Research - Biophotonics
A tightly focused laser beam can be used
to trap micrometer sized objects at the focal point due to the difference
in refractive index of the object and its surrounding medium. This
technique, so called laser trapping, is used to probe and manipulate
micro-object in a non-contact mode, which presents many significant
advantages over many conventional micro-manipulation techniques.
The aims of this project are to develop novel optical trapping probes
for optical imaging, sensing and manipulation, particularly in the
near-field region, and achieve technological advances in the following
aspects:
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Engineering of optical momentum and angular momentum including
the development of FDTD simulation model for near-field scattering
and trapping.
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Optical nanometry system for measuring ultra-weak force and
torque.
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Optical trapping with ultrashort pulsed lasers and morphology
dependent resonance.
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Dynamic control of focal spots for near-field imaging, sensing
and manipulation.
Ultimately, this project is aimed to novel techniques for cutting-edge research fields including single molecule detection,
molecule assembly, micro-fluidic systems and cell manipulation and dynamics.
Figure left: Schematic illustrations of the
focused evanescent trapping.
Figures right: Dynamic control and manipulation of a red blood cell
using a near field trap.
References
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Min Gu, Jean-Baptiste Haumonte, James W. M. Chon, Xiaosong
Gan, Laser trapping and manipulation under focused evanescent
wave illumination
Appl. Phys. Lett., 84, 4236-4238 (2004).
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Smitha Kuriakose, Xiasong Gan, James W. M. Chon, and Min
Gu, Optical lifting force under focused evanescent wave illumination:
a ray-optics model
J. Appl. Phys. 97, 083103 (2005).
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Baohua Jia, Xiaosong Gan, and Min Gu. Direct measurement
of a radially polarized focused evanescent field facilitated
by a single LCD.
Optics Express, 13:6821–6827, (2005).
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Baohua Jia, Xiaosong Gan, and Min Gu. Anomalous phenomenon
of a focused evanescent Laguerre-Gaussian beam.
Optics Express 13:10360–10366, (2005).
- Min Gu, Smitha Kuriakose, and Xiaosong Gan, A single beam
near-field laser trap for optical stretching, folding and rotation
of erythrocytes,
Opt. Express 15 (3), 1369-1375 (2007)
Nonlinear optical imaging based on multi-photon absorption and higher harmonic generation has emerged as one of the best
non-invasive means of optical imaging techniques. The aim of the project is to develop a nonlinear optical endoscope that uses the nonlinear interactions
of laser with tissues to image internal organ sites in vivo, providing tools for the better detection of early cancer. A miniaturized nonlinear optical
microscope based on flexible fibre-optic devices would be the soul instrumentation to permit the cellular imaging within hollow tissue tracts or solid
organs that are inaccessible to a conventional optical microscope. An ultra-small probe head is designed to fit the working channel of a flexible endoscope
and connect to the bulk optical components via a single piece of fibre (see figure). A double-clad photonic crystal fibre is adopted to improve the detection
efficiency of the imaging system by delivering the near infrared laser beam in the central core and collecting visible light through the inner cladding. A
microelectromechanical system (MEMS) mirror is built in the probe to steer the light at the fibre tip. The technology will enable in vivo visualizations of
functional and morphological changes of tissues at the microscopic level rather than direct observations with a traditional instrument at the macroscopic level.
 
Figure: (Left) Nonlinear
fibre optical endoscope. (Right) Z projection of 8 slices through
the rat esophagus tissue stained with Acridine Orange imaged with
nonlinear optical endoscopy. Two-photon fluorescence (red) and SHG
(green) visualize cell nuclei and connective tissue, respectively.
