CONFOCAL LASER SCANNING MICROSCOPY

Confocal laser scanning microscopy (CLSM or LSCM) is a technique for obtaining high-resolution optical images. The key feature of confocal microscopy is its ability to produce in-focus images of thick specimens, a process known as optical sectioning. Images are acquired point-by-point and reconstructed with a computer, allowing three-dimensional reconstructions of topologically-complex objects.



DESCRIPTION

In a confocal laser scanning microscope, a laser beam passes through a light source aperture and then is focused by an objective lens into a small (ideally diffraction limited) focal volume within a fluorescent specimen. A mixture of emitted fluorescent light as well as reflected laser light from the illuminated spot is then recollected by the objective lens. A beam splitter separates the light mixture by allowing only the laser light to pass through and reflecting the fluorescent light into the detection apparatus. After passing a pinhole, the fluorescent light is detected by a photodetection device (a photomultiplier tube (PMT) or avalanche photodiode), transforming the light signal into an electrical one that is recorded by a computer.

The detector aperture obstructs the light that is not coming from the focal point, as shown by the dotted gray line in the image. The out-of-focus light is suppressed: most of their returning light is blocked by the pinhole, resulting in sharper images than those from conventional fluorescence microscopy techniques, and permits one to obtain images of various z axis planes (also known as z stacks) of the sample.



The detected light originating from an illuminated volume element within the specimen represents one pixel in the resulting image. As the laser scans over the plane of interest, a whole image is obtained pixel-by-pixel and line-by-line, whereas the brightness of a resulting image pixel corresponds to the relative intensity of detected fluorescent light. The beam is scanned across the sample in the horizontal plane by using one or more (servo controlled) oscillating mirrors. This scanning method usually has a low reaction latency and the scan speed can be varied. Slower scans provide a better signal-to-noise ratio, resulting in better contrast and higher resolution. Information can be collected from different focal planes by raising or lowering the microscope stage. The computer can generate a three-dimensional picture of a specimen by assembling a stack of these two-dimensional images from successive focal planes.





CONFOCAL IMAGE OF GFP FUSION PROTEIN




Confocal microscopy also provides a substantial improvement in lateral resolution and the capacity for direct, noninvasive, serial optical sectioning of intact, thick, living specimens with a minimum of sample preparation. Because CLSM depends on fluorescence, a sample usually needs to be treated with fluorescent dyes to make objects visible. However, the actual dye concentration can be low to minimize the disturbance of biological systems: some instruments can track single fluorescent molecules. Also, transgenic techniques can create organisms that produce their own fluorescent chimeric molecules (such as a fusion of GFP, green fluorescent protein with the protein of interest).

RESOLUTION OF IMAGES

CLSM is a scanning imaging technique in which the resolution obtained is best explained by comparing it with another scanning technique like that of the scanning electron microscope (SEM). Do not confuse CLSM with phonograph-like imaging—AFM or STM, for example, where the image is obtained by scanning with an atomic tip over a conducting surface.

In CLSM a fluorescent specimen is illuminated by a point laser source, and each volume element is associated with a discrete fluorescence intensity. Here, the size of the scanning volume is determined by the spot size (close to diffraction limit) of the optical system because the image of the scanning laser is not an infinitely small point but a three-dimensional diffraction pattern. The size of this diffraction pattern and the focal volume it defines is controlled by the numerical aperture of the system's objective lens and the wavelength of the laser used. This can be seen as the classical resolution limit of conventional optical microscopes using wide-field illumination. However, with confocal microscopy it is even possible to overcome this resolution limit of wide-field illuminating techniques because only light generated in a small volume element is detected at a given time. Here the effective volume of light generation is usually smaller than the volume of illumination; that is, the diffraction pattern of detectable light creation is sharper and smaller than the diffraction pattern of illumination. The resolution limit in confocal microscopy depends not only on the probability of illumination but also on the probability of creating enough detectable photons, so that the actual addressable volume being associated with a generated light intensity is smaller than the illuminated volume. Depending on the fluorescence properties of the used dyes, there is a more or less subtle improvement in lateral resolution compared to conventional microscopes. However, with light creation processes with much lower probabilities of occurrence such as second harmonic generation (SHG), the volume of addressing is reduced to a small region of highest laser illumination intensity, substantially improving lateral resolution. Unfortunately, the probability decrease in creation of detectable photons negatively affects the signal-to-noise ratio. One can compensate for this effect by using more sensitive photodetectors or by increasing the intensity of the illuminating laser point source. Increasing the intensity of illumination later risks excessive bleaching or other damage to the specimen of interest, especially for experiments in which comparison of fluorescence brightness is required.

