Confocal Laser Scanning Microscopy
Confocal microscopy offers several advantages over conventional widefield optical microscopy, including the ability to control depth of field, elimination or reduction of background information away from the focal plane (that leads to image degradation), and the capability to collect serial optical sections from thick specimens. The basic key to the confocal approach is the use of spatial filtering techniques to eliminate out-of-focus light or glare in specimens whose thickness exceeds the immediate plane of focus. There has been a tremendous explosion in the popularity of confocal microscopy in recent years, due in part to the relative ease with which extremely high-quality images can be obtained from specimens prepared for conventional fluorescence microscopy, and the growing number of applications in cell biology that rely on imaging both fixed and living cells and tissues. In fact, confocal technology is proving to be one of the most important advances ever achieved in optical microscopy. Current instruments are highly evolved from the earliest versions, but the principle of confocal imaging is employed in all modern confocal microscopes. In a conventional widefield microscope, the entire specimen is bathed in light from a mercury or xenon source, and the image can be viewed directly by eye or projected onto an image capture device or photographic film. In contrast, the method of image formation in a confocal microscope is fundamentally different. Illumination is achieved by scanning one or more focused beams of light, usually from a laser or arc-discharge source, across the specimen. This point of illumination is brought to focus in the specimen by the objective lens, and laterally scanned using some form of scanning device under computer control. The sequences of points of light from the specimen are detected by a photomultiplier tube (PMT) through a pinhole (or in some cases, a slit), and the output from the PMT is built into an image and displayed by the computer. Although unstained specimens can be viewed using light reflected back from the specimen, they usually are labeled with one or more fluorescent probes.
The Bio-Rad confocal microscope we have is fitted to an Olympus BX51 upright microscope. This allows a wide variety of specimen geometries and sizes to
- The main confocal system comprises at its heart, two lasers, a standard HeNe laser of wavelength 543nm and a second argon laser, with lines at 457, 476, 488, 514nm.
- The lasers are mounted externally to the microscope and the light is transmitted via fibreoptics to the microscope.
- The microscope is equipped with a Solent Scientific fully enclosed incubator system to allow long term cultures and studies to be performed.
- Coupled with the incubator and to minimise and reduce the need to access the chamber, the microscope is equipped with a fully motorised ProScan II X-Y stage supplied by Prior to allow manipulation and also for producing large area composite images.
The Main Unique Aspects Of The Machine
- The two laser system allows a number of different dyes to be used simultaneously for example for colocalisation experiments or live dead staining to be carried out. The image below on the right is an example of a live-dead stain of human oral fibroblasts embedded in a collagen-glass composite, with live cells stained green and dead are in red.
- The incubator and X-Y manipulation allow operation without disturbing the sample. Couple with the software, it is simple to produce time dependent studies of cells and materials.
- Data interpretation is via a large suite of software, including LaserVox for 3D volume rendering. Data is also analysed using the NIH developed ImageJ software and associated plug-ins and macros.
Examples Of Work Undertaken At The Institute
A wide variety of projects have utilised this technique for qualitative and quantitative measurement of a wide variety of parameters.
The image below in the centre is of bone marrow-derived human mesenchymal stem cells cultured upon a biomimetic calcium phosphate surface and differentiated to the osteogenic lineage and stained with phalloidin in green and a red nuclear stain.
The image below on the right is an example of a live-dead stain of human oral fibroblasts embedded in a collagen-glass composite, with live cells stained green and dead cells red.