Shreyash Tandon M.S. III Year 20091015
Confocal microscopy is a powerful tool for generating high-resolution images and 3-D reconstructions of a specimen by using point illumination and a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane.
A laser light beam is focused onto a fluorescent specimen through the objective lens. The mixture of reflected and emitted light is captured by the same objective and is sent to the dichroic mirror. The reflected light is deviated by the mirror while the emitted fluorescent light passes through a confocal aperature (pinhole) to reduce the out of focus light. The focused light then passes through the emission filter and proceeds to the photomultiplier. In order to generate an entire image, the single point is scanned in an X-Y manner as the laser focus is moved over the specimen.
Less Cross Talk In most applications, fluorophores have overlapping emission spectra. Hence, the emission signals cannot be separated completely into different detection channels resulting in bleed through or cross talk. However, if the fluorophores have distinct excitation spectra, they can be excited sequentially using one excitation wavelength at a time. This is only possible with confocal systems that offer the multitracking feature.
Optical Sectioning of Objects Without Physical Contact Zebra fish embryo Neurons (green) Cell adhesion molecule (red)
Three-Dimensional Visualization Data gathered from a series of optical sections imaged at short and regular intervals along the optical axis are used to create the 3D reconstruction. 3D shadow projection Tight junctions (red) Cytoskeletal structures (green)
Animated 3-Dimensional Reconstruction Laser Scanning Microscopy LSM510 3D for LSM www.zeiss.com
Improved Resolution Rat Cerebellum Astrocytes (green) Mn dismutase (red) Images of cells of spirogyra generated with and without optical sectioning.
1. Resolution Inherent resolution limitations due to diffraction. Although it is assumed that the point source used produces a point of light on the specimen, it appears in the focal plane as an Airy disk, whose size depends on the wavelength of the light source and the numerical aperture of the objective lens. Airy disk similar to that of an image of a very small particle
2. Pinhole Size The strength of the optical sectioning (the rate at which the detected intensity drops off in the axial direction) depends strongly on the size of the pinhole. 3. Intensity of Incident Light 4. Choice of Fluorophores 5. Photobleaching
1. FRAP (Fluorescence Recovery After Photobleaching) A region with fluorescent molecules is irradiated or photo bleached with laser light. This results in the fluorescent molecules inside that region to become non-fluorescent. The recovery part of this experiment is the subsequent redistribution of fluorescent and bleached molecules throughout the volume. This gives information on their mobility. Using FRAP, one can determine the mobility of fluorescently tagged proteins in living cells.
2. FRET (Fluorescence Resonance Energy Transfer) The high resolution of a confocal microscope allows us to study the physical interaction of protein partners. FRET is the non-radioactive transfer of photon energy from an excited fluorophore (the donor) to another fluorophore (the acceptor) when both are located within close proximity (1-10nm).
Using FRET one can resolve the relative proximity of molecules beyond the optical limit of a light microscope to reveal (1) molecular interactions between two protein partners, (2) structural changes within one molecule (eg. enzymatic activity or DNA/RNA conformation), (3) ion concentrations using special FRET-tools like the CFP-YFP cameleon No FRET Signal CFP is excited by light and emits light CFP is more than 10nm from YFP YFP is not excited and does not emit light FRET Signal CFP is excited by light and emits light CFP is in close proximity to YFP YFP emits light
3. Colocalization To distinguish between small features such as proteins within a cell it is useful to tag them with different fluorophores and image them as separate colors. There are two ways to do this: (a) fluorophores are selected to correspond with the wavelengths of a multiline laser (b) their response to the same excitation wavelength causes emission at different wavelengths.
Colocalization of insulin and calcitonin receptor-like receptor Colocalization of up to 4 different proteins
4. FLIM (Fluorescence Lifetime Imaging) The fluorescence lifetime is defined as the average time that