Light microscopy digital imaging.
Authors: Joubert J, Sharma D
Abstract
This unit presents an overview of digital imaging hardware used in light microscopy. CMOS, CCD, and EMCCDs are the primary sensors used. The strengths and weaknesses of each define the primary applications for these sensors. Sensor architecture and formats are also reviewed. Color camera design strategies and sensor window cleaning are also described in the unit.
PMID: 21965107 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cytometry)
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Assessing mitochondrial redox status by flow cytometric methods: vascular response to fluid shear stress.
Authors: Li R, Jen N, Yu F, Hsiai TK
Abstract
Mitochondria are an important source of superoxide production contributing to physiological and pathological responses, including vascular oxidative stress that is relevant to cardiovascular diseases. Vascular oxidative stress is intimately linked with pro-inflammatory states and atherosclerosis. Oxidized low-density lipoprotein (OxLDL) modulates intracellular redox status and induces apoptosis in endothelial cells. Hemodynamic, specifically, fluid shear stress imparts both biomechanical and metabolic effects on vasculature. Mitochondria are an important source...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Practical Methods for Molecular In Vivo Optical Imaging.
Authors: Chen H, Thorne SH
Abstract
Traditional approaches for translating observations of molecular events into the context of a living organism have suffered from the requirements for either sacrificing animals at multiple time points prior to labor-intensive analyses of multiple tissues, or have relied on subjective observations or measurements of the animals over time. Recently an explosion of dedicated animal imaging modalities and the release of modified clinical imaging devices dedicated for animal imaging have allowed for the design of quantitative real time experiments incorporating fewer animals ...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Capture of Fluorescence Decay Times by Flow Cytometry.
Authors: Houston JP, Naivar MA, Jenkins P, Freyer JP
Abstract
In flow cytometry, the fluorescence decay time of an excitable species has been largely underutilized and is not likely found as a standard parameter on any imaging cytometer, sorting, or analyzing system. Most cytometers lack fluorescence lifetime hardware mainly owing to two central issues. Foremost, research and development with lifetime techniques has lacked proper exploitation of modern laser systems, data acquisition boards, and signal processing techniques. Secondly, a lack of enthusiasm for fluorescence lifetime applications in cells and...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Fountain flow cytometry.
Authors: Johnson P
Abstract
Fountain Flow Cytometry (FFC) is a simple and inexpensive technology that is adaptable to situations requiring detection and enumeration of cells/organisms at low concentrations, but is limited to particles of relatively high fluorescence intensity. This work presents the basic physics behind the novel scheme Fountain Flow Cytometry employs for the detection of target particles, a hybrid of conventional flow cytometry and video epifluorescence microscopy. The method is based on LED-induced fluorescence of labeled particles and requires no filtration step. Unlike conventional flo...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Two-photon imaging of the immune system.
Authors: Dzhagalov IL, Melichar HJ, Ross JO, Herzmark P, Robey EA
Abstract
Two-photon microscopy is a powerful method for visualizing biological processes as they occur in their native environment in real time. The immune system uniquely benefits from this technology as most of its constituent cells are highly motile and interact extensively with each other and with the environment. Two-photon microscopy has provided many novel insights into the dynamics of the development and function of the immune system that could not have been deduced by other methods and has become an indispensible tool in the arsenal...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Near-infrared molecular probes for in vivo imaging.
Authors: Zhang X, Bloch S, Akers W, Achilefu S
Abstract
Cellular and tissue imaging in the near-infrared (NIR) wavelengths between 700 and 900 nm is advantageous for in vivo imaging because of the low absorption of biological molecules in this region. This unit presents protocols for small animal imaging using planar and fluorescence lifetime imaging techniques. Included is an overview of NIR fluorescence imaging of cells and small animals using NIR organic fluorophores, nanoparticles, and multimodal imaging probes. The development, advantages, and application of NIR fluorescent probes that have been used ...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Live imaging of the lung.
