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International Journal of Legal Medicine Nov 2010In forensic sciences, short tandem repeat (STR) analysis has become the prime tool for DNA-based identification of the donor(s) of biological stains and/or traces. Many... (Review)
Review
In forensic sciences, short tandem repeat (STR) analysis has become the prime tool for DNA-based identification of the donor(s) of biological stains and/or traces. Many traces, however, contain cells and, hence, DNA, from more than a single individual, giving rise to mixed genotypes and the subsequent difficulties in interpreting the results. An even more challenging situation occurs when cells of a victim are much more abundant than the cells of the perpetrator. Therefore, the forensic community seeks to improve cell-separation methods in order to generate single-donor cell populations from a mixed trace in order to facilitate DNA typing and identification. Laser capture microdissection (LCM) offers a valuable tool for precise separation of specific cells. This review summarises all possible forensic applications of LCM, gives an overview of the staining and detection options, including automated detection and retrieval of cells of interest, and reviews the DNA extraction protocols compatible with LCM of cells from forensic samples.
Topics: Biometric Identification; Cell Separation; DNA Fingerprinting; Equipment Design; Female; Flow Cytometry; Forensic Pathology; Humans; Lasers; Male; Microdissection; Research Design; Semen; Sex Offenses; Spermatozoa; Violence
PubMed: 20680318
DOI: 10.1007/s00414-010-0499-4 -
Scientific Reports Dec 2017The blood-nerve barrier (BNB), formed by tight junction-forming microvessels within peripheral nerve endoneurium, exists to regulate its internal microenvironment...
The blood-nerve barrier (BNB), formed by tight junction-forming microvessels within peripheral nerve endoneurium, exists to regulate its internal microenvironment essential for effective axonal signal transduction. Relatively little is known about the unique human BNB molecular composition. Such knowledge is crucial to comprehend the relationships between the systemic circulation and peripheral nerves in health, adaptations to intrinsic or extrinsic perturbations and alterations that may result in disease. We performed RNA-sequencing on cultured early- and late-passage adult primary human endoneurial endothelial cells and laser-capture microdissected endoneurial microvessels from four cryopreserved normal adult human sural nerves referenced to the Genome Reference Consortium Human Reference 37 genome browser, using predefined criteria guided by known transcript or protein expression in vitro and in situ. We identified 12881 common transcripts associated by 125 independent biological networks, defined as the normal adult BNB transcriptome, including a comprehensive array of transporters and specialized intercellular junctional complex components. These identified transcripts and their interacting networks provide insights into peripheral nerve microvascular morphogenesis, restrictive barrier formation, influx and efflux transporters with relevance to understanding peripheral nerve homeostasis and pharmacology, including targeted drug delivery and the mediators of leukocyte trafficking in peripheral nerves during normal immunosurveillance.
Topics: Adult; Blood-Nerve Barrier; Cells, Cultured; Endothelial Cells; Female; Gene Expression Profiling; Humans; Laser Capture Microdissection; Male; Middle Aged; Primary Cell Culture; Sciatic Nerve; Sequence Analysis, RNA; Sural Nerve; Transcriptome
PubMed: 29234067
DOI: 10.1038/s41598-017-17475-y -
Lab on a Chip Jul 2022Spatial proteomics holds great promise for revealing tissue heterogeneity in both physiological and pathological conditions. However, one significant limitation of most...
Spatial proteomics holds great promise for revealing tissue heterogeneity in both physiological and pathological conditions. However, one significant limitation of most spatial proteomics workflows is the requirement of large sample amounts that blurs cell-type-specific or microstructure-specific information. In this study, we developed an improved sample preparation approach for spatial proteomics and integrated it with our previously-established laser capture microdissection (LCM) and microfluidics sample processing platform. Specifically, we developed a hanging drop (HD) method to improve the sample recovery by positioning a nanowell chip upside-down during protein extraction and tryptic digestion steps. Compared with the commonly-used sitting-drop method, the HD method keeps the tissue pixel away from the container surface, and thus improves the accessibility of the extraction/digestion buffer to the tissue sample. The HD method can increase the MS signal by 7 fold, leading to a 66% increase in the number of identified proteins. An average of 721, 1489, and 2521 proteins can be quantitatively profiled from laser-dissected 10 μm-thick mouse liver tissue pixels with areas of 0.0025, 0.01, and 0.04 mm, respectively. The improved system was further validated in the study of cell-type-specific proteomes of mouse uterine tissues.
