| 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
Publications
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.
with Drs. David Brenner and Sally Amundson of Center
of Radiological Research, Columbia University Medical Center
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