Authors: Thornton EE, Krummel MF, Looney MR
Abstract
Live imaging is critical to determining the dynamics and spatial interactions of cells within the tissue environment. In the lung, this has proven to be difficult due to the motion incurred by ventilation and cardiac contractions. In this chapter, we report protocols for imaging ex vivo live lung slices and the intact mouse lung.
PMID: 22470155 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cytometry)
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Preparing a Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) compliant manuscript using the International Society for Advancement of Cytometry (ISAC) FCS file repository (FlowRepository.org).
Authors: Spidlen J, Breuer K, Brinkman R
Abstract
FlowRepository.org is a Web-based flow cytometry data repository provided by the International Society for Advancement of Cytometry (ISAC). It supports storage, annotation, analysis, and sharing of flow cytometry datasets. A fundamental tenet of scientific research is that published results should be open to independent validation and refutation. With FlowRepository, researchers can annotate their datasets in compliance with the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard, thus greatly facilitating third-party interpretation o...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Total internal reflection fluorescence (TIRF) microscopy illuminator for improved imaging of cell surface events.
Authors: Johnson DS, Jaiswal JK, Simon S
Abstract
Total internal reflection fluorescence (TIRF) microscopy is a high-contrast imaging technique suitable for observing biological events that occur on or near the cell membrane. The improved contrast is accomplished by restricting the thickness of the excitation field to over an order of a magnitude narrower than the z-resolution of an epi-fluorescence microscope. This technique also increases signal-to-noise, making it a valuable tool for imaging cellular events such as vesicles undergoing exocytosis or endocytosis, viral particle formation, cell signaling, ...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Three-dimensional second-harmonic generation imaging of fibrillar collagen in biological tissues.
Authors: Xie J, Ferbas J, Juan G
Abstract
Multiphoton-induced second-harmonic generation (SHG) has developed into a very powerful approach for in depth visualization of some biological structures with high specificity. In this unit, we describe the basic principles of three-dimensional SHG microscopy. In addition, we illustrate how SHG imaging can be utilized to assess collagen fibrils in biological tissues. Some technical considerations are also addressed.
PMID: 22752952 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cytometry)
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Ex vivo imaging of excised tissue using vital dyes and confocal microscopy.
Authors: Johnson S, Rabinovitch P
Abstract
Vital dyes routinely used for staining cultured cells can also be used to stain and image live tissue slices ex vivo. Staining tissue with vital dyes allows researchers to collect structural and functional data simultaneously and can be used for qualitative or quantitative fluorescent image collection. The protocols presented here are useful for structural and functional analysis of viable properties of cells in intact tissue slices, allowing for the collection of data in a structurally relevant environment. With these protocols, vital dyes can be applied as a res...
Source: Current Protocols in Cytometry - November 20, 2015 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 - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Super-resolution microscopy: a comparative treatment.
Authors: Kasuboski JM, Sigal YJ, Joens MS, Lillemeier BF, Fitzpatrick JA
Abstract
One of the fundamental limitations of optical microscopy is that of diffraction, or in essence, how small a beam of light can be focused by using an optical lens system. This constraint, or barrier if you will, was theoretically described by Ernst Abbe in 1873 and is roughly equal to half the wavelength of light used to probe the system. Many structures, particularly those within cells, are much smaller than this limit and thus are difficult to visualize. Over the last two decades, a new field of super-resolution imaging has ...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Quantitative fluorescent speckle microscopy (QFSM) to measure actin dynamics.
Authors: Mendoza MC, Besson S, Danuser G
Abstract
Quantitative fluorescent speckle microscopy (QFSM) is a live-cell imaging method to analyze the dynamics of macromolecular assemblies with high spatial and temporal resolution. Its greatest successes were in the analysis of actin filament and adhesion dynamics in the context of cell migration and microtubule dynamics in interphase and the meiotic/mitotic spindle. Here, focus is on the former application to illustrate the procedures of FSM imaging and the computational image processing that extracts quantitative information from these experiments. QFSM is ad...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
