Group 6 Project - Confocal Microscopy
Confocal Microscopy is a technique which has revolutionized the light microscope in its ability to create more detailed images of cells and their components. In combination with immunohistochemistry, the microscope has evolved to allow images and real time movies of in vitro and even some in vivo cells which has allowed cell biologists to examine the structure and function of many cellular components and cells as a whole.
- 1 Basic Mechanism
- 2 Timeline and Development
- 3 Today's Microscopes
- 4 Selected Useful Applications
- 5 Evaluation
- 6 Glossary
- 6.1 Aperture
- 6.2 Artefacts
- 6.3 CCD Camera
- 6.4 Contrast Media
- 6.5 Deconvolution
- 6.6 Dichroic Beam Splitter
- 6.7 Digital Micro-mirror Device (DMD)
- 6.8 Emission Light
- 6.9 Excitation Light
- 6.10 Fluorescence Recovery After Photobleaching (FRAP)
- 6.11 Fluorescent probes
- 6.12 Fluorescent tagging
- 6.13 Fluorophores
- 6.14 Focal plane
- 6.15 Galvanometer
- 6.16 Interference (DIC) microscopy
- 6.17 Multi-photon
- 6.18 Noise
- 6.19 Optical sectioning
- 6.20 Out-of-focus light
- 6.21 Photobleaching
- 6.22 Photon
- 6.23 Phototoxicity
- 6.24 Pseudorandom Sequence
- 6.25 Raster Scan
- 6.26 Refraction Index
- 6.27 Resolution
- 6.28 Resonant Scanner
- 6.29 Spatial Light Modulator (SLM)
- 6.30 Spherical aberrations
- 6.31 Superposition
- 6.32 Temporal resolution
- 6.33 Wavelength
- 6.34 Wide Field Imaging
- 6.35 Zirconium Arc
- 7 References
When looking through a conventional microscope, the light that is not in focus will be transmitted into the lens and visible as a blur for the viewer. However, confocal microscopy aims to limit the amount of light that enters the lens as well as limiting the illumination to a particular point as to eliminate the noise from the light of surrounding cells that creates blurring. A pinhole or aperture is designed to limit the focal plane to the cell required while a light source passes through the same convex lens on the same focal path (hence the name, confocal) and uses a dichroic beam splitter, to illuminate the same point on a sample.
The following diagram explains the whole process very simply:
The microscope will then move the lasers to examine different points and planes so that the final image can be formed. Essentially, one pixel on a computer equals the amount of light seen at a particular focused spot on the specimen.
Timeline and Development
The initial patent for a confocal microscope was granted to Marvin Minsky in 1961, whilst he was working at Harvard University. Minsky configured his microscope so that the point source of light was attained by placing a pinhole in front of a zirconium arc. Minsky scanned the light beam across his image not by moving his beam of light, but by moving the specimen on a vibrating stage. The main problems with confocal microscopy at this time were that the systems used to view the image did not have the resolution needed to show the full potential of confocal microscopy and that the system was too slow and vibration sensitive for biological uses. Petrán solved this problem independently by including a Nipkow disk in his arrangement instead of having just a single point of light. This was essentially the same as having many single points running simultaneously.
Slowly, additional improvements have become available to researchers, but the basic principle of the confocal microscope has remained unchanged and the same problems were ever present. These were the wavelength of light, the objective lens and the properties if the specimen itself. Advances such as better lasers for point accuracy, better mirrors, video displays and improved software for image analysis to help reduce noise were developed independently from the confocal microscope, but have all contributed to improving the imaging capabilities and resolution of the device.
And therefore increase resolution of the image recovered. A major increase in the usefulness of confocal microscopy came in the 1980’s, where confocal microscopy and fluorescence microscopy were used together at the MRC in Oxford to create a prototype that had addressed many of the problems associated with confocal microscopy earlier to that date. Companies such as Zeiss and Nikon quickly produced copies of this new form of confocal microscope.
The massive improvement in image that was given over a conventional fluorescence microscope was the main attraction for confocal microscopes and attributed greatly to its popularity.
The next great advancement of confocal microscopy came in the 1990’s, with the confocal microscope laid the foundation for two-photon induced fluorescence imaging. 
