-
Nature Biotechnology Aug 2022Despite the availabilty of imaging-based and mass-spectrometry-based methods for spatial proteomics, a key challenge remains connecting images with...
Despite the availabilty of imaging-based and mass-spectrometry-based methods for spatial proteomics, a key challenge remains connecting images with single-cell-resolution protein abundance measurements. Here, we introduce Deep Visual Proteomics (DVP), which combines artificial-intelligence-driven image analysis of cellular phenotypes with automated single-cell or single-nucleus laser microdissection and ultra-high-sensitivity mass spectrometry. DVP links protein abundance to complex cellular or subcellular phenotypes while preserving spatial context. By individually excising nuclei from cell culture, we classified distinct cell states with proteomic profiles defined by known and uncharacterized proteins. In an archived primary melanoma tissue, DVP identified spatially resolved proteome changes as normal melanocytes transition to fully invasive melanoma, revealing pathways that change in a spatial manner as cancer progresses, such as mRNA splicing dysregulation in metastatic vertical growth that coincides with reduced interferon signaling and antigen presentation. The ability of DVP to retain precise spatial proteomic information in the tissue context has implications for the molecular profiling of clinical samples.
Topics: Humans; Laser Capture Microdissection; Mass Spectrometry; Melanoma; Proteome; Proteomics
PubMed: 35590073
DOI: 10.1038/s41587-022-01302-5 -
Nature Neuroscience Aug 2021The human neonatal cerebellum is one-fourth of its adult size yet contains the blueprint required to integrate environmental cues with developing motor, cognitive and...
The human neonatal cerebellum is one-fourth of its adult size yet contains the blueprint required to integrate environmental cues with developing motor, cognitive and emotional skills into adulthood. Although mature cerebellar neuroanatomy is well studied, understanding of its developmental origins is limited. In this study, we systematically mapped the molecular, cellular and spatial composition of human fetal cerebellum by combining laser capture microscopy and SPLiT-seq single-nucleus transcriptomics. We profiled functionally distinct regions and gene expression dynamics within cell types and across development. The resulting cell atlas demonstrates that the molecular organization of the cerebellar anlage recapitulates cytoarchitecturally distinct regions and developmentally transient cell types that are distinct from the mouse cerebellum. By mapping genes dominant for pediatric and adult neurological disorders onto our dataset, we identify relevant cell types underlying disease mechanisms. These data provide a resource for probing the cellular basis of human cerebellar development and disease.
Topics: Cerebellum; Fetus; Humans; Laser Capture Microdissection; Neurogenesis; Single-Cell Analysis; Transcriptome
PubMed: 34140698
DOI: 10.1038/s41593-021-00872-y -
Nature Methods Oct 2023Single-cell proteomics by mass spectrometry is emerging as a powerful and unbiased method for the characterization of biological heterogeneity. So far, it has been...
Single-cell proteomics by mass spectrometry is emerging as a powerful and unbiased method for the characterization of biological heterogeneity. So far, it has been limited to cultured cells, whereas an expansion of the method to complex tissues would greatly enhance biological insights. Here we describe single-cell Deep Visual Proteomics (scDVP), a technology that integrates high-content imaging, laser microdissection and multiplexed mass spectrometry. scDVP resolves the context-dependent, spatial proteome of murine hepatocytes at a current depth of 1,700 proteins from a cell slice. Half of the proteome was differentially regulated in a spatial manner, with protein levels changing dramatically in proximity to the central vein. We applied machine learning to proteome classes and images, which subsequently inferred the spatial proteome from imaging data alone. scDVP is applicable to healthy and diseased tissues and complements other spatial proteomics and spatial omics technologies.
Topics: Animals; Mice; Proteome; Mass Spectrometry; Proteomics; Laser Capture Microdissection
PubMed: 37783884
DOI: 10.1038/s41592-023-02007-6 -
Kidney International Apr 2019Bulk-tissue RNA-Seq is increasingly being used in the study of physiological and pathophysiological processes in the kidney; however, the presence of multiple cell types...
Bulk-tissue RNA-Seq is increasingly being used in the study of physiological and pathophysiological processes in the kidney; however, the presence of multiple cell types in kidney tissue complicates data interpretation. We addressed the question of which cell types are represented in whole-kidney RNA-Seq data in order to identify circumstances in which bulk-kidney RNA-Seq can be successfully interpreted. We carried out RNA-Seq in mouse whole kidneys and in microdissected renal tubule segments. To aid in the interpretation of the data, we compiled a database of cell-type selective protein markers for 43 cell types believed to be present in kidney tissue. The whole-kidney RNA-Seq analysis identified transcripts corresponding to 17,742 genes, distributed over 5 orders of magnitude of expression level. Markers for all 43 curated cell types were detectable. Analysis of the cellular makeup of mouse and rat kidney, calculated from published literature, suggests that proximal tubule cells account for more than half of the mRNA in a kidney. Comparison of RNA-Seq data from microdissected proximal tubules with data from whole kidney supports this view. RNA-Seq data for cell-type selective markers in bulk-kidney samples provide a valid means to identify changes in minority-cell abundances in kidney tissue. Because proximal tubules make up a substantial fraction of whole-kidney samples, changes in proximal tubule gene expression can be assessed presumptively by bulk-kidney RNA-Seq, although results could potentially be complicated by the presence of mRNA from other cell types.
