Fundamentals of Acoustic Cytometry.
Authors: Ward MD, Kaduchak G
Abstract
Acoustic cytometry uses radiation pressure forces instead of or in addition to hydrodynamic focusing to position cells or particles in a flowing stream for analysis. Commercial implementations to date combine both hydrodynamic and acoustic focusing together to enable high precision analysis of a broad dynamic range of volumetric sample input rates up to an order of magnitude higher than is practical with hydrodynamic focus alone. This capability allows great flexibility in reducing assay time or modifying or eliminating concentration requirements or concentration steps...
Source: Current Protocols in Cytometry - July 25, 2018 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Overview of Flow Cytometry and Microbiology.
Authors: Robinson JP
Abstract
Although in recent years flow cytometry has become commonplace in hematology and immunology laboratories, application of the technology to microbiology remains largely unrealized. This overview presents the historical background, discusses applications in various areas of the field, and speculates on the directions of future developments. The availability of high-quality methods should be a prime factor in convincing microbiologists that flow cytometry may have certain advantages over traditional methods and that it does indeed have much to contribute to microbiology. © 2018 ...
Source: Current Protocols in Cytometry - July 25, 2018 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Clearing for Deep Tissue Imaging.
This article describes four popular clearing protocols that are relevant to a wide variety of scenarios across biologic disciplines: CUBIC, CLARITY, 3DISCO, and SeeDB. © 2018 by John Wiley & Sons, Inc.
PMID: 30005145 [PubMed - as supplied by publisher] (Source: Current Protocols in Cytometry)
Source: Current Protocols in Cytometry - July 15, 2018 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Resolution of Viable and Membrane-Compromised Free Bacteria in Aquatic Environments by Flow Cytometry.
Authors: Grégori G, Denis M, Sgorbati S, Citterio S
Abstract
In aquatic environments, free heterotrophic bacteria play an extremely important role due to their high biomass, wide panel of metabolisms, and ubiquity, as well as the toxicity of certain species. This unit presents a nucleic-acid double-staining protocol (NADS) for flow cytometry that can distinguish fractions of viable, damaged, or membrane-compromised cells within the free-bacterial community. The NADS protocol is based on the simultaneous utilization of two nucleic acid stains-membrane-permeant SYBR Green and membrane-impermeant propidium i...
Source: Current Protocols in Cytometry - July 1, 2018 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Mitochondrial Subtype Identification and Characterization.
Authors: Daniele JR, Heydari K, Dillin A
Abstract
Healthy, functional mitochondria are central to many cellular and physiological phenomena, including aging, metabolism, and stress resistance. A key feature of healthy mitochondria is a high membrane potential (Δψ) or charge differential (i.e., proton gradient) between the matrix and inner mitochondrial membrane. Mitochondrial Δψ has been extensively characterized via flow cytometry of intact cells, which measures the average membrane potential within a cell. However, the characteristics of individual mitochondria differ dramatically even within a singl...
Source: Current Protocols in Cytometry - June 28, 2018 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Multiphoton Intravital Calcium Imaging.
Authors: Cheetham CEJ
Abstract
Multiphoton intravital calcium imaging is a powerful technique that enables high-resolution longitudinal monitoring of cellular and subcellular activity hundreds of microns deep in the living organism. This unit addresses the application of 2-photon microscopy to imaging of genetically encoded calcium indicators (GECIs) in the mouse brain. The protocols in this unit enable real-time intravital imaging of intracellular calcium concentration simultaneously in hundreds of neurons, or at the resolution of single synapses, as mice respond to sensory stimuli or perform behavioral t...
Source: Current Protocols in Cytometry - June 28, 2018 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Modern Laser Scanning Confocal Microscopy.
We describe the components of a CLSM system and assess how modern implementations of the approach have further expanded the use of the technique. Finally, we briefly outline some practical considerations to take into account when acquiring data using a CLSM system. © 2018 by John Wiley & Sons, Inc.
PMID: 29927100 [PubMed - as supplied by publisher] (Source: Current Protocols in Cytometry)
Source: Current Protocols in Cytometry - June 22, 2018 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Live-Animal Imaging of Renal Function by Multiphoton Microscopy.
Authors: Dunn KW, Sutton TA, Sandoval RM
Abstract
Intravital microscopy, microscopy of living animals, is a powerful research technique that combines the resolution and sensitivity found in microscopic studies of cultured cells with the relevance and systemic influences of cells in the context of the intact animal. The power of intravital microscopy has recently been extended with the development of multiphoton fluorescence microscopy systems capable of collecting optical sections from deep within the kidney at subcellular resolution, supporting high-resolution characterizations of the structure and functi...