BIOMEDICAL APPLICATIONS

1.)CLSM is widely-used in numerous biological science disciplines, from cell biology and genetics to microbiology and developmental biology.



(Organization of actin arrays in mitosis)

2.)Clinically, CLSM is used in the evaluation of various eye diseases, and is particularly useful for imaging, qualitative analysis, and quantification of endothelial cells of the cornea. It is used for localizing and identifying the presence of filamentary fungal elements in the corneal stroma in cases of keratomycosis, enabling rapid diagnosis and thereby early institution of definitive therapy.

3.)Research into CLSM techniques for endoscopic procedures is also showing promise.

4.)CLSM is also used as the data retrieval mechanism in some 3D optical data storage systems and

5.)It has helped determine the age of the Magdalen papyrus.

CONFOCAL MICROSCOPY

Confocal microscopy is an optical imaging technique used to increase micrograph contrast and/or to reconstruct three-dimensional images by using a spatial pinhole to eliminate out-of-focus light or flare in specimens that are thicker than the focal plane. This technique has been gaining popularity in the scientific and industrial communities. Typical applications include life sciences and semiconductor inspection.



Confocal microscopy enables the visualization and imaging of fixed as well as living cells and tissues that contain fluorescent probes (antibodies, green fluorescent proteins, dyes, substrates). This technique allows sharply defined optical sections to be collected, from which three dimensional rendering and movies can be created.


(Human cytomegalovirus infected human endothelial cells. Multicolor Immunofluorescence (IF). Blue: DAPI = cellular DNA. Green = GFP (green fluorescence protein). Red + Magenta = two different viral proteins. Captured with a Zeiss LSM510 laser scanning confocal microscope)

CONCEPT OF CONFOCAL MICROSCOPY

In a conventional fluorescence microscope, the entire specimen is flooded in light from a light source. Due to the conservation of light intensity transportation, all parts of the specimen throughout the optical path will be excited and the fluorescence detected by a photodetector or a camera. In contrast, a confocal microscope uses point illumination and a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus information. Only the light within the focal plane can be detected, so the image quality is much better than that of wide-field images. As only one point is illuminated at a time in confocal microscopy, 2D or 3D imaging requires scanning over a regular raster (i.e. a rectangular pattern of parallel scanning lines) in the specimen. The thickness of the focal plane is defined mostly by the square of the numerical aperture of the objective lens, and also by the optical properties of the specimen and the ambient index of refraction.





TYPES OF CONFOCAL MICROSCOPES



1.)Confocal laser scanning microscopes



2.)Spinning-disk (Nipkow disk) confocal microscopes

AFFINITY CHROMATOGRAPHY

Affinity chromatography is a chromatographic method of separating biochemical mixtures, based on a highly specific biologic interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand. Affinity chromatography combines the size fractionation capability of gel permeation chromatography with the ability to design a stationary phase that reversibly binds to a known subset of molecules.



Uses

1.)Affinity chromatography can be used to:
2.)Purify and concentrate a molecule from a mixture into a buffering solution
3.)Reduce the amount of a molecule in a mixture
4.)Discern what biological compounds bind to a particular molecule, such as drugs

Principle

Usually the starting point is an undefined heterogeneous group of molecules in solution, such as a cell lysate, growth medium or blood serum. The molecule of interest will have a well known and defined property which can be exploited during the affinity purification process. The process itself can be thought of as an entrapment, with the target molecule becoming trapped on a solid or stationary phase or medium. The other molecules in solution will not become trapped as they do not possess this property. The solid medium can then be removed from the mixture, washed and the target molecule released from the entrapment in a process known as elution.



