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Methods in Molecular Biology (Clifton,... 2018The lung is ideally suited to the application of histological methods to study its structure, cellular composition, and molecular characteristics of more than 30 types...
The lung is ideally suited to the application of histological methods to study its structure, cellular composition, and molecular characteristics of more than 30 types of cells. The key in these endeavors are proper tissue preservation/fixation, well-established protocols aimed at sectioning and staining, and understanding of lung morphology. Molecular studies can be performed in laser-captured cells and microscopic structures.
Topics: Histological Techniques; Humans; Immunohistochemistry; Laser Capture Microdissection; Lung
PubMed: 29987797
DOI: 10.1007/978-1-4939-8570-8_20 -
Infection, Genetics and Evolution :... Jun 2020Single-cell sequencing (SCS) is a next-generation sequencing method that is mainly used to analyze differences in genetic and protein information between cells, to... (Review)
Review
Single-cell sequencing (SCS) is a next-generation sequencing method that is mainly used to analyze differences in genetic and protein information between cells, to obtain genetic information on microorganisms that are difficult to cultivate at a single-cell level and to better understand their specific roles in the microenvironment. By sequencing the whole genome, transcriptome and epigenome of a single cell, the complex heterogeneous mechanisms involved in disease occurrence and progression can be revealed, further improving disease diagnosis, prognosis prediction and monitoring of the therapeutic effects of drugs. In this study, we mainly summarized the methods and application fields of SCS, which may provide potential references for its future clinical applications, including the analysis of embryonic and organ development, the immune system, cancer progression, and parasitic and infectious diseases as well as stem cell research, antibody screening, and therapeutic research and development.
Topics: Epigenesis, Genetic; Genome; Genomics; High-Throughput Nucleotide Sequencing; Humans; Immunomagnetic Separation; Microdissection; Microfluidics; Organ Specificity; Sequence Analysis, DNA; Single-Cell Analysis; Species Specificity; Transcriptome
PubMed: 31958516
DOI: 10.1016/j.meegid.2020.104198 -
Fertility and Sterility Jun 2024
Topics: Humans; Male; Testis; Testicular Neoplasms; Microdissection
PubMed: 38554764
DOI: 10.1016/j.fertnstert.2024.03.023 -
The Analyst Jun 2021The human body comprises rich populations of cells, which are arranged into tissues and organs with diverse functionalities. These cells exhibit a broad spectrum of... (Review)
Review
The human body comprises rich populations of cells, which are arranged into tissues and organs with diverse functionalities. These cells exhibit a broad spectrum of phenotypes and are often organized as a heterogeneous but sophisticatedly regulated ecosystem - tissue microenvironment, inside which every cell interacts with and is reciprocally influenced by its surroundings through its life span. Therefore, it is critical to comprehensively explore the cellular machinery and biological processes in the tissue microenvironment, which is best exemplified by the tumor microenvironment (TME). The past decade has seen increasing advances in the field of spatial proteomics, the main purpose of which is to characterize the abundance and spatial distribution of proteins and their post-translational modifications in the microenvironment of diseased tissues. Herein, we outline the achievements and remaining challenges of mass spectrometry-based tissue spatial proteomics. Exciting technology developments along with important biomedical applications of spatial proteomics are highlighted. In detail, we focus on high-quality resources built by scalpel macrodissection-based region-resolved proteomics, method development of sensitive sample preparation for laser microdissection-based spatial proteomics, and antibody recognition-based multiplexed tissue imaging. In the end, critical issues and potential future directions for spatial proteomics are also discussed.
Topics: Ecosystem; Humans; Laser Capture Microdissection; Mass Spectrometry; Proteins; Proteomics
PubMed: 34042124
DOI: 10.1039/d1an00472g -
Seminars in Reproductive Medicine Nov 2023Nonobstructive azoospermia (NOA) is among the most common causes of male infertility. For men with NOA seeking fertility treatment, microdissection testicular sperm...