By excluding much of the light surrounding the targeted cell(s), the image from the confocal microscope will appear sharper with higher contrast. If we examine the images from a conventional microscope, we will see that the internal structures of a cell are very blurred and almost impossible to make out. This is because the light has not been excluded in any way and the out-of-focus light is still viewed, making the image appear blurred. The blurriness or noise from conventional microscopes attributes to its lower resolution compared to confocal microscopes. This makes confocal microscopy very useful in the examination of the internal structures of cells, especially in a thick sample. 
The depth at which confocal microscopy is able to image depends on the type of specimen being scanned. The typical maximum depth of imaging is currently 40 micrometers which can be increased to 100 micrometers if spherical aberrations are eliminated from the laser pathway.
The use of the confocal microscope really owes much of its success to fluorescent tagging, where a targeted protein can be expressed through the attachment of a fluorophore. Without this technique, the internal structures of a cell wouldn’t be examinable with light microscopes. fluorescent tagging allows us to target nearly any protein inside a cell and view its structure, distribution and movement within a cell. The cells are excited at a specific wavelength and emit a different wavelength which is recorded by a CCD camera.
Confocal microscopy has also been used in conjunction with multiphoton imaging. Typical fluorescence imaging will use a short excitation wavelength to produce a larger emission wavelength whereas multiphoton involves the use of two long wavelengths (ie. Infra-red) to excite the fluorophore and produce a shorter wavelength. This produces a sharper image as out of focus light will not be present as the fluorophore is excited only at the point of focus. For this reason, a pinhole isn't needed at the detector. 
As new and faster ways of scanning different locations on a sample, the confocal microscope is able to examine all three dimensions of a cell at various times so that a three dimensional, time-lapse “movie” is possible. The relative size of an image can be adjusted by altering the magnification or by adjusting the size of the scanning field.
The whole process is very much dependent on computers. For images which involve a Z plane (the 3rd dimension), the microscope will scan a 2D layer within a thick section and then move onto the next thin section. The computer then has to deconvolute the images where it uses complex algorithms to remove some of the processes of optical illusion. This whole process is known as optical sectioning and it is very useful as it allows thick specimens of living cells or tissues in culture to be examined without having to physically slice them into thinner sections and prepare each section.
The following clip is of an S2 cell undergoing mitosis using confocal microscopes which is fluorescence tagged for tubulin. The movie time-lapsed over about 9 minutes which indicates just how fast the microscope is working. Tubulin in mitotic cell
Types of Confocal Microscopes
Confocal Laser Scanning Microscopy (CLSMs)
The laser scanning microscope acts exactly the way you would assume from the name. It seems logical to move the lasers rather than the specimen simply for the speed at which laser scanning can operate. The scanner will operate in a raster pattern (like in a television) until the whole field is scanned. Typically, a 500x500 pixel image in one dimension will take 2.5 seconds which limits the frame rate for time-lapse.
The movement of the lasers and subsequent emissions from the specimen are controlled by the internal mirrors which are moved by galvanometers controlled by computers. This is what limits the speed of laser scanning microscopes. However, new resonant scanner and high resolution galvanometers can achieve speeds of 30 milliseconds per 512x512 pixel images (translates to 30 frames per second) which now makes it applicable to live cell imaging. 
Another use of the CLSM is the aperture size which is adjustable. By narrowing the pinhole, the thinner the individual sections will be for optical sectioning and vice versa. Narrowing the pinhole size can also produce sharper images but these will contain less light. A balance between blurriness and illumination must be chosen. 
A number of images taken from a CLSM are available on the Nikon Website
Spinning Disk (Nipkow Disk) Confocal Microscopy
The spinning disk confocal microscope works by having numerous pinholes by which both the lasers and light from the specimen will pass. The disk typically has a spiral pattern of about 20,000 pinholes which will spin at up to 5,000 revolutions per minute. A column of excitation light will pass through about 1,000 pinholes to image the entire field every millisecond! This is the microscope that is able to create real-time movies with the possibility of 2000 frames per second which is necessary for examining some of the cellular processes which occur in milliseconds. Additionally, the excitation light will pass through an identical spinning disk which has micro-lenses instead of pinholes. This must rotate on the same axis as the pinholes disk.