A GRIN lens used for imaging has a diameter of 0.5 mm and a NA of
0.5. The excitation power on the sample resulting in two-photon
fluorescence and SHG signals is 10 mW and 25 mW, respectively. Slice
spacing is 5 ?m. Scale bar represents 20 ?m (cover taken from Optics
Express 14 1027-1032 (2006))
Another aspect of this project is to use a femtosecond laser for
cellular medication and engineering. The nature of femtosecond pulse
lasers is such that they can deliver very precise and highly localised
energy to target cells or tissues with little or no heating damage
to the targeted specimen or surrounding environment. A finely focused
period of femtosecond pulses can alter individual cell characteristics
without leading to the destruction of the cell. The use of femtosecond
pulse lasers has already been demonstrated as a method for tissue
dissection, cell photo-disruption, cell microinjection and cell
transfection. Previously, cell mechanics have been investigated
by using micropipette tips to exert localised forces on cells, or
by the use of atomic force microscopy (AFM), magnetic twisting cytometry,
optical tweezing and the like. In this project we describe a new
method of biomechanical research by using the power of focused targeting
with femtosecond pulse lasers to induce precise mechanical strains
and stresses to various cells, whilst simultaneously being able
to image cells using confocal microscopy. Because it is well established
that bone cells respond to mechanical strain application by increasing
bone mineralization, the technology proposed could also prove a
worthwhile approach for bone tissue engineering.
References
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L. Fu, A. Jain, H. Xie, C. Cranfield, and M. Gu, “Nonlinear
optical endoscopy based on a double-clad photonic crystal fiber
and a MEMS mirror,”
Optics Express 14 1027-1032 (2006).
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M. Gu and L. Fu, “Three-dimensional image formation in fiber-optical
second-harmonic-generation microscopy,”
Optics Express 14, 1175-1181(2006).
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L. Fu, X. Gan, M. Gu, "Characterization of the GRIN lens-fiber
spacing toward applications in two-photon fluorescence endoscopy,"
Applied Optics 44, 7270-7274 (2005).
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L. Fu, X. Gan, M. Gu, "Nonlinear optical microscopy based on
double-clad photonic crystal fibers,"
Optics
Express 13, 5528-5534 (2005).
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L. Fu, X. Gan, M. Gu, "Use of a single-mode fiber coupler for
second-harmonic-generation microscopy,"
Optics Letters. 30, 385-387 (2005).
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Daniel Day, Charles Cranfield, and Min Gu, High-Speed Fluorescence
Imaging and Intensity Profiling of Femtosecond-Induced Calcium
Transients,
Int. J. Biomed. Imag. 2006, 1-6 (2006)
Modern biology has benefited from the recent development of microfluidic
devices that have the ability to integrate several macroscopic
biological processes into a single microscopic chip, otherwise
known as “Lab-on-a-chip”. When studying biological systems one
of the greatest challenges is to investigate the systems in their
natural states, with little or no interference from the observation
or manipulation tools.
Recent research at the Centre for Micro-Photonics has demonstrated
two key fabrication technologies required in order to fabricate
3D micro-environments for cellular engineering:
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Two-photon photopolymerisation polymerisation of micro-structures
using two-photon induced photopolymerisation allows the fabrication
of arbitrary three- dimensional structures.
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Multi-photon ionisation - Etching on the surface or within
the volume of a substrate can be achieved with femtosecond
pulses with mJ pulse energy. Multi-photon ionisation or multi-photon
localised heating can be used to fabricate the interconnecting
channels and other functional microfluidic components as well
as complex 3D structures.
The design, fabrication and simulation of different optical components,
such as waveguides, couplers, microfluidic fluorescent light sources
and microfluidic lasers are being investigated. The generation,
coupling and detection of intensity and spectroscopic changes
in the light as a result of changes in the cellular and fluidic
environment is a key technology development.
The current research projects in this area are:
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Fabrication of three-dimensional microstructures for cell
confinement and proliferation.
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Fabrication and simulation of three-dimensional structures
for mixing in microfluidic devices.
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Femtosecond fabrication of optical sensors for microfluidic
applications.
Figure: Microfluidic flow in fabricated microfluidic
channels in a PMMA substrate before and after the in-situ fabrication
of a three-dimensional mixing structure.
