain the sperm cells. Thus, the present study demonstrated that SA and BM dyes are able to stain the spermatozoa and with further modification could be used as alternative dyes for sperm staining in the study of sperm morphology, but not viability. Staining with these dyes can be an alternative to current costly chemical staining methods.
Topics: Animals; Carthamus tinctorius; Cattle; Coloring Agents; Male; Morus; Naphthoquinones; Plant Preparations; Semen Analysis; Spermatozoa; Staining and Labeling
PubMed: 32323476
DOI: 10.1002/vms3.268 -
Gut Dec 1964
Topics: Colon; Coloring Agents; Crohn Disease; Enteritis; Granuloma; Humans; Ileum; Pathology; Sarcoidosis; Staining and Labeling
PubMed: 14244024
DOI: 10.1136/gut.5.6.510 -
Journal of Anatomy Apr 1965
Topics: Acrylic Resins; Animals; Arachnoid; Capillaries; Coloring Agents; Dura Mater; Histological Techniques; Histology; Nerve Tissue; Paraffin; Research; Sheep; Staining and Labeling; Subarachnoid Space
PubMed: 14327176
DOI: No ID Found