Batch and Column Chromatography



Binding to the solid phase may be achieved by column chromatography, whereby the solid medium is packed onto a chromatography column, the initial mixture run through the column to allow binding, a wash buffer run through the column and the elution buffer subsequently applied to the column and collected. These steps are usually done at ambient pressure.



Alternatively binding may be achieved using a batch treatment, by adding the initial mixture to the solid phase in a vessel, mixing, separating the solid phase (by centrifugation for example), removing the liquid phase, washing, re-centrifuging, adding the elution buffer, re-centrifuging and removing the eluate.



Sometimes a hybrid method is employed, the binding is done by the batch method, then the solid phase with the target molecule bound is packed onto a column and washing and elution are done on the column.

A third method, expanded bed adsorption, which combines the advantages of the two methods mentioned above, has also been developed. The solid phase particles are placed in a column where liquid phase is pumped in from the bottom and exits at the top. The gravity of the particles ensure that the solid phase does not exit the column with the liquid phase.





Specific uses

Affinity chromatography can be used in a number of applications, including nucleic acid purification, protein purification from cell free extracts and antibody purification from blood serum.



1.)Antibody affinity

Another use for the procedure is the affinity purification of antibodies from blood serum. If serum is known to contain antibodies against a specific antigen (for example if the serum comes from an organism immunized against the antigen concerned) then it can be used for the affinity purification of that antigen. For example if an organism is immunised against a GST-fusion protein it will produce antibodies against the fusion-protein, and possibly antibodies against the GST tag as well. The protein can then be covalently coupled to a solid support such as agarose.

For thoroughness the GST protein and the GST-fusion protein can each be coupled separately. The serum is initially allowed to bind to the GST affinity matrix. This will remove antibodies against the GST part of the fusion protein. The serum is then separated from the solid support and allowed to bind to the GST-fusion protein matrix. This allows any antibodies that recognize the antigen to be captured on the solid support. Elution of the antibodies of interest is most often achieved using a low pH buffer such as glycine pH 2.8. The eluate is collected into a neutral tris or phosphate buffer, to neutralize the low pH elution buffer and halt any degradation of the antibody's activity. This is a nice example as affinity purification is used to purify the initial GST-fusion protein, to remove the undesirable anti-GST antibodies from the serum and to purify the target antibody.

2.)Immobilized metal ion affinity chromatography

Immobilized metal ion affinity chromatography (IMAC) is based on the specific coordinate covalent binding of amino acids, allowing proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions, such as cobalt, nickel, copper, zinc, or iron ions. Many naturally occurring proteins do not have an affinity for metal ions, therefore recombinant DNA techniques are used to introduce this property into a protein of interest. Methods used to elute the protein of interest include changing the pH, or adding a specific molecule, such as imidazole. Similarly, the plant protein concanavalin A is able to bind with glucose immobilised on a column matrix. The concavalin molecule is separated from the glucose by increasing the glucose concentration, displacing the concavalin from the immobilised glucose.

3.)Recombinant proteins

Possibly the most common use of affinity chromatography is for the purification of recombinant proteins. Proteins with a known affinity are tagged in order to aid their purification. The protein may have been genetically modified so as to allow it to be selected for affinity binding, this is known as a fusion protein. Tags include His-tags and GST (glutathione-S-transferase) tags. His6-tags have an affinity for nickel or cobalt ions which are coordinated with NTA for the purposes of solid medium entrapment. For elution, an excess amount of a compound able to act as a nickel ligand, such as imidazole, is used. GST has an affinity for glutathione - commercially available immobilized as glutathione agarose. For elution, excess glutathione is used to displace the tagged protein.