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Robotic Preparation
of cDNA from Single Cells (funded by NIH,
Publications)
| The current state of the
art for optical microscopy of living cells provides an array of
techniques of extraordinary power. Living cells can now be studied in
multiple dimensions (three spatial dimensions, time, multiple
wavelengths, and multiple stage positions). A unique feature of
microscopic approaches is the capability of observing transient,
ephemeral structures and interactions on a cell-by-cell basis. This
includes the ability to monitor subcellular processes and to follow cell
movements as well as cell-cell interactions over time. We propose to
develop an instrument that will couple the power of multidimensional
microscopy with that of DNA array technology. Specifically, we
envision an instrument in which individual cells selected on the basis
of optically detectable features at critical time points in dynamic
processes can be rapidly and robotically micromanipulated into reaction
chambers to permit amplified cDNA synthesis and subsequent array
analysis. In this way, "snapshots" of gene expression in single cells
can be related to information obtained with multidimensional microscopy.
The proposed instrument will incorporate an inverted research
microscope capable of widefield deconvolution microscopy as well as a
robotic system for manipulation of cells and reagents. An environmental
chamber will provide conditions for optimal maintenance of cells. A
laser ablation system will provide for automated cell lysis. Algorithms
will be developed for automatic recognition and manipulation of cells, a
requirement for high throughput. The planned system is expected to
process 500- 1 000 cells per day.
We believe that the proposed instrument will represent a genuine
advance in technology that will be of great benefit to cell biology and
the study of cancer cells. For example, the progression to malignancy
involves the gradual accumulation of genetic changes in single cells,
leading to heterogeneity among malignant cells. Studies of gene
expression at the single-cell level will permit an orderly dissection of
this heterogeneity.
In summary, we believe that an instrument which couples
multidimensional microscopy with DNA array technology will be a
spectacular toot that will be useful to many laboratories.
with Dr. Lou Cleveland of St. Luke's-Roosevelt Hospital Center
Automated
Robotically-Based High-Throughput Radiation Biodosimetry (funded by
NIH)
Our goal is to develop a fully
automated ultra-high throughput radiation biodosimetry workstation,
using purpose-built robotics and advanced high-speed automated image
acquisition. Maximum throughput will be 30,000 samples / day, compared
with throughputs in current devices of a few hundred samples / day.
The basic system involves the
well-characterized micronucleus assay in lymphocytes, with all the
assays being carried out in-situ in multi-well plates.
 | By calling up pre-programmed options in
timing, liquid handling, and image analysis, the device will also
measure gamma-H2AX foci yields, and micronucleus yields in
reticulocytes, both providing "same-day answer" dose estimates. |
| By calling up pre-programmed options in
liquid handling steps, the device will also measure micronuclei in
other readily-accessible tissues, such as exfoliated cells from urine
or buccal smears. |
A key option of the system
will be that each lymphocyte sample will be split in two, with one of
the two split samples being irradiated to a dose of 1.8 Gy, before being
analyzed. This will allow a positive control for each individual,
providing an internal calibration to take into account inter-individual
variability in radiosensitivity.
We will develop both a Phase 1
and a Phase 2 device, with a 12-18 month lag between them:
| The Phase 1 device, using 96-well plates,
will have a throughput of 6,000 samples (3,000 individuals) per 15
hour day, and will use monochrome imaging. Peripheral blood drawn by
venipuncture will be used. |
| The Phase 2 device, using 384-well plates,
will have a throughput of 30,000 samples (15,000 individuals) per 15
hour day, and will use color imaging. Capillary blood from a finger
stick or a high-throughput laser skin perforator will be used. . |
Our Specific Aims, which will
run in parallel throughout the project are 1) product development, and
2) the optimization / calibration / testing of biological protocols.
Mass radiological triage will
be critical after a large-scale event because of the need to identify,
at an early stage, those individuals who will benefit from medical
intervention, and those who will not. Our goal is to develop a fully
automated ultra-high throughput biodosimetry workstation product (30,000
samples/day), using purpose-built robotics and advanced high-speed
automated image acquisition.
Publications:
with Drs. David Brenner and Sally Amundson of
Center of Radiological Research, Columbia University Medical Center
Salerno, A., Zhang, J., Bhatla, A., Lyulko, O.
V., Nie, J., Dutta, A., Garty, G., Simaan, N., Randers-Pehrson, G., Yao,
Y. L. and Brenner, D. J. “Design Considerations for a Minimally
Invasive High-Troughput Automation System for Radiation Biodosimetry“
Proceedings of the 3rd Annual IEEE Conference on Automation Science and Engineerng Scottsdale, AZ, USA, Sept 22-25, 2007, pp. 846-852
PDF (2112 kB)
Guy Garty,
Gerhard Randers-Pehrson, Nabil Simaan, Alessio Salerno, Aparajita Dutta,
Jing Nie, Giuseppe Schettino, Sasha Lyulko, Jian Zhang, Anubha Bhathala,
Lawrence Yao, David J. Brenner, “Development of an ultrahigh-throughput
robotically-based biodosimetry workstation using in-situ assays,” The
13th International Congress on Radiation Research, San
Francisco, CA, July 8-12, 2007, abstract only.
PDF (18 kB)
Guy Garty,
Youhua Chan, Alessio Salerno, Helen Turner, Jian Zhang, Oleksandra V
Lyulko, Yanping Xu, Hongliang Wang, Nabil Simaan, Gerhard Randers-Pehrson,
Y. Lawrence Yao, Sally Admundson, David J. Brenner, “The RABIT: A Rapid
Automated Biodosimetry Tool for radiological Triage,” Health Physics
Journal, 2009, accepted. PDF (2,221
kB)
Youhua Chen,
Jian Zhang, Hongliang Wang, Guy Garty, Yanping Xu, Oleksandra Victorovna
Lyulko, Helen Turner, Gerhard Randers-Pehrson, Nabil Simaan, Y. Lawrence
Yao and D. J. Brenner “RABiT - Rapid Automated Biodosimetry Tool for
High Throughput Radiological Triage,” IDEC09, accepted.
PDF (668 kB)
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