Nonobstructive azoospermia (NOA) is among the most common causes of male infertility. For men with NOA seeking fertility treatment, microdissection testicular sperm extraction (microTESE) is the best option for retrieving sperm, which can be used with in vitro fertilization-intracytoplasmic sperm injection to achieve pregnancy in their partner. With the aid of the operating microscope, microTESE allows for thorough evaluation of the testis tissue and selection of seminiferous tubules that appear most capable of sperm production. Rates of success with microTESE vary depending on the underlying cause of NOA and the center at which the procedure is performed. Not all patients are candidates for microTESE, and those who are candidates should be counseled on the likelihood of sperm retrieval and the potential for changes in postoperative testis function.
Topics: Pregnancy; Female; Humans; Male; Testis; Microdissection; Retrospective Studies; Semen; Azoospermia; Spermatozoa
PubMed: 38262439
DOI: 10.1055/s-0043-1777833 -
Journal of Visualized Experiments : JoVE Apr 2023The ocular micro-dissection of the rodent eye involves the segmentation of the enucleated eyeball with the attached nictitating membrane, or third eyelid, to obtain the...
The ocular micro-dissection of the rodent eye involves the segmentation of the enucleated eyeball with the attached nictitating membrane, or third eyelid, to obtain the anterior and posterior eyecups. With this technique, the sub-parts of the eye, including the corneal tissue, neural tissue, retinal pigment epithelial (RPE) tissue, and lens, can be obtained for wholemounts, cryo-sectioning, and/or single-cell suspensions of a specific ocular tissue. The presence of the third eyelid presents unique and significant advantages, as it benefits the maintenance of the orientation of the eye, which is important for understanding eye physiology following any localized intervention or in studies involving ocular analysis relating to the eye's spatial topography. In this method, we enucleated the eyeball at the socket along with the third eyelid by carefully and slowly cutting through the extraocular muscles and severing the optic nerve. The eyeball was pierced through the corneal limbus using a microblade. The incision was used as the point of entry, allowing for cutting along the corneal-scleral junction by inserting micro-scissors through the incision point. Small and continuous cuts along the circumference were made until the cups separated. These could be further dissected by gently peeling the translucent layer of the neural retina using Colibri suturing forceps to obtain the neural retina and RPE layers. Further, three/four equidistant cuts were made from the periphery perpendicularly to the optic center until the optic nerve was reached. This opened the hemispherical cups into a floret shape so that they fell flat and could be easily mounted. This technique has been used in our lab for corneal wholemounts and retinal sections. The presence of the third eyelid delineates the nasal-temporal orientation, which allows for the study of various cell therapy interventions post-transplantation and, thus, the targeted physiological validation vital for visualization and accurate representation in such studies.
Topics: Animals; Microdissection; Eye; Retina; Retinal Pigment Epithelium; Lens, Crystalline; Cornea
PubMed: 37154568
DOI: 10.3791/64414 -
Journal of the American Society of... Jan 2024Laser capture microdissection and mass spectrometry (LCM/MS) is a technique that involves dissection of glomeruli from paraffin-embedded biopsy tissue, followed by... (Review)
Review
Laser capture microdissection and mass spectrometry (LCM/MS) is a technique that involves dissection of glomeruli from paraffin-embedded biopsy tissue, followed by digestion of the dissected glomerular proteins by trypsin, and subsequently mass spectrometry to identify and semiquantitate the glomerular proteins. LCM/MS has played a crucial role in the identification of novel types of amyloidosis, biomarker discovery in fibrillary GN, and more recently discovery of novel target antigens in membranous nephropathy (MN). In addition, LCM/MS has also confirmed the role for complement proteins in glomerular diseases, including C3 glomerulopathy. LCM/MS is now widely used as a clinical test and considered the gold standard for diagnosis and typing amyloidosis. For the remaining glomerular diseases, LCM/MS has remained a research tool. In this review, we discuss the usefulness of LCM/MS in other glomerular diseases, particularly MN, deposition diseases, and diseases of complement pathways, and advocate more routine use of LCM/MS at the present time in at least certain diseases, such as MN, for target antigen detection. We also discuss the limitations of LCM/MS, particularly the difficulties faced from moving from a research-based technique to a clinical test. Nonetheless, the role of LCM/MS in glomerular diseases is expanding. Currently, LCM/MS may be used to identify the etiology in certain glomerular diseases, but in the future, LCM/MS can play a valuable role in determining pathways of complement activation, inflammation, and fibrosis.
