[2] Practical considerations in acquiring biological signals from confocal microscope

Kong, Siu Kai; Ko, Sandy S.; Lee, C.Y.; Lui, P.Y.

doi:10.1016/s0076-6879(99)07004-4

Cited by 3 publications

(2 citation statements)

References 11 publications

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“…Temperature control is also vital to maintain a constant focus. Small fluctuations in the temperature change the refractive index of the immersion oil, resulting in changes in the focal plane (27). Elevated temperatures increase the evaporation rate, which can quickly result in toxic salt concentrations.…”

Section: Microscopy Of Live and Fixed Cellsmentioning

confidence: 99%

The cytoplasmic escape and nuclear accumulation of endocytosed and microinjected HPMA copolymers and a basic kinetic study in hep G2 cells

et al. 2001

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The development of macromolecules as drugs and drug carriers requires knowledge of their fate in cells. To this end, we studied the internalization and subcellular fate of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers in Hep G2 (human hepatocellular carcinoma) cells. Semiquantitative fluorometry confirmed that galactose was an effective ligand for receptormediated endocytosis for Hep G2 cells. The rate of internalization of a galactose-targeted copolymer was almost 2 orders of magnitude larger than that of the nontargeted copolymer. Confocal fluorescent microscopy of both fixed and live cells revealed that the polymer entered the cells by endocytosis. After longer incubation times (typically >8 hours), polymer escaped from small vesicles and distributed throughout the cytoplasm and nuclei of the cells. Polymer that entered the cytoplasm tended to accumulate in the nucleus. Microinjection of the HPMA copolymers into cells' cytoplasm and nuclei indicated that the polymers partitioned to the nucleus. The data from fixed cells was confirmed by microscopy of live, viable cells. To examine the effect of the fluorescent dye on the intracellular fate, polymers with fluorescein, Oregon Green 488, Lissamine rhodamine B, and doxorubicin were tested; no significant differences were observed.

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Section: Microscopy Of Live and Fixed Cellsmentioning

confidence: 99%

The cytoplasmic escape and nuclear accumulation of endocytosed and microinjected HPMA copolymers and a basic kinetic study in hep G2 cells

et al. 2001

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“…This can happen in many different ways, including condensation of water on the inside surface of dye vials. Water contamination leads to labeling artifacts, especially clumps of intracellular fluorescence (Kong et al, 1999;Lee et al, 1996;Lui et al, 1997). To minimize contamination, fluorescent dyes should be dissolved in dry DMSO (Lee et al, 1996).…”

Section: Cells and Protocolsmentioning

confidence: 99%

Fluorescence microscopy: Established and emerging methods, experimental strategies, and applications in immunology

Petty

2007

Microscopy Res & Technique

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Cutting-edge biophysical technologies including total internal reflection fluorescence microscopy, single molecule fluorescence, single channel opening events, fluorescence resonance energy transfer, high-speed exposures, two-photon imaging, fluorescence lifetime imaging, and other tools are becoming increasingly important in immunology as they link molecular events to cellular physiology, a key goal of modern immunology. The primary concern in all forms of microscopy is the generation of contrast; for fluorescence microscopy contrast can be thought of as the difference in intensity between the cell and background, the signal-to-noise ratio. High information-content images can be formed by enhancing the signal, suppressing the noise, or both. As improved tools, such as ICCD and EMCCD cameras, become available for fluorescence imaging in molecular and cellular immunology, it is important to optimize other aspects of the imaging system. Numerous practical strategies to enhance fluorescence microscopy experiments are reviewed. The use of instrumentation such as light traps, cameras, objectives, improved fluorescent labels, and image filtration routines applicable to low light level experiments are discussed. New methodologies providing resolution well beyond that given by the Rayleigh criterion are outlined. Ongoing and future developments in fluorescence microscopy instrumentation and technique are reviewed. This review is intended to address situations where the signal is weak, which is important for emerging techniques stressing super-resolution or live cell dynamics, but is less important for conventional applications such as indirect immunofluorescence. This review provides a broad integrative discussion of fluorescence microscopy with selected applications in immunology. Microsc. Res. Tech. 70:687-709, 2007. V

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Recent advances in quantitative colocalization analysis: Focus on neuroscience

Zinchuk

Grossenbacher-Zinchuk

2009

Progress in Histochemistry and Cytochemistry

157

137

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[2] Practical considerations in acquiring biological signals from confocal microscope

Cited by 3 publications

References 11 publications

The cytoplasmic escape and nuclear accumulation of endocytosed and microinjected HPMA copolymers and a basic kinetic study in hep G2 cells

The cytoplasmic escape and nuclear accumulation of endocytosed and microinjected HPMA copolymers and a basic kinetic study in hep G2 cells

Fluorescence microscopy: Established and emerging methods, experimental strategies, and applications in immunology

Recent advances in quantitative colocalization analysis: Focus on neuroscience

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