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Annals of the New York Academy of... Nov 2020The use of chemical warfare agents (CWAs) in military conflicts and against civilians is a recurrent problem. Despite ongoing CWA research using in vitro or in vivo... (Review)
Review
The use of chemical warfare agents (CWAs) in military conflicts and against civilians is a recurrent problem. Despite ongoing CWA research using in vitro or in vivo models, progress to elucidate mechanisms of toxicity and to develop effective therapies, decontamination procedures, and general countermeasures is still limited. Novel scientific approaches to address these questions are needed to expand perspectives on existing knowledge and gain new insights. To achieve this, the use of ex vivo techniques like precision-cut tissue slices (PCTSs) can be a valuable approach. Existing studies employing this economical and relatively easy to implement method show model suitability and comparability with the use of in vitro and in vivo models. In this article, we review research on CWAs in PCTSs to illustrate the advantages of the approach and to promote future applications.
Topics: Animals; Chemical Warfare Agents; Humans; Microdissection
PubMed: 32808309
DOI: 10.1111/nyas.14459 -
Sheng Wu Gong Cheng Xue Bao = Chinese... Jan 2019Basic research in life science and medicine has dug into single cell level in recent years. Single-cell analysis offers to understand life from diverse perspectives and... (Review)
Review
Basic research in life science and medicine has dug into single cell level in recent years. Single-cell analysis offers to understand life from diverse perspectives and is used to profile cell heterogeneity to investigate mechanism of diseases. Single cell technologies have also found applications in forensic medicine and clinical reproductive medicine, while the techniques are rapidly evolving and have become more and more sophisticated. In this article, we reviewed various single cell isolation techniques and their pros and cons, including manual cell picking, laser capture microdissection and microfluidics, as well as analysis methods for DNA, RNA and protein in single cell. In addition, we summarized major up-to-date single cell research achievements and their potential applications.
Topics: Animals; Cell Separation; DNA; Laser Capture Microdissection; RNA; Single-Cell Analysis
PubMed: 30756532
DOI: 10.13345/j.cjb.180102 -
International Journal of Molecular... Nov 2022The advancement in molecular techniques has been attributed to the quality and significance of cancer research. Pancreatic cancer (PC) is one of the rare cancers with... (Review)
Review
The advancement in molecular techniques has been attributed to the quality and significance of cancer research. Pancreatic cancer (PC) is one of the rare cancers with aggressive behavior and a high mortality rate. The asymptomatic nature of the disease until its advanced stage has resulted in late diagnosis as well as poor prognosis. The heterogeneous character of PC has complicated cancer development and progression studies. The analysis of bulk tissues of the disease was insufficient to understand the disease, hence, the introduction of the single-cell separating technique aided researchers to decipher more about the specific cell population of tumors. This review gives an overview of the Laser Capture Microdissection (LCM) technique, one of the single-cell separation methods used in PC research.
Topics: Humans; Laser Capture Microdissection; Pancreatic Neoplasms; Pancreas; Carcinoma, Pancreatic Ductal
PubMed: 36498893
DOI: 10.3390/ijms232314566 -
Cytometry. Part a : the Journal of the... Dec 2019Glass needle-based chromosome microdissection (midi) is a standard approach developed in the 1980s and remains more frequently applied in testing than the comparable...
Glass needle-based chromosome microdissection (midi) is a standard approach developed in the 1980s and remains more frequently applied in testing than the comparable technique using laser-based platforms. As the amount of DNA extracted by this technique is minimal and often in the range of picograms, the isolated DNA must be further amplified prior to use; the isolated amplified product can be readily utilized in multiple molecular research and diagnostic investigation. DNA libraries created by midi are either chromosome- or chromosome-region-specific. However, a critical component to this process is the need for timely chromosome preparation via the air-drying method not to exceed a ~2-3 h before midi is performed. Failure of this time-sensitive step often results in the chromosomes drying out after dropping, and upon initiation of the midi technique, the dissected material can jump away while touching by the needle, and collection of a suitable sample is inhibited. Herein, we demonstrate with a simple adaptation of the standard procedure, midi can be performed on semi-archived material stored for longer periods at -20°C. Thus, the critical step to obtain well-spread chromosome preparations can be completed under established conditions, for example, in the primary laboratory, stored at -20°C, and sent directly to specialized reference laboratories offering midi. In our study, we were able to obtain high-quality DNA libraries, as verified by gel electrophoreses and reverse fluorescence in situ hybridization, via midi extracted chromosome spreads derived from human, fish, snake, lampbrush, and insect stored for up to 6 months. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.