What is useful is that to make a wide field fluorescence image, the disk can be removed from the light path. Some slits can also be closed so that only certain areas are illuminated and this is used for Fluorescence Recovery After Photobleaching (FRAP).
A number of movies taken with spinning disk confocal microscopes are available on the Nikon Website.
Reflectance Confocal Microscopy (RCM)
This is a relatively new concept which was developed for in-vivo examination of the skin or eye since it is relatively transparent. It works on the practice of reflected light rather than fluorescence. In the skin, reflection will occur where two cellular structures have differing refraction indexes such as a cell membrane or melanosomes. Reflectance can also occur when the laser’s wavelength is similar to the object being viewed (700-1400nm). The power of this laser must be controlled to avoid tissue damage. The technique can be applied with both laser scanning and spinning disk confocal microscopes.
The following image is a RCM image of keratinocytes in vivo.  What is noticeable is that the sharpness and resolution of the image is reduced compared to fluorescence confocal microscopy.
Programmable Array Microscopes (PAM)
Programmable Array Microscopes (PAM) function using a spatial light modulator (SLM) placed to generate an arbitrary pattern of illumination and detection elements.  Generally, imaging with the PAM is done in one of two ways. The first way is to use a grid of points is shifted to simply scan the whole image. The other way, which is faster uses what is called a pseudorandom sequence to scan the image. Yet, if the latter approach is used, then processing is required to retrieve the confocal image.
A commonly used from of PAM is as a reflection microscope. What is interesting about this type of microscopy is how the light is split by a digital micro-mirror device (DMD) to form to images, referred to as the conjugate image and the non-conjugate image. This each light paths are captured on different cameras, and separation is achieved by moving the DMD to an ‘on’ and ‘off’ positions. The non-conjugated light image consists of light deflected by the DMD in the ‘off’ position and corresponds to the emission light that is blocked by the aperture of the confocal microscope. 
Development in this area goes in the way of dual path PAM, as opposed to single sided PAM, as it has significantly less noise and sharper focus. 
Total Internal Reflectance Microscopy (TIRF)
Total internal reflection fluorescence microscopy has the advantages of being able to give images that have very low background fluorescence and virtually no out of focus light, reducing the amount of noise in the image and increasing clarity. 
The theory behind the TIRF microscope relies on a principle of light known as total internal reflection. This is a special case of refraction that occurs when the beam of light hits the changing medium (solid/liquid solid/gas etc), it actually reflects back into the prism. This angle is called the critical angle.
This property of light means that there is only a small plane that is fluorescent, and the light does not actually go through the section. This means that there is a massive decrease in the chance of cells suffering from phototoxicity. This is because the light only generates a “field of evanescence” which is about 100nm in height, which can be less than the width of some cells. 
Selected Useful Applications
- Microtubules: Confocal microscopy can be used to visualise elements within the cell such as microtubules. For example, in a study done by Azakir et al., it was found that the transmembrane protein "dysferlin" interacts with microtubular structures in rat cells. Dysferlin mutations are known to lead to disorders such as Miyoshi Myopathy, however, its mechanism has not been well understood. Due to advancements in confocal microscopy, it has now been suggested that microtubules play a role in dysferin trafficking.
- Microvessels: Confocal microscopy can also be used for researching physiological aspects such as microvessel diameter and blood-brain barrier disruption. This was the case in a study completed by Lu et al., where they used laser scanning confocal microscopy to find that ‘dynamic contrast enhanced perfusion MR imaging’ can indicate post-ischemic hyperemia and microvascular damage. Thus, confocal microscopy can be used both directly and indirectly where it determines the effectiveness of another system.
- Diseases: Confocal microscopy can be used to visualise the interactions of pathogens with their host e.g. it was found that Salmonella type III interacts with the host via ‘Slrp’ with either thioredoxin (in the cytosol) or ERdj3 (in the endoplasmic reticulum).
- Enzymes: Using fluorescence confocal microscopy, enzymes can be visualised and their role hypothesised. For instance, in the study completed by Tyteca et al., it was shown via confocal microscopy that the enzyme NAT8 is associated with the endoplasmic reticulum in rat cells.