Topics: Humans; Kidney Diseases; Kidney Glomerulus; Mass Spectrometry; Laser Capture Microdissection; Glomerulonephritis, Membranous; Amyloidosis
PubMed: 37749770
DOI: 10.1681/ASN.0000000000000221 -
Systems Biology in Reproductive Medicine Jun 2016Non-obstructive azoospermia (NOA) is a severe form of infertility accounting for 10% of infertile men. Microdissection testicular sperm extraction (microTESE) includes a... (Review)
Review
Non-obstructive azoospermia (NOA) is a severe form of infertility accounting for 10% of infertile men. Microdissection testicular sperm extraction (microTESE) includes a set of clinical protocols from which viable sperm are collected from patients (suffering from NOA), for intracytoplasmic sperm injection (ICSI). Clinical protocols associated with the processing of a microTESE sample are inefficient and significantly reduce the success of obtaining a viable sperm population. In this review we highlight the sources of these inefficiencies and how these sources can possibly be removed by microfluidic technology and single-cell Raman spectroscopy.
Topics: Clinical Protocols; Humans; Male; Microdissection; Microfluidics; Spectrum Analysis, Raman; Sperm Retrieval
PubMed: 27104311
DOI: 10.3109/19396368.2016.1159748 -
International Journal of Urology :... Jan 2024Approximately 1% of the general male population has azoospermia, and nonobstructive azoospermia accounts for the majority of cases. The causes vary widely, including... (Review)
Review
Approximately 1% of the general male population has azoospermia, and nonobstructive azoospermia accounts for the majority of cases. The causes vary widely, including chromosomal and genetic abnormalities, varicocele, drug-induced causes, and gonadotropin deficiency; however, the cause is often unknown. In azoospermia caused by hypogonadotropic hypogonadism, gonadotropin replacement therapy can be expected to produce sperm in the ejaculate. In some cases, upfront varicocelectomy for nonobstructive azoospermia with varicocele may result in the appearance of ejaculated spermatozoa; however, the appropriate indication should be selected. Each guideline recommends microdissection testicular sperm extraction for nonobstructive azoospermia in terms of successful sperm retrieval and avoidance of complications. Sperm retrieval rates generally ranged from 20% to 70% but vary depending on the causative disease. Various attempts have been made to predict sperm retrieval and improve sperm retrieval rates; however, the evidence is insufficient. Further evidence accumulation is needed for salvage treatment in cases of failed sperm retrieval. In Japan, there is inadequate provision on the right to know the origin of children born from artificial insemination of donated sperm and the rights of sperm donors, as well as information on unrelated family members, and the development of these systems is challenging. In the future, it is hoped that the pathogenesis of nonobstructive azoospermia with an unknown cause will be elucidated and that technology for omics technologies, human spermatogenesis using pluripotent cells, and organ culture methods will be developed.
Topics: Child; Humans; Male; Azoospermia; Varicocele; Microdissection; Semen; Retrospective Studies; Gonadotropins; Testis
PubMed: 37737473
DOI: 10.1111/iju.15301 -
Hepatology International Mar 2019Routine transcriptomic and proteomic analysis are usually performed at a whole organ or tissue level. These approaches provide an average readout of all cell types... (Review)
Review
Routine transcriptomic and proteomic analysis are usually performed at a whole organ or tissue level. These approaches provide an average readout of all cell types present within the tissue but do not allow differentiating the profile of specific cell populations. Laser capture microdissection (LCM) constitutes an excellent tool to isolate cell populations or areas of interest within a tissue. By direct visualization, the selected area is excised by a laser and can be further processed for a variety of downstream analyses. This technology has been widely used in the study of liver diseases, from DNA and RNA sequencing to mass spectrometry. However, LCM also has important limitations. To ensure the best integrity of the molecule of interest, optimal tissue preservation, careful tissue sectioning, and optimization of the staining procedure are required. The present review provides a description of the LCM technology, including tips and technical recommendations to perform the procedure, as well as an overview of studies using LCM technology in the field of liver disease.
Topics: Humans; Laser Capture Microdissection; Liver Diseases; Sequence Analysis, DNA; Sequence Analysis, RNA; Staining and Labeling; Tissue Preservation
PubMed: 30600479
DOI: 10.1007/s12072-018-9917-3