Topics: Animals; Biomarkers; Gene Expression Profiling; Kidney; Mice; Microdissection; RNA, Messenger; RNA-Seq; Rats; Transcriptome
PubMed: 30826016
DOI: 10.1016/j.kint.2018.11.028 -
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 -
Methods in Molecular Biology (Clifton,... 2011Over the past 15 years, laser-based microdissection has improved the precision by which scientists can procure cells of interest from a heterogeneous tissue section....
Over the past 15 years, laser-based microdissection has improved the precision by which scientists can procure cells of interest from a heterogeneous tissue section. However, for studies that require a large amount of material (e.g., proteomics) or for cells that are scattered and difficult to identify by standard histological stains, an immunostain-based, automated approach becomes essential. In this chapter, we discuss the use of immunohistochemistry (IHC) and immunofluorescence (IF) to guide the microdissection process via manual and software-driven auto-dissection methods. Although technical challenges still exist with these innovative approaches, we present here methods and protocols to successfully perform immuno-based microdissection on commercially available laser dissection systems.
Topics: Cell Separation; Cryopreservation; Humans; Image Processing, Computer-Assisted; Immunohistochemistry; Keratins; Lasers; Male; Microdissection; Paraffin Embedding; Prostate; Software; Tissue Fixation
PubMed: 21761293
DOI: 10.1007/978-1-61779-163-5_4 -
Asian Journal of Andrology Jan 2013Patients with non-obstructive azoospermia (NOA) were once considered to be infertile with few treatment options due to the absence of sperm in the ejaculate. In the last... (Review)
Review
Patients with non-obstructive azoospermia (NOA) were once considered to be infertile with few treatment options due to the absence of sperm in the ejaculate. In the last two decades, the advent of intracytoplasmic sperm injection (ICSI), and the application of various testicular sperm retrieval techniques, including fine needle aspiration (FNA), conventional testicular sperm extraction (TESE) and microdissection testicular sperm extraction (micro-TESE) have revolutionized treatment in this group of men. Because most men with NOA will have isolated regions of spermatogenesis within the testis, studies have illustrated that sperm can be retrieved in most men with NOA, including Klinefelter's syndrome (KS), prior history of chemotherapy and cryptorchidism. Micro-TESE, when compared with conventional TESE has a higher sperm retrieval rate (SRR) with fewer postoperative complications and negative effects on testicular function. In this article, we will compare the efficacy of the different procedures of sperm extraction, discuss the medical treatment and the role of testosterone optimization in men with NOA and describe the micro-TESE surgical technique. Furthermore, we will update our overall experience to allow counseling on the prognosis of sperm retrieval for the specific subsets of NOA.
Topics: Azoospermia; Biopsy; Humans; Klinefelter Syndrome; Male; Microdissection; Sperm Injections, Intracytoplasmic; Sperm Retrieval; Testosterone
PubMed: 23241638
DOI: 10.1038/aja.2012.141 -
Neurosurgery Jan 2009Proteins are the primary components of cells and are vital constituents of any living organism. The proteins that make up an organism (proteome) are constantly changing... (Review)
Review
OBJECTIVE
Proteins are the primary components of cells and are vital constituents of any living organism. The proteins that make up an organism (proteome) are constantly changing and are intricately linked to neurological disease processes. The study of proteins, or proteomics, is a relatively new but rapidly expanding field with increasing relevance to neurosurgery.
METHODS
We present a review of the state-of-the-art proteomic technology and its applications in central nervous system diseases.
RESULTS
The technique of "selective microdissection" allows an investigator to selectively isolate and study a pathological tissue of interest. By evaluating protein expression in a variety of central nervous system disorders, it is clear that proteins are differentially expressed across disease states, and protein expression changes markedly during disease progression.
CONCLUSION
Understanding the patterns of protein expression in the nervous system has critical implications for the diagnosis and treatment of neurological disease. As gatekeepers in the diagnosis, evaluation, and treatment of central nervous system diseases, it is important for neurosurgeons to develop an appreciation for proteomic techniques and their utility.
Topics: Central Nervous System Diseases; Humans; Microdissection; Molecular Diagnostic Techniques; Proteomics
PubMed: 19145153
DOI: 10.1227/01.NEU.0000335776.93176.83 -
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 -
Histology and Histopathology Nov 2015Any tissue is made up of a heterogeneous mix of spatially distributed cell types. In response to any (patho) physiological cue, responses of each cell type in any given... (Review)
Review
Any tissue is made up of a heterogeneous mix of spatially distributed cell types. In response to any (patho) physiological cue, responses of each cell type in any given tissue may be unique and cannot be homogenized across cell-types and spatial co-ordinates. For example, in response to myocardial infarction, on one hand myocytes and fibroblasts of the heart tissue respond differently. On the other hand, myocytes in the infarct core respond differently compared to those in the peri-infarct zone. Therefore, isolation of pure targeted cells is an important and essential step for the molecular analysis of cells involved in the progression of disease. Laser capture microdissection (LCM) is powerful to obtain a pure targeted cell subgroup, or even a single cell, quickly and precisely under the microscope, successfully tackling the problem of tissue heterogeneity in molecular analysis. This review presents an overview of LCM technology, the principles, advantages and limitations and its down-stream applications in the fields of proteomics, genomics and transcriptomics. With powerful technologies and appropriate applications, this technique provides unprecedented insights into cell biology from cells grown in their natural tissue habitat as opposed to those cultured in artificial petri dish conditions.
Topics: Animals; Biomarkers; Cell Separation; Gene Expression Profiling; Genetic Markers; Genomics; Humans; Laser Capture Microdissection; Proteomics
PubMed: 25892148
DOI: 10.14670/HH-11-622