Laser scanning confocal microscopy (LSCM)

Laser scanning confocal microscopy (usually shortened to just confocal microscopy) uses the principle of fluorescence excitation to investigate the structural properties of cells and the location of particular structures or protein populations within those cells in fixed tissue. We can also use confocal microscopy to investigate the  movement of biological entities in live cells, be they vesicles or even individual proteins.

Light source

In confocal microscopes, the light source is generally one or more laser(s). Unlike in wide-field fourescence microscopy, the excitation light bandwidth is thus determined by the source, not the excitation filter and so is much narrower (2-3 nm rather than 20 - 30 nm). The laser beam, as the name suggests, is a narrow beam of light and so, in order to illuminate the whole visual field, the laser beam has to be rapidly scanned across the area in a series of lines, much like a TV image is generated. The fluorescence detected at each point is measured in a photomultiplier tube (PMT) and an image built up. This method of illumination has enormous  advantages over wide-field imaging in that it is possible to illuminate selected regions of the visual field allowing complex photobleaching protocols to be carried out to investigate the rates of lateral travel of fluorophores ... etc and for the excitation of different fluorophores in different regions of the same cell.

The Pinhole

The major difference between fluorescence microscopy and LSCM is the pinhole. What's the pinhole? This is a device that removes unwanted, out-of-focus fluorescence, giving an optical slice of a 3-dimensional image.

So, how does this pinhole work? In an ideal imaging protocol, excitation light incident on the sample would excite the object of interest, and give a good, sharp fluorescent image (Image in figure). However, in the real world, this is not the case (Wide Field in figure). Excitation light is scattered as it passes through tissue, solutions bathing the sample, glass coverslips - even air itself. Fluorescence is also emitted from ALL the material that is excited by the incident light. Thus there is a vertical 'barrel' of fluorescnce from the focal plane of the objective and all the tissue above and below. This gives a 'fuzzy-edged' image.

The pinhole is placed in front of the PMT (Confocal in figure). It blocks the passage of this out-of-focus light into the PMT. This means that the only light to enter the PMT, and thus detected, comes from near the focal plane of the objective lens of the microscope. As this is taken across the area of the sample, it produces a an image that is a slice through the object and surrounding material. This is known as optical slicing and allows the observer to see inside the object of interest. This gives clearer images, with more fine detail observable.

3-Dimensional Imaging

3D reconstruction of part of the dendritic tree of a cultured hippocampal neuron showing interleaved axons from neighbouring neurons. In addition to the above advantages, by altering the focus of the microscope, images can be obtained at different depths. Each image is called a z-section, and can be used to reconstruct an image of the 3-dimensional object. Imagine a hard boiled egg that has been cut into slices. If the slices are piled back on top of each other, then the shape of the egg can be reconstructed. The same thing can be achieved using multiple z-sections. If images are stacked on top of one another in the correct order, a single image of the object can be generated. This technique is illustrated here with a cultured hippocam Image of an en-passant synapsepal neuron that has been modified to express Green Fluorescent Protein (GFP - click the Play button to see the image rotate). The long, thick structures are dendrites while the fine, thread-like structures are axons. The knobbly structures seen along the dendrites are dendritic spines. These are the sites of neuronal communication, the synapses, seen in more detail in the zoomed image on the right. The ability to perform these analyses is vital in work involving the co-localisation of two objects in 3-D space.