The following weblink provides a range of fascinating confocal images and allows you to move through the specimen (in order to visualise the various layers contributing to its overall structure): http://www.microscopyu.com/galleries/confocal/
- Image quality: Since the early stages of microscopy, artefacts have limited the quality of microscopic images. Thus, future uses will be in familiar areas where the goal is to increase image quality.
- Resolution: Additionally, resolution is important as it effects the measured time of an event e.g. if temporal resolution is insufficient then the time-course of events wouldn’t be measured accurately.
- Correlative microscopy: Even when significant care is taken to minimise optical aberrations and other artefacts, incorrect interpretation of images can still occur. Correlative microscopy becomes vital in these situations since it uses two or more types of microscopy to reduce incorrect interpretations. For instance, a sample can be viewed with both confocal and interference (DIC) microscopy. When multiple microscope techniques show similar structures, it is a good indication that the images correspond to the actual structure of the specimen. In time, the use of correlated microscopy will increase and thus, increase the accuracy of observations and measurements in research studies.
- Multimodal microscopy: Further developments could also be within the realm of multimodal microscopes which contain several imaging modes e.g. a confocal microscope with both single-photon mode and multi-photon excitation mode. Another possibility is the combination of interference (DIC) microscopy and confocal microscopy which has the advantage of minimising artefacts and obtaining additional information. Unfortunately, with increasing numbers of operating modes, the image quality in each mode is reduced.
- Clinical: Further clinical developments are leading to confocal endoscopes and other devices for live cell and tissue imaging. For example, in ophthalmology there are clinical confocal microscopes being used for visualising the in vivo human anterior eye. There have also been developments in dermatology, however, there has yet to be significant development in clinical diagnostic medicine. This is a possible area of development for applications in the future.
- Light sources: Other developments will be in light sources that are cheaper, more compact, have easier maintenance and have a wide range of tunability.
- Far-field optical microscopy: Developments will also be in the manufacture of microscope objectives that have longer working distances and accordingly, allow deeper penetration into thick tissues.
- New probes and contrast media: An active area in confocal microscopy is in the development of new fluorescent probes and contrast media. This would lead to the reduction in bleaching of the specimen and the greater localisation of the probes in the appropriate areas of interest.
Confocal microscopy is a widely utilised tool within biological and biomedical investigation. Though there are some limitations to confocal microscopy, there are also numerous advantages which have lead to its increasing popularity.
|Live tissue: Confocal microscopy allows the non-destructive observation of living cells and tissues. This means that changes within the cell due to various cellular processes can be noted, unlike in electron microscopy.||Limited fluorophores: Confocal microscopy utilises lasers which emit 1 or a few narrow lines of light at one instant. This means that there is a relatively small choice of laser wavelengths and consequently, a relatively limited choice of fluorophores that can be used.|
|Wide protein range: Due to advancements in fluorescent tagging, virtually any cellular protein can be tagged e.g. by green fluorescent protein and visualised through confocal microscopy.||Laser cost: Maintenance costs of some lasers can be high due to frequent tube replacement e.g. the argon-krypton laser. However, this can be overcome by using multiple lasers e.g. the relatively robust and inexpensive argon and helium-neon lasers.|
|3D image: Unlike regular light microscopes, confocal microscopes provide a 3D picture so that structures can be seen as in real life and changes in response to stimuli can be visualised. This is done by taking serial (rather than singular) images to create the overall structure of the cell.||Restriction of field of view: Though confocal microscopy effectively excludes light, it also is restricted by the size of the pinhole. The field of view is basically a spot the size of the remagnified pinhole.|
|Observation of heterogeneity: Cells respond to stimuli differently. This means that the bulk-type biochemical and physiological measurements don’t accurately represent the time-course and magnitude of individual cellular changes. In confocal microscopy, singular cells can be visualised and thus, their differing responses can be noted.||Dimness: Confocal images are formed from thin sections of a thick specimen and accordingly, their images are considerably dimmer than, for instance, wide-field fluorescence images. To make up for this, the specimens are exposed to stronger light and are thus vulnerable to photobleaching and damage.|
|Increased contrast: The images obtained through confocal microscopy are of better depth and quality in comparison with regular light microscopes and similar. This is due to both the smaller depth of field and the rejection of out-of-focus light. For example, in thick specimens using light microscopy, the superposition of out-of-focus light from specimen features causes image deterioration. In fluorescence microscopy, out-of-focus fluorescence leads to diffuse halos surrounding the in-focus structures of interest. Confocal microscopy eliminates such artefacts through taking a series of very thin optical slices of specimen and the pinhole feature. In-focus light is focused precisely at the pinhole while that from above and below the focal plane is spread out at the pinhole and selectively blocked.|
|Thick specimens: Even in a very thin specimen of 5 um, 80% of the light may be coming from out-of-focus regions. However, for the reasons previously discussed, confocal microscopes are able to emit out-of-focus light and thus, make it possible to view details within thick specimens without physically sectioning the tissue (and often introducing artefacts).|
Another word for the pinhole through which light is limited.
