Bioelectrophysics--Biophotonics--Real-time Imaging System
Bioeletrophysics
Electric field pulses of relatively long duration
(microseconds) with relatively long rise and fall times (tens
of nanoseconds) and relatively low amplitude (100 kilovolts
per meter) produce biological effects at the cellular level
primarily as a result of the formation of openings in the
external membrane of the cell through the process of electroporation.
High-field pulses with widths substantially less than the
charging time of the plasma membrane and nanosecond or sub-nanosecond
rise times are expressed across intracellular structures and
can affect cells in dramatically different ways. Here
is a schematic showing differences of electroperturbation
and electroporation.
Membrane Electrophysics --
Nanosecond, megavolt-per-meter pulses cause a rearrangement
of phospholipids in the cytoplasmic membrane.
Intracellular Calcium Distribution -- Nanoelectropulses
induce release of calcium from intracellular compartments
within milliseconds of pulse exposure.
Perturbation of Physiological Homeostasis
-- Disturbance by ultra-short pulsed electric fields of cytoplasmic
compartments, cytoskeletal attachments, nuclear, mitochondrial,
and plasma membranes, and the configuration of the endoplasmic
reticulum may have specific, dose-dependent effects that are
complex and largely uncharacterized functions of pulse width,
amplitude, frequency, and pattern.
Caspase Activation -- Unique proteolytic
enzymes are activated in cells after exposure to nanoelectropulse
exposure. These caspases (cysteine aspartate-specific proteases)
are the effectors and executioners of apoptosis (programmed
cell death).
Cytological Changes -- Nanoelectropulse
exposure causes changes in cytoplasmic volume, granulation
of the nucleus, and blebbing of the plasma membrane.
Biophotonics
Quantum dot probes are fluorescent colloidal
semiconductor nanocrystals. Compared with organic fluorophores,
quantum dots have excellent photostability, strong fluorescence,
size-tunable emission wavelengths, low toxicity, and long-term
chemical stability. These advantages make them extremely proper
for long-term cell observations and in vivo tracking. However
delivering nanoscale particles into cells and tissues is a
challenge for biology research and applications. Two delivery
methodes are in progress.
Electropermeabilization --
quantum dots were transferred into various cells by the technique
of electroporation. Experimental results suggested that the
transfer process was partical-size dependent.
Streptavidin conjugated
QDs enter the cells through anti-EGFR mediated endocytosis
-- Despite aggressive treatment by surgery, radiotherapy,
and chemotherapy, the median survival of patients diagnosed
with malignant gliomas is less than 12 months. A key variable
affecting the survival and quality of life of patients with
intracranial gliomas is the extent of tumor resection. In
vivo fluorescent spectroscopy and imaging using endogenous
and exogenous sources of contrast can provide new approaches
for enhanced demarcation of tumor margins and infiltration.
The epidermal growth factor receptor (EGFR) is a member of
a family of four receptors, which include EGFR (HER1 or ErbB1),
ErbB2 (HER2/neu), ErbB3 (HER3), and ErbB4 (HER4). EGFR has
been implicated in the development and progression of a number
of human solid tumors including brain tumors. Since EGFR is
overexpressed in human gliomas, it’s a promising molecular
target for quantum dots for tumor demarcation. we investigate
the potential use of distinct class of fluorescent molecular
probe, quantum dots (QDs), for enhanced optical imaging of
brain tumors and intraoperative surgical guidance.
QD-Streptavidin-biotin-anti-EGFR complex can
enter human U87 glioma cells and concentrate in the cytoplasm,
suggesting that QDs get into cells through anti-EGFR specifically
finding to EGFR.
Facility -- Real-time
Imaging System
A fluorescence microscopy imaging system has been built up
for the real-time investigations of electroperturbation and
electroporation. An inverted epi-fluorescence microscope coupled
with a nanosecond high-voltage pulser, micrometer-scale electrode
chambers, and a sensitive CCD camera enable the real-time
imaging of intracellular processes during exposures of cells
to megavolt per meter electric pulses on a standard microscope
glass slide. Custom programs and commercial image acquisition
and processing software permit real-time fluorescence imaging
and flexible, automated control of pulse parameters. Biological
transient responses of Jurkat cells — calcium bursts, phosphatidylserine
externalization, and nuclear condensation after nanosecond
megavolt-per-meter electric pulses — have been recorded with
this system.
Related courses
Cell biology (BISC)
Molecular biology (BISC)
Tissue engineering (CHE)
Biochemistry (CHEM)
Methods in experimental pathology (PATH): Each topic has different
instructor.
Biotechnology (BISC): Lab class. Not allowed to sit in.
Materials characterization (MASC)
Instrumental analysis (CHEM)
Transmittance electron microscope (MASC)
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