Back to Purdue
University Cytometry Labs
Cytometry Laboratories Areas of Interest
Engineering Interactions
Considering that the specialty instrumentation in this facility
consists basically of optical components, computers, lasers, and
hydrodynamically controlled devices, it is not surprising that
biologists and engineers must interact. There are currently
several projects in which direct
interactions exist at a high level between biological scientists
in the facility and engineering scientists.
Recently the Graduate School at Purdue awarded a special initiative grant for a graduate student to enter the program known as the Optical Engineering Instrumentation and Cell Biology Program. This initiative will be expanded to allow students to interact on any engineering project.
Advanced Concept Spectroscopic Cytometer Project
This project is designed to utilize state-of-the-art spectroscopic
ideas in flow cytometry. The use of time-resolved flow cytometry has
some advantages over traditional techniques currently used. Primary
interactions are with the Schools of Mechanical and Chemical
Engineering. Associate Faculty include Professor Fred Lytle (Chemistry)
Professor Galen King (Mechanical Engineering), Professor Eva Sevick
(Chemical Engineering).
Expert Systems for Diagnosis of
Hematological Diseases
Although an enormous amount of data is derived from flow
cytometric evaluations of patients, it is not readily analyzed.
Further, without new developments in computer technology, computer
programming, and analysis algorithms, it is too difficult to
utilize available data in a timely manner. It can often take 2 or
3 days for complete analysis of flow cytometry data by hospital
laboratories. Unfortunately, because of the complexity of the
data, it is impossible to extract all of the useful information
from the present data structure. New developments by the Cytometry
Laboratories have enabled experts from the School of Mechanical
Engineering Department of Control Systems to apply the concepts of
expert systems and neural networks for rapid analysis. It should
be possible to determine in minutes many of the diagnostic
features from within complex multiparameter data produced on
modern day flow cytometers. Associate Faculty include
Professor Galen King (Mechanical Engineering), Professor Okan
Ersoy (Electrical Engineering).
Fluid Sorting and Distribution
Using well known principles of fluid switching, scientists from
the School of Mechanical Engineering are attempting to develop new
applications in sorting single cells in flow cytometry. Since all
measurements made in flow cytometry are based upon cells moving
within a stream, separating individual cells at a high sort rate
and with a high degree of precision should be possible. This
project may have significance to chemical engineers interested in
isolating specific organisms used in biotechnology applications.
Kinetic Measurements of Viable Human Blood Cells
When white blood cells perform their normal functions, they
operate within the body under control of the immune system. Each
cell type has a specific function, and measurement of these
functions in vitro is difficult. Concepts envisioned by scientists
from both biological and engineering disciplines would allow a
replication of the in vivo environment of a blood vessel and be
able to monitor many hundreds of operating blood cells within that
environment. This is termed the kinetic environmental module and
is a high-tech environment in which is a close replication of a
closed blood vessel. Solutions can be infused or removed from the
cell and cells can be added or removed. Functioning cells within
the vessel can be monitored continuously as individual cells and
biochemical pathways monitored. While this sounds a relatively
simple task, it is enormously complex and requires many advanced
technologies, including laser beam-controlled signal measurement,
complex timing and signal identification, hydrodynamic systems,
and computer data analysis operations.
New Blue-Green Diode Lasers
PUCL is working with professors from the School of Electrical
Engineering to apply the newly developed blue-green diode lasers
to flow cytometry. This concept will allow instruments to be
designed as small modules rather than very large laboratory
instruments. Associate Faculty include Professors Kevin
Webb, Gerald Neudack, Andrew Weiner (Electrical Engineering)
Agricultural / Veterinary / Biomedical Interactions
There are a number of ongoing agriculture / veterinary /
biomedical projects in the cytometry laboratory. (Projects menu)
Immune Dysfunction & Endotoxemia
Patients who become infected with certain micro-organisms or who
undergo surgery or suffer significant burns can develop a very
serious condition termed endotoxic shock. The syndrome has severe
and even fatal consequences for many patients. A similar problem
occurs in many farm animals, resulting in severe economic cost to
the nation. Studies using flow cytometry as a major tool are being
performed to try to understand what are the critical factors in
endotoxic shock and how we may more readily alleviate these
symptoms. Associate Faculty include Professor Geral Bottoms
(Basic Medical Sciences).
Trauma and Thermal Injury
Scientists within the facility are attempting to design a cell
culture model that will replicate the symptoms of endotoxic shock
resulting from trauma such as surgery, thermal injury, or severe
accidents. Certain highly responsive immune cells are grown in
culture and then subjected to the same biochemical stress as in a
human trauma. If this program can demonstrate the importance of
this modality, it may be most useful in assisting us to determine
the most appropriate methods for treatment of shock. We are
currently working with cultures of pulmonary artery endothelial
cells which can be manipulated to identify some of the mechanisms
important for oxidative tissue damage in trauma.
Prognosis of Malignancies
Several scientists are involved in studies of cancers in an
attempt to predict the course of the disease at an early stage.
This would be most useful in determining treatment regimens and
would be more accurate in predicting the outcome of the disease.
Techniques used include measuring the exact cell cycle for the
replication of malignant cells. Sometimes this information
determines the type and dosage of chemotherapeutic drugs to combat
the disease. These studies have application to both human and
veterinary medicine and the economic impact could be pronounced in
both areas.