Properties in the image of microscopic objects that are not inherent to the object in its natural state.
A Charge-coupled device camera is a high quality imaging device used in light micrscopy.
Agents which enhance the contrast of a specimen of interest and accordingly, visually enhance imaging.
The reversing of the processes of optical distortion done by software algorithms. It removes out-of-focus light from planes of interest. 
Dichroic Beam Splitter
An optical mirrored device which is able to split light into separate wavelengths. Longer wavelengths will be transmitted through a longpass filter whereas shorter wavelengths are reflected.
Digital Micro-mirror Device (DMD)
The light emitted from an excited fluorophore that travels back to the camera for imaging. This wavelength will be different to the wavelength used to excite the molecule. 
The light (laser in confocal microscopy) that is used to illuminate/excite the fluorescence molecules in a specimen.
Fluorescence Recovery After Photobleaching (FRAP)
A value given that refers to how well a cell recovers it fluorescence over time. This value can be used to investigate the movement of molecules inside of a cell.
Substances labelled with a flurophore that are able to detect particular substances of interest. 
Utilised to aid in the detection of a molecule via chemically attaching a fluorophore to the molecule of interest.
Structural regions within molecules that are able to emit fluorescent light. 
The place in an optical device where the image of interest is focused.
Consists of a coil of wire (with attached pointer) suspended in the magnetic field of a permanent magnet. Measures current in an electric circuit. 
Interference (DIC) microscopy
A type of phase-contrast microscopy which is useful in visualising transparent specimens.
Microscopy that uses two or more photons that have differing wavelengths. 
An undesirable by-product of imaging resulting from random variation of brightness from unwanted background light.
The image slicing of thin, 2D sections of a specimen using a pinhole and deconvolution techniques in confocal microscopy.
The usually irreversible photon-induced damage of specimens via the formation of free oxygen radicals.
A quantum of light. The basic unit of light.
Cell death as the result of high levels of exposure to light. This mainly occurs from the production of reactive oxygen species. 
A scan which forms a 2D image by scanning along a horizontal plane in one direction before scanning the next horizontal plane in the other direction until the whole vertical plane is complete. Essentially, it scans in a zig-zag pattern.  see image
A measurement which reflects the ability of a medium to bend light as the light passes through it. 
In an optical system, the resolution is the ability of the system to distinguish between two points of a certain distance. 
A type of galvanometer which is able to scan multiple fields without altering optical zoom. This allows for rapid scanning. 
Spatial Light Modulator (SLM)
A blur circle which is generated at a focal point due to the fact that a circle is not perfect for imagery on a spot on an axis (or a field point). 
The addition of multiple light waves to form a resultant wave.
The frequency at which images are captured. The higher the frequency, the higher the temporal resolution.
The distance of one cycle/oscillation of an electromagnatic wave. 
Wide Field Imaging
The normal light microscope in which the image is obtained by simple transmission of light through the object being viewed.
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Fluorescent-PCR | RNA Interference | Immunohistochemistry | Cell Culture | Electron Microsopy | Confocal Microscopy | Monoclonal Antibodies | Microarray | Fluorescent Proteins | Somatic Cell Nuclear Transfer