Source: Current Protocols in Cytometry - January 19, 2018 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Basics of Digital Microscopy.
Authors: Wallace CT, Jessup M, Bernas T, Peña KA, Calderon MJ, Loughran PA
Abstract
Modern digital microscopy combines the equipment of classical light microscopy with a computerized imaging system. The technique comprises image formation by optics, image registration by a camera, and saving of image data in a computer file. This chapter describes limitations that are particular to each of these processes, including optical resolution, efficiency of image registration, characteristics of image file formats, and data management. Further suggestions are given which serve, in turn, to help construct a set of...
Source: Current Protocols in Cytometry - January 19, 2018 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Lasers for Flow Cytometry: Current and Future Trends.
Authors: Shapiro HM, Telford WG
Abstract
Lasers are the principal light sources for flow cytometers. Virtually all cytometers are equipped with at least one (and often many more) lasers. This unit covers the various types of lasers available and the qualities that make them suitable or unsuitable for use in flow cytometers. Also included is a discussion of future directions, particularly in the area of tunable laser development. Practical tips are provided for building multilaser cytometer systems. © 2018 by John Wiley & Sons, Inc.
PMID: 29345328 [PubMed - in process] (Source: Current Protocols in Cytometry)
Source: Current Protocols in Cytometry - January 19, 2018 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Generating Quantitative Cell Identity Labels with Marker Enrichment Modeling (MEM).
Authors: Diggins KE, Gandelman JS, Roe CE, Irish JM
Abstract
Multiplexed single-cell experimental techniques like mass cytometry measure 40 or more features and enable deep characterization of well-known and novel cell populations. However, traditional data analysis techniques rely extensively on human experts or prior knowledge, and novel machine learning algorithms may generate unexpected population groupings. Marker enrichment modeling (MEM) creates quantitative identity labels based on features enriched in a population relative to a reference. While developed for cell type analysis, MEM labels can be g...
Source: Current Protocols in Cytometry - January 19, 2018 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Non-Parametric Comparison of Single Parameter Histograms.
Authors: Wood JCS
Abstract
A number of methods have been developed to compare single parameter histograms. Some perform a channel-by-channel analysis and others give a single statistic about how the histograms may or may not differ. If they do differ, then the significance of the difference or confidence limit is usually provided. The specific location(s) for the greatest deviations may also be given. Some are more effective at resolving severely overlapping populations and others work poorly when there is any significant overlap. Each method makes certain assumptions about the data. It is important to und...
Source: Current Protocols in Cytometry - January 19, 2018 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Live-animal imaging of renal function by multiphoton microscopy.
Authors: Dunn KW, Sutton TA, Sandoval RM
Abstract
Intravital microscopy, microscopy of living animals, is a powerful research technique that combines the resolution and sensitivity found in microscopic studies of cultured cells with the relevance and systemic influences of cells in the context of the intact animal. The power of intravital microscopy has recently been extended with the development of multiphoton fluorescence microscopy systems capable of collecting optical sections from deep within the kidney at subcellular resolution, supporting high-resolution characterizations of the structure and functi...
Source: Current Protocols in Cytometry - October 17, 2017 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
CyTOF Measurement of Immunocompetence Across Major Immune Cell Types.
Authors: Subrahmanyam PB, Maecker HT
Abstract
The central role of the immune system is becoming appreciated in a wide variety of diseases. Cancer immunotherapy is one area that has yielded much recent success, although not all patients benefit equally. At the same time, recent studies have highlighted the heterogeneity of the human immune system. Despite this heterogeneity, we do not routinely measure immune competence in clinical practice, and there are no consensus assays of healthy immune function. Using mass cytometry (CyTOF), we can simultaneously detect ∼40 markers to identify various cell subsets ...
Source: Current Protocols in Cytometry - October 4, 2017 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Staining of Frozen and Formalin-Fixed, Paraffin-Embedded Tissues with Metal-Labeled Antibodies for Imaging Mass Cytometry Analysis.
Authors: Chang Q, Ornatsky O, Hedley D
Abstract
This unit describes protocols for labeling tissue sections using combinations of metal-tagged antibodies and an iridium-containing DNA intercalator for analysis by imaging mass cytometry. Imaging mass cytometry (IMC) allows the labeling of up to 40 individual markers simultaneously using antibody cocktails. We discuss labeling of both cryostat sections and sections from formalin-fixed, paraffin-embedded (FFPE) tissue blocks. The protocols are similar to those used for optical microscopy techniques, while allowing much higher complexity of analysis. © 2017 by...
Source: Current Protocols in Cytometry - October 4, 2017 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
