KEYWORDS: Microscopes, Holography, New and emerging technologies, Cancer, Diffusion, Phase contrast, Microscopy, Image processing, Prototyping, Time lapse microscopy
Recognizing the value and unmet need for label-free kinetic cell analysis, Phase Holograhic Imaging defines its market segment as automated, easy to use and affordable time-lapse cytometry. The process of developing new technology, meeting customer expectations, sources of corporate funding and R&D adjustments prompted by field experience will be reviewed. Additionally, it is discussed how relevant biological information can be extracted from a sequence of quantitative phase images, with negligible user assistance and parameter tweaking, to simultaneously provide cell culture characteristics such as cell growth rate, viability, division rate, mitosis duration, phagocytosis rate, migration, motility and cell-cell adherence without requiring any artificial cell manipulation.
Live-cell imaging enables studying dynamic cellular processes that cannot be visualized in fixed-cell assays. An increasing number of scientists in academia and the pharmaceutical industry are choosing live-cell analysis over or in addition to traditional fixed-cell assays. We have developed a time-lapse label-free imaging cytometer HoloMonitorM4. HoloMonitor M4 assists researchers to overcome inherent disadvantages of fluorescent analysis, specifically effects of chemical labels or genetic modifications which can alter cellular behavior. Additionally, label-free analysis is simple and eliminates the costs associated with staining procedures.
The underlying technology principle is based on digital off-axis holography. While multiple alternatives exist for this type of analysis, we prioritized our developments to achieve the following: a) All-inclusive system – hardware and sophisticated cytometric analysis software; b) Ease of use enabling utilization of instrumentation by expert- and entrylevel researchers alike; c) Validated quantitative assay end-points tracked over time such as optical path length shift, optical volume and multiple derived imaging parameters; d) Reliable digital autofocus; e) Robust long-term operation in the incubator environment; f) High throughput and walk-away capability; and finally g) Data management suitable for single- and multi-user networks.
We provide examples of HoloMonitor applications of label-free cell viability measurements and monitoring of cell cycle phase distribution.
A fully integrated instrument for cereal grain quality assessment is presented. Color video images of grains fed onto a belt are digitized. These images are then segmented into kernel entities, which are subject to the analysis. The number of degrees of freedom for each such object is decreased to a suitable level for Artificial Neural Network (ANN) processing. Feed- forward ANN's with one hidden layer are trained with respect to desired features such as purity and flour yield. The resulting performance is compatible with that of manual human ocular inspection and alternative measuring methods. A statistical analysis of training and test set population densities is used to estimate the prediction reliabilities and to set appropriate alarm levels. The instrument containing feeder belts, balance and CCD video camera is physically separated from the 90 MHz Pentium PC computer which is used to perform the segmentation, ANN analysis and for controlling the instrument under the Unix operating system. A user-friendly graphical user interface is used to operate the instrument. The processing time for a 50 g grain sample is approximately 2 - 3 minutes.
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