Confocal microscopy is an optical imaging technique used to increase optical resolution and contrast of a micrograph by using a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane.
It enables the reconstruction of three-dimensional structures from the obtained images
Light microscopes use visible light sources including white light, fluorescent light and lasers to image live and fixed samples.
There are many different microscopy techniques including brightfield, phase contrast, differential interference contrast (DIC), reflected light microscopy, darkfield, fluorescence and confocal. The Centre has a number of instruments covering these modes.
Common techniques performed at the Centre are listed below.
Fluorescent microscopes use a specific wavelength of light to excite fluorochromes. In the biological field, antibodies are labelled with fluorochomes and used to tag cellular structures. Emitted fluoresence is passed through an emission filter and collected using a cooled charge-coupled device (CCD) camera. Thick samples may cause out-of-focus light to blur images making it difficult to resolve fine detail.
Digital slide scanning
Using medium-resolution robotic and-high resolution manual scanning technology, brightfield microscope images can be collected and stored for online visualisation and image analysis. This technology is particularly suited to online conferencing and histology and pathology teaching. A high-resolution 100x oil immersion scanner has been added which will particularly facilitate high-resolution scanning of heamatology slides and single-cell preparations.
Confocal microscopy uses a pinhole to block out-of-focus light and therefore increase optical resolution. The light source is usually a single wavelength laser allowing tight illumination focus. Samples can be optically sectioned (z stacks) and the resulting images can then be reconstructed into a 3D data set.
Multiphoton fluorescence microscopy allows optical sectioning of thick samples using two photons of light and leads to excitation only at the focal point. Therefore, all light collected by the system must be from the plane of focus. Using longer wavelengths (near infra red) provides several benefits including less photoxicity and deeper penetration, allowing imaging around 500 microns into a sample.
Live cell imaging
Imaging cells in real time can provide critical insights into the nature of biological cell and tissue function. The Tokai Hit incubation chamber can tightly control the environment of the cells and maintain living cells in a healthy state during long-term imaging. The chamber is portable and can be used on all the microscopes at the QEII campus. The Olympus IX81 and Nikon A1 microscopes have hardware components (ZDC and PFS respectively) to prevent focal drift during long-term imaging.
Total internal reflection fluorescence
Total internal reflection fluorescence (TIRF) imaging allows very high signal-to-noise imaging of structures adjacent to a coverslip. A laser is bounced off the surface of the coverslip extending approximately 200nm into the sample. This extremely thin region of excitation allows imaging of shallow structures with no background, and higher resolution in the Z-plane compared to confocal imaging. Applications include adherence structures of cells, and visualisation of fluorescently tagged single-membrane channels/receptors.
Laser Microdissection and Laser Tweezers
Laser microdissection uses a focused 337nm laser beam to dissect cells and sub-cellular components from paraffin-embedded and frozen sections. These individual cells can then be pressure catapulted onto microscope slides or containers for micro-cloning or DNA/RNA analyses. Laser tweezers 'trap' small specimens using a near-infrared laser beam and can be used for in vitro fertilisation, cell fusion and performing molecular force measurements.
Microinjector and Micromanipulator
Micro-mechanical operations on cells or tissues (like holding cells in one place or collecting or transferring individual cells) can be performed using the micro-manipulation equipment. The microinjector can be used to inject femtolitre amounts of dyes, bioactive agents or DNA constructs into individual cells or organisms. Microinjection tips should be supplied by the researcher wishing to use this instrument.
Both the microinjector and micromanipulator are installed on the same microscope platform and, if necessary, can be transferred between an Olympus IMT-2 inverted fluorescence microscope and a Nikon dissecting microscope.