We house a wide range of equipment that are used in conjunction with many optimised
techniques to study the relationships between form, function and disease.
Below are a few pieces of technology that are used regularly by us, followed by
a brief description of how and why they are used.
2-Photon
Microscopy (2-PM)
Fluorescence emission by 2-photon excitation (left) compared to large cone around
plane of focus in conventional fluorescence microscopy (right). Image by Christian
Soeller and Mark Cannell.
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2-photon microscopy is an imaging modality, similar to confocal microscopy, in which
a high-powered, femto-second pulsed infra-red laser is scanned across a sample.
The simultaneous absorption of two photons stimulates fluorescence from fluorescent
molecules, and this absorption only occurs at the point-of-focus, allowing 3-D imaging
(optical sectioning) of the sample. Advantages of 2PM include better depth penetration,
less photobleaching and phototoxicity, higher photon efficiency and the ability
to photolyse caged molecules at a 3-D resolved point in the sample.
- Written by Angus McMorland.
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Laser Scanning Confocal
Microscopy (LSCM)
Laser scanning confocal micrograph (raw data) of healthy cardiac ventricular tissue
stained for F-actin. Staining shows clear striations, which are indicative of the
sarcomeric structure of myocytes. Large, intensely staining bands are most likely
to be the intercalating discs that join longitudinally-adjacent cells. Image by
David Crossman.
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We have a Zeiss Axiovert LSM410 inverted confocal microscope for use and also access
to the Biomedical Imaging Resource Unit (BIRU) confocals. Fluorescence microscopy
takes advantage of fluorescent molecules that absorb light of a particular wavelength
and release light again of a longer wavelength. Thus, we can excite specifically-localised
fluorescent molecules and collect the light to find out where they are located.
However, conventional fluorescent microscopy suffers from lower contrast due to
light from outside the focal plane being captured. Laser scanning confocal microscopy
aims to minimise capture of this light. In essence, confocal microscopy aims to
eliminate detection of z-axis light scatter by means of a pinhole in front of the
photomultiplier. Typically, this technique enables us to view optical sections as
thin as 1 micron. z-stacks are able to be taken and rendered into voxels for 3D
interpretation.
- Written by Cherrie Kong
- For more information, visit
Wikipedia
- Pawley, JB. Handbook of Biological Confocal Microscopy 3rd Ed. New York: Springer;
2006.
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Total Internal Reflection Fluorescence Microscopy (TIRFM)
TIRF takes advantage of the behaviour of light as it passes between media of different
refractive indices. The angle at which the incident light interacts with the barrier
between these two media determines what the light will do. At angles greater than
some critical angle (determined by the refractive indices), the light will reflect
back into the first medium. Though most of the light energy is reflected, a portion
of the electric field at the barrier crosses into the second medium. This tiny bit
of energy is called the 'evanescent wave' and its intensity decreases exponentially
with z-distance away from the barrier. Due to the properties of this evanescent
wave, we are able to visualise specimens at a field depth of approximately 150nm,
thus allowing us to see structures and events occurring at the cell membrane without
contamination by fluorescence from the rest of the cell.
- Cherrie Kong
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Magnetic Resonance Imaging (MRI)
MRI is also known as magnetic resonance tomography (MRT) or nuclear magnetic resonance
imaging (NMRI) and is an imaging system routinely used in a clinical setting. MRI
can be used to obtain images in any 2D plane, which can be reconstructed into a
3D array. Typically, MRI involves exciting hydrogen nuclei (for example, of water
molecules in a patient) while they are in a magnetic field. This allows acquisition
of images because the hydrogen nuclei align with (and against) the magnetic field.
Once perturbed at the Lamour frequency, the axis of spin tilts towards the XY plane.
This shift in axis induces a current that is detected. We use the facilities at
the Centre for Advanced MRI, here within the faculty. Here, tagged MRI is used to
locate normal and abnormally contracting regions of the heart for further study.
- Cherrie Kong
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Whole-Cell patch clamping
A setup for electrophysiological recordings. Shown is an Axopatch 200A with a customised
Axioskop 2FS Mot. Setup by Angus McMorland. Image taken by Cherrie Kong.
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Currently, our lab holds two functional patch-clamping setups. These are invested
with specialisations that can be used simultaneously with patch-clamping protocols.
One is suited for ratiometric fluorescent imaging, while the other is suited for
2-photon microscopy. Whole-cell patch-clamping is an electrophysiological technique
where a microelectrode is sealed on to the outside of a cell and a hole made in
the membrane trapped within the tip, making the inside of the electrode continuous
with the inside of the cell. This technique allows high-sensitivity recording of
electrical activity inside single cells, and is also used in research to introduce
intracellular fluorescent dyes to the intracellular space, labeling structures or
ions of interest.
- Angus McMorland and Cherrie Kong
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Immunocytochemistry / Immunohistochemistry
Immunolabelling techniques describe the use of specific antibodies to identify and
localise proteins or structures (for example, cell membranes) of interest. Immunocytochemistry
is the term used when these techniques are used to label cellular preparations,
while immunohistochemistry refers to tissue preparations. Antibodies specific to
the item of interest are used for localisation. Detection of these antibodies can
involve a range of methods. In our lab, we tend to use secondary antibodies that
have been conjugated to fluorescent markers (indirect immunolabellng). However,
we also use primary antibodies that are conjugated to fluorescent markers (called
direct immunolabelling). The fluorescent signal can be detected using any of the
fluorescent imaging modalities described here.
- Cherrie Kong
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Image processing
We use many sophisticated image processing techniques to extract and quantify image
data. Techniques include deconvolution and autocorrlelation. Most image processing
and analysis is achieved in Interactive Data Language (IDL).
- Cherrie Kong
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