Topics: Animals; Laser Capture Microdissection; Mice; Proteome; Proteomics; Specimen Handling; Workflow
PubMed: 35838077
DOI: 10.1039/d2lc00384h -
Alcohol Research & Health : the Journal... 2008When analyzing alcohol's effects on the brain, researchers often want to look at small clusters of cells that can be studied in isolation from the surrounding brain... (Review)
Review
When analyzing alcohol's effects on the brain, researchers often want to look at small clusters of cells that can be studied in isolation from the surrounding brain tissue rather than at the entire brain or larger brain areas. This implies that relatively small numbers of cells have to be retrieved from the brain and studied in culture or subjected to biochemical analyses. The challenge then becomes how to isolate small numbers of cells from a specific brain region without including unwanted cells. One approach to solving this problem is to use a technology known as laser-assisted microdissection (LMD). This article reviews some of the principles of LMD and its use in alcohol research.
Topics: Animals; Brain; Gene Expression Profiling; Humans; Laser Capture Microdissection; Neurons; Substance-Related Disorders
PubMed: 23584869
DOI: No ID Found -
Molecular & Cellular Proteomics : MCP Dec 2022Despite their diminutive size, islets of Langerhans play a large role in maintaining systemic energy balance in the body. New technologies have enabled us to go from...
Despite their diminutive size, islets of Langerhans play a large role in maintaining systemic energy balance in the body. New technologies have enabled us to go from studying the whole pancreas to isolated whole islets, to partial islet sections, and now to islet substructures isolated from within the islet. Using a microfluidic nanodroplet-based proteomics platform coupled with laser capture microdissection and field asymmetric waveform ion mobility spectrometry, we present an in-depth investigation of protein profiles specific to features within the islet. These features include the islet-acinar interface vascular tissue, inner islet vasculature, isolated endocrine cells, whole islet with vasculature, and acinar tissue from around the islet. Compared to interface vasculature, unique protein signatures observed in the inner vasculature indicate increased innervation and intra-islet neuron-like crosstalk. We also demonstrate the utility of these data for identifying localized structure-specific drug-target interactions using existing protein/drug binding databases.
Topics: Islets of Langerhans; Proteomics; Proteins; Laser Capture Microdissection
PubMed: 36244662
DOI: 10.1016/j.mcpro.2022.100426 -
Expert Review of Proteomics Oct 2021Laser Capture Microdissection (LCM) uses a laser to isolate, or capture, specific cells of interest in a complex heterogeneous tissue section, under direct microscopic... (Review)
Review
INTRODUCTION
Laser Capture Microdissection (LCM) uses a laser to isolate, or capture, specific cells of interest in a complex heterogeneous tissue section, under direct microscopic visualization. Recently, there has been a surge of publications using LCM for tissue spatial molecular profiling relevant to a wide range of research topics.
AREAS COVERED
We summarize the many advances in tissue Laser Capture Proteomics (LCP) using mass spectrometry for discovery, and protein arrays for signal pathway network mapping. This review emphasizes: a) transition of LCM phosphoproteomics from the lab to the clinic for individualized cancer therapy, and b) the emerging frontier of LCM single cell molecular analysis combining proteomics with genomic, and transcriptomic analysis. The search strategy was based on the combination of MeSH terms with expert refinement.
EXPERT OPINION
LCM is complemented by a rich set of instruments, methodology protocols, and analytical A.I. (artificial intelligence) software for basic and translational research. Resolution is advancing to the tissue single cell level. A vision for the future evolution of LCM is presented. Emerging LCM technology is combining digital and AI guided remote imaging with automation, and telepathology, to a achieve multi-omic profiling that was not previously possible.