Topics: Animals; Biological Specimen Banks; Chromosomes; DNA Probes; Humans; In Situ Hybridization, Fluorescence; Microdissection
PubMed: 31532073
DOI: 10.1002/cyto.a.23896 -
Journal of Genetics and Genomics = Yi... Sep 2023Spatial omics technologies have become powerful methods to provide valuable insights into cells and tissues within a complex context, significantly enhancing our... (Review)
Review
Spatial omics technologies have become powerful methods to provide valuable insights into cells and tissues within a complex context, significantly enhancing our understanding of the intricate and multifaceted biological system. With an increasing focus on spatial heterogeneity, there is a growing need for unbiased, spatially resolved omics technologies. Laser capture microdissection (LCM) is a cutting-edge method for acquiring spatial information that can quickly collect regions of interest (ROIs) from heterogeneous tissues, with resolutions ranging from single cells to cell populations. Thus, LCM has been widely used for studying the cellular and molecular mechanisms of diseases. This review focuses on the differences among four types of commonly used LCM technologies and their applications in omics and disease research. Key attributes of application cases are also highlighted, such as throughput and spatial resolution. In addition, we comprehensively discuss the existing challenges and the great potential of LCM in biomedical research, disease diagnosis, and targeted therapy from the perspective of high-throughput, multi-omics, and single-cell resolution.
Topics: Laser Capture Microdissection; Multiomics; Biomedical Research
PubMed: 37544594
DOI: 10.1016/j.jgg.2023.07.011 -
Molecular Aspects of Medicine Feb 2018Laser microdissection (LM) has become widely democratized over the last fifteen years. Instruments have evolved to offer more powerful and efficient lasers as well as... (Review)
Review
Laser microdissection (LM) has become widely democratized over the last fifteen years. Instruments have evolved to offer more powerful and efficient lasers as well as new options for sample collection and preparation. Technological evolutions have also focused on the post-microdissection analysis capabilities, opening up investigations in all disciplines of experimental and clinical biology, thanks to the advent of new high-throughput methods of genome analysis, including RNAseq and proteomics, now globally known as microgenomics, i.e. analysis of biomolecules at the cell level. In spite of the advances these rapidly developing methods have allowed, the workflow for sampling and collection by LM remains a critical step in insuring sample integrity in terms of histology (accurate cell identification) and biochemistry (reliable analyzes of biomolecules). In this review, we describe the sample processing as well as the strengths and limiting factors of LM applied to the specific selection of one or more cells of interest from a heterogeneous tissue. We will see how the latest developments in protocols and methods have made LM a powerful and sometimes essential tool for genomic and proteomic analyzes of tiny amounts of biomolecules extracted from few cells isolated from a complex tissue, in their physiological context, thus offering new opportunities for understanding fundamental physiological and/or patho-physiological processes.
Topics: Animals; Gene Expression Profiling; Genomics; Humans; Lasers; Microdissection; Proteomics
PubMed: 28927943
DOI: 10.1016/j.mam.2017.09.003 -
Operative Neurosurgery (Hagerstown, Md.) Jan 2021The cerebellum is one of the most primitive and complex parts of the human brain. The fiber microdissection technique can be extremely useful for neurosurgeons to...
BACKGROUND
The cerebellum is one of the most primitive and complex parts of the human brain. The fiber microdissection technique can be extremely useful for neurosurgeons to understand the topographical organization of the cerebellum's important contents, such as the deep cerebellar nuclei and the cerebellar peduncles, and their relationship with the brain stem.
OBJECTIVE
To dissect the deep cerebellar nuclei and the cerebellar peduncles using the fiber microdissection technique.
METHODS
Under the operating microscope, 5 previously frozen, formalin-fixed human cerebellums and brain stems were dissected from the superior surface, and 5 were dissected from the inferior surface. Each stage of the process is described. The primary dissection tools were handmade, thin, wooden spatulas with tips of various sizes, toothpicks, and a fine regulated suction.
RESULTS
In 15 simplified dissection steps (6 for the superior surface and 9 for the inferior surface), the deep cerebellar nuclei (dentate, interpositus, and fastigial) and the cerebellar peduncles (inferior, middle, and superior) are delineated. Their anatomical relationships with each other and other neighboring structures are demonstrated.