Determination of Tetraploidy in Cultivated Fish
Tetraploid fish are particularly useful in situations where it is
not desirable to have rapidly breeding fish introduced into a new
environment. Further, because these fish are sterile, they grow
more rapidly and contain a higher amount of protein than regular
diploid fish. The process involved in producing tetraploid fish is
of interest to scientist in the School of Agriculture. A new
technique has been developed with the Cytometry Laboratories to
determine ploidy of these fish within a couple of days of
hatching, eliminating the need to wait several weeks for the fish
to grow to sufficient size. This project has other implications -
it is also possible to distinguish many strains of fish very
rapidly by their DNA content. Associate Faculty include
Professor Chris Bidwell (Animal Science).
Identification of Bovine Leukotoxins
Scientists from the Animal Diseases Diagnostic Laboratories
together with staff from the Cytometry Laboratories are developing
new ways of evaluating the effects of leukotoxins on bovine
neutrophils. Using flow cytometry and multivariate analysis
techniques it is possible to monitor in real time the effects of
these toxins. New understandings of the mechanism of action of
such toxins will result from these studies. Associate Faculty include
Professor Terry Bowersock (Pathobiology).
Sorting & Identification of Plant Chromosomes
Scientists from the Department of Horticulture are using modern
laser-based flow cytometry techniques for isolating plant
protoplasts. Such applications are at the leading edge in this
field. The approach allows identification and physical isolation
of single plant chromosomes.
Confocal Microscopy
The facility has 2 confocal microscopes, plus a significant assortment of image analysis
equipment. We are interested in using these
systems for both basic image analysis as well as in new application
development. Current areas of interest include development of imaging systems for
detections of tissue injury, tumors, and related areas.
a. Bio-Rad MRC 1024 UV/Vis System: This system is a 2 laser system with
UV lines at 353 & 361, 488 & 514 nm. In addition several other lines are
available from an Ar-Kr laser (488 nm, 568 nm, & 647 nm). Simultaneous excitation
of almost any combination is possible. There are 3 PMT detectors as well
as a transmission detector. The system is mounted on a Nikon 300 inverted
microscope, which has heated stages and objectives to assist in
physiological studies.
b. DVC 250 Direct View Confocal Microscope: This system is a direct view
scope mounted on a Nikon 200 inverted microscope. An intensified video
camera collects signals via an image analysis computer. The system is
very useful for fast fluorescence studies. For more details on the
facilities go to Confocal Capabilities. Associate
Faculty include Professor Kevin Webb (Electrical Engineering), Professor Eva
Sevick (Chemical Engineering), Professor Galen King (Mechanical Engineering).
Educational Activities
The Facility has a strong interest in developing
teaching tools, particularly interactive computer tools.
"Handbook of Flow Cytometry Methods"
EDITOR:
J.Paul Robinson
ASSOCIATE EDITORS:
Zbigniew Darzynkiewicz
Phillip Dean
Lynn Dressler
Hans Tanke
Leon Wheeless
The Handbook contains 250 pages of basic and advanced flow cytometry methods. In addition, suppliers and chemicals used are listed. The Handbook is a compilation of methods from many research scientists throughtout the world, and provides some useful insights into the practical problems of running flow cytometry assays. More information can be found about the Handbook by going to [References Areas in Flow Cytometry].
The Handbook is available from:
Wiley-Liss, Inc.,
605 Third Avenue
New York, NY, 10158-0012
ISBN 0-471-59634-5 The cost is $39.95.
The current edition was published in 1993.
STEP - Specialty Training and Education Program of ISAC
This program is currently under development. It was initiated by Dr. Zbigniew Darzynkiewicz during his Presidency of the International Society for Analytical Cytology. The purpose of the program is to provide information regarding training and educational opportunities in the field of cytometry. We now have an Introductory Homepage for STEP which you can visit. If you feel your laboratory might be able to participate as a provider of training or educational materials, please email me directly now at
robinson@flowcyt.purdue.edu .
Current Protocols in Cytometry
Current Protocols in Cytometry is under development as a project between the International Society for Analytical Cytology and the publisher Wiley-Liss. The project is designed to provide up-to-date, comprehensive protocols for use with cytometry technologies such as flow cytometry and image analysis. It is possible to submit suggestions for protocols for inclusion in the volume by sending them to me at the address below or email me directly now with your suggestions:
Send an email message now to suggest protocols to J.Paul Robinson: robinson@flowcyt.cyto.purdue.edu
J.Paul Robinson, Ph.D.
Current Protocols in Cytometry Editorial Office
1515 Hansen
Hall
Purdue University
West Lafayette, IN 47907-1515
Phone: (317) 494-0757
FAX (317) 494-0517
email current-protocols@flowcyt.cyto.purdue.edu
Instructions are under development.
Back to Purdue
University Cytometry LabsFor information contact J.Paul Robinson, Ph.D., Director PUCL, 1515 Hansen B050, Purdue University, West Lafayette, IN 47907-1515, U.S.A. Phone: (317) 494-0757; Fax: (317) 494-0517 email: robinson@flowcyt.cyto.purdue.edu