Topics: Artificial Intelligence; Laser Capture Microdissection; Lasers; Precision Medicine; Proteomics
PubMed: 34607525
DOI: 10.1080/14789450.2021.1984886 -
Hypertension (Dallas, Tex. : 1979) Oct 2020
Topics: Denervation; Humans; Hypertension; Kidney; Microdissection; Nervous System
PubMed: 32903105
DOI: 10.1161/HYPERTENSIONAHA.120.15834 -
Journal of Medicine and Life Jan 2023A limited number of individuals with non-obstructive azoospermia (NOA) may recover spermatozoa through traditional testicular sperm extraction (TESE) techniques. There...
A limited number of individuals with non-obstructive azoospermia (NOA) may recover spermatozoa through traditional testicular sperm extraction (TESE) techniques. There is an ongoing debate over the effectiveness of microdissection TESE compared to standard TESE methods. Microdissection TESE (micro-TESE) techniques enable the identification of spermatogenesis foci in non-obstructive forms of azoospermia. Only histological examination can provide an objective and definitive assessment of the testicular phenotype. This study aimed to evaluate the correlation between histopathological findings after microdissection TESE (micro-TESE) and the predictive role of various factors in determining the success of sperm retrieval. We evaluated 24 patients with azoospermia who underwent micro-TESE and considered the patient's hormonal profile, testis ultrasound, genetic evaluation, histology, and immunohistology (PLAP antibody) of collected testis biopsies. The preoperative blood FSH level, in conjunction with other parameters, may aid in the prediction of micro-TESE success. Sensitivity increases, and specificity decreases with higher FSH levels. Furthermore, testicular volume and FSH levels are typically normal in patients with maturation arrest. In conclusion, hormones, ultrasound evaluation of the testicles, testis volume, and available genetic tests have a predictive value in differentiating obstructive azoospermia (OA) from NOA with various sensitivity and specificity rates. Histological and immunohistochemical evaluation establishes the testicular phenotype accurately and guides patient management.
Topics: Male; Humans; Testis; Azoospermia; Microdissection; Semen; Spermatozoa; Follicle Stimulating Hormone
PubMed: 36873127
DOI: 10.25122/jml-2022-0336 -
Journal of Visualized Experiments : JoVE Nov 2019Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such...
Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.
Topics: Animals; Cochlea; Epithelium; Hair Cells, Auditory; Hair Cells, Auditory, Inner; Mice; Microdissection; Organ of Corti
PubMed: 31762458
DOI: 10.3791/60299 -
Journal of Biomolecular Techniques : JBT Sep 2010An important need of many cancer research projects is the availability of high-quality, appropriately selected tissue. Tissue biorepositories are organized to collect,... (Review)
Review
An important need of many cancer research projects is the availability of high-quality, appropriately selected tissue. Tissue biorepositories are organized to collect, process, store, and distribute samples of tumor and normal tissue for further use in fundamental and translational cancer research. This, in turn, provides investigators with an invaluable resource of appropriately examined and characterized tissue specimens and linked patient information. Human tissues, in particular, tumor tissues, are complex structures composed of heterogeneous mixtures of morphologically and functionally distinct cell types. It is essential to analyze specific cell types to identify and define accurately the biologically important processes in pathologic lesions. Laser capture microdissection (LCM) is state-of-the-art technology that provides the scientific community with a rapid and reliable method to isolate a homogeneous population of cells from heterogeneous tissue specimens, thus providing investigators with the ability to analyze DNA, RNA, and protein accurately from pure populations of cells. This is particularly well-suited for tumor cell isolation, which can be captured from complex tissue samples. The combination of LCM and a tissue biorepository offers a comprehensive means by which researchers can use valuable human biospecimens and cutting-edge technology to facilitate basic, translational, and clinical research. This review provides an overview of LCM technology with an emphasis on the applications of LCM in the setting of a tissue biorepository, based on the author's extensive experience in LCM procedures acquired at Fox Chase Cancer Center and Hollings Cancer Center.
Topics: DNA; Humans; Lasers; Microdissection; Proteins; RNA; Specimen Handling
PubMed: 20808641
DOI: No ID Found