CONCLUSION
The anatomy of the deep cerebellar nuclei and the cerebellar peduncles are clearly defined and understood through the use of the fiber microdissection technique. These stepwise dissections will guide the neurosurgeon in acquiring a topographical understanding of these complex and deep structures of the cerebellum. This knowledge, along with radiological information, can help in planning the most appropriate surgical strategy for various lesions of the cerebellum.
Topics: Brain Stem; Cerebellar Nuclei; Cerebellum; Humans; Microdissection; White Matter
PubMed: 33047123
DOI: 10.1093/ons/opaa318 -
Current Protocols Jul 2023To study the transcriptome of individual plant cells at specific points in time, we developed protocols for fixation, embedding, and sectioning of plant tissue followed...
To study the transcriptome of individual plant cells at specific points in time, we developed protocols for fixation, embedding, and sectioning of plant tissue followed by laser capture microdissection (LCM) and processing for RNA recovery. LCM allows the isolation of individual cell types from heterogeneous tissue sections and is particularly suited to plant processing because it does not require the breakdown of cell walls. This approach allows accurate separation of a small volume of cells that can be used to study gene expression profiles in different tissues or cell layers. The technique requires neither separation of cells by enzymatic digestion of any kind nor cell-specific reporter genes, and it allows storage of fixed and embedded tissue for months before capture. The methods for fixation, embedding, sectioning, and capturing of plant cells that we describe yield high-quality RNA suitable for making libraries for RNASeq. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Tissue Preparation for Laser Capture Microdissection Basic Protocol 2: Tissue Sectioning Basic Protocol 3: Laser Capture Microdissection of Embedded Tissue Basic Protocol 4: RNA Extraction from Laser Capture Microdissection Samples.
Topics: Laser Capture Microdissection; RNA; Plants; Transcriptome; Paraffin Embedding
PubMed: 37486164
DOI: 10.1002/cpz1.844 -
Microdissection Methods Utilizing Single-Cell Subtype Analysis and the Impact on Precision Medicine.Methods in Molecular Biology (Clifton,... 2022Improving the utilization of tumor tissue from diagnostic biopsies is an unmet medical need. This is especially relevant today in the rapidly evolving precision oncology...
Improving the utilization of tumor tissue from diagnostic biopsies is an unmet medical need. This is especially relevant today in the rapidly evolving precision oncology field where tumor genotyping is often essential for the indication of many advanced and targeted therapies. National Comprehensive Cancer Network (NCCN) guidelines now mandate molecular testing for clinically actionable targets in certain malignancies. Utilizing advanced stage lung cancer as an example, an improved genotyping approach for solid tumors is possible. The strategy involves optimization of the microdissection process and analysis of a large number of identical target cells from formalin-fixed paraffin-embedded (FFPE) specimens sharing similar characteristics, in other words, single-cell subtype analysis. The shared characteristics can include immunostaining status, cell phenotype, and/or spatial location within a histological section. Synergy between microdissection and droplet digital PCR (ddPCR) enhances the molecular analysis. We demonstrate here a methodology that illustrates genotyping of a solid tumor from a small tissue biopsy sample in a time- and cost-efficient manner, using immunostain targeting as an example.
Topics: Formaldehyde; Humans; Microdissection; Neoplasms; Paraffin Embedding; Polymerase Chain Reaction; Precision Medicine; Tissue Fixation
PubMed: 35094324
DOI: 10.1007/978-1-0716-1811-0_7 -
Oral Diseases Sep 2017Laser capture microdissection (LCM) is a high-end research and diagnostic technology that helps in obtaining pure cell populations for the purpose of cell- or... (Review)
Review
Laser capture microdissection (LCM) is a high-end research and diagnostic technology that helps in obtaining pure cell populations for the purpose of cell- or lesion-specific genomic and proteomic analysis. Literature search on the application of LCM in oral tissues was made through PubMed. There is ample evidence to substantiate the utility of LCM in understanding the underlying molecular mechanism involving an array of oral physiological and pathological processes, including odontogenesis, taste perception, eruptive tooth movement, oral microbes, and cancers of the mouth and jaw tumors. This review is aimed at exploring the potential application of LCM in oral tissues as a high-throughput tool for integrated oral sciences. The indispensable application of LCM in the construction of lesion-specific genomic libraries with emphasis on some of the novel molecular markers thus discovered is also highlighted.
Topics: Genomics; Humans; Laser Capture Microdissection; Mouth; Mouth Diseases; Proteomics; Specimen Handling
PubMed: 27580277
DOI: 10.1111/odi.12578