Biomedical Engineering Reference
In-Depth Information
without sample removal, facilitating the tracking of rapidly changing events such as
calcium release. The convenience of the unusually large dynamic range of the C6
system has been exploited in the simultaneous analysis of plant species whose nuclear
content DNA values (C-values) span at least two orders of magnitude and reflect
almost the entire described angiosperms [28]. The incorporation of a fully digital data
collection system facilitates post-collection analysis and highlights the potential
value for data mining in screening applications.
The Amnis ImageStreamX combines the advantages of flow cytometry in sample
handling and fluorescence sensitivity with the imaging of cells directly in suspension
at the resolution of a 60
microscope. The EDF (extended depth of field) capacity
increases image quality and ensures that the whole cell stays in focus [29]. This
platform permits phenotypic and functional studies with data extraction from 12
images per cell. Analysis performance is in the range of 1000 cells/s with a software
package to quantify predefined fluorescence and morphologic parameters for multiple
applications [26, 29-36]. The instrument adds a complementary technology for coping
with the problem of rare event analysis. For example, the mobilization of nonhema-
topoietic very small embryonic-like stem cells (VSELs) in acute myocardial infarction
has been studied by VSELs isolated using FACSAria
, and expression of pluripotent
markers, early cardiac and endothelial markers, and chemokine receptor CXCR4 then
confirmed using an ImageStream system [36]. Given that flow cytometry is restricted
when attempting to address morphological or subcellular spatial details, image
cytometry has the combined advantages of both, for example, in the multispectral
imaging of stages of erythroid maturation by measuring changes in Ter119 mean
intensity and area, DNA (DRAQ5 stain) mean intensity and area, and RNA content
(thiazole orange stain) [34]. The ImageStream has the capacity to approach difficult
forms of in-flow analysis such as receptor internalization and nuclear translocation,
providing a route tohighlymultiplexed assays, compound screening, andprofiling [37].
The imaging cytometer also highlights the potential for the definition of new complex
analytical parameters that can be exploited to identify a change in a cell state of interest.
Of course, this still applies for the reexamination of conventional parameters, such as
light scatter, as recently demonstrated in the tracking of changes in prostate cancer
cellular granularity, monitored on the HyperCyt high-throughput flow cytometer
system, to screen for small molecule inducers of intracellular granularity revealing
a candidate class of aryl-oxazoles that increased intracellular granularity in both
androgen-sensitive and androgen-independent cell lines [38].
Multiple approaches to increasing throughput are now available with robotics and
automated sample processing interfaces being important components in workflows.
Solutions are available for many instrument platforms. For example, the Hudson Flow
Cytometry Workcell from Hudson Robotics, Inc. enables automated sample acquisi-
tion system in an integrated platform combining the Guava EasyCyte
Plus Flow
s PlateCrane EX microplate robot arm and microvolume
sampling, multiplex analysis, and up to six detection parameters. The allied software
package facilitates data tracking and scheduling. Millipore
Cytometer with Hudson
s Guava instrument
platform provides a microcapillary flow cytometry operating without sheath fluid,
reducing sample volumes, waste, and maintenance requirements. The new easyCyte
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