References
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Daniel Day and Min Gu, “Formation of voids in a doped polymethylmethacrylate
polymer”
Appl. Phys. Lett., 80, 2404-2406 (2002)
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D. Day, M. Gu, “Microchannel fabrication in PMMA based on
localized heating by nanojoule high repetition rate femtosecond
pulses”
Opt. Express
13, 5939-5946 (2005)
- Daniel Day and Min Gu, "Femtosecond fabricated photomasks
for fabrication of microfluidic devices"
Opt. Express
14, 10753-10758 (2006)
The Cell Biology Laboratory was established in July 2005 to allow
the wealth of novel technologies developed by the Centre for Micro-Photonics
to be utilized in cutting edge biological experiments. An important
attribute of this collaboration is the strong links between the
Cell Biology Laboratory at Swinburne and the Immune Signalling
Laboratory at the Peter MacCallum Cancer Centre (both run by Sarah
Russell), which enable a fluid exchange between top quality photonics
and biological research. We have established molecular biology
and tissue culture facilities at the PeterMac, and begun a number
of exciting projects involving collaborations between researchers
at the PeterMac and many staff at the CMP. Many CMP technologies,
such as microfabrication and laser tweezing, will be utilized
in this work.
A number of projects have been initiated that will elucidate
the mechanisms of action and physiological functions of a network
of proteins that regulate cell shape, the “polarity network”.
The polarity network includes two proteins called Discs large
(Dlg) and Scribble, which are also tumour suppressors in certain
circumstances. Understanding how these proteins work will lead
to important diagnostic and therapeutic opportunities in a number
of diseases. Examples of two such projects are described below.
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We are developing new approaches for quantitative, high resolution,
tracking and manipulation of proteins during the establishment
of cell polarity (in both T cells and epithelial cells). This
project involves genetically engineering proteins to tag them
with fluorescent markers, expressing them in cells, and imaging
their movements while the cells undergo polarity changes.
We will combine improvements in both sensitivity and computational
analysis with genetic manipulation of the individual components
of the polarity network, to elucidate the hierarchy of molecular
events required for cell polarisation.
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It is becoming evident that for a number of cancers, disease
progression is dramatically influenced by the surrounding
normal tissue, and the polarity network plays an important
role in communications between the cancer and its surrounding
tissue. It has recently come to light that tissue rigidity
and cell tension have important influences on cancer progression.
We have initiated a program of research to investigate possible
molecular links between the regulation of cell polarity, cell
tension and cell division. This project involves the utilization
of both microfabricated cell supports and laser tweezing to
manipulate tension in cells, imaging of polarity proteins
as in project 1, and correlation with activities associated
with cancer, such as cell proliferation and death.
Figure: Reorganization of polarity proteins
during killing by a T lymphocyte.
References
-
Ludford-Menting, MJ, Oliaro, J, Sacirbegovic, F, Cheah, E,
Pedersen, N, Thomas, SJ, Pasam, A,, Iazzolino, R, Dow, LE,
Waterhouse, NJ, Murphy, A, Ellis, S, Smyth, MJ, Kershaw, MH,
Darcy, PK, Humbert, PO, and SM. Russell “A network of PDZ-containing
proteins regulates T cell polarity and morphology in motility
and immunological synapse formation.”
Immunity,
22: 737-748 (2005)
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Russell, SM and J.Oliaro, Compartmentalization in T cell
signalling: Membrane microdomains and polarity orchestrate
signalling and morphology.
Immunol. Cell Biol (invited review). 84: 107-113 (2006).
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L.E. Dow, J. S. Kauffman, J. Caddy, A. S. Peterson, S. M.
Jane, S. M. Russell, and P O Humbert, The tumour-suppressor
Scribble dictates cell polarity during directed epithelial
migration: regulation of Rho GTPase recruitment to the leading
edge
Oncogene
26, 2272–2282 (2007)
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John T. Chang, Vikram R. Palanivel, Ichiko Kinjyo, Felix
Schambach, Andrew M. Intlekofer, Arnob Banerjee, Sarah A.
Longworth, Kristine E. Vinup, Paul Mrass, Jane Oliaro, Nigel
Killeen, Jordan S. Orange, Sarah M. Russell, Wolfgang Weninger,
Steven L. Reiner, "Asymmetric T Lymphocyte Division in
the Initiation of Adaptive Immune Responses"
Science
315, 1687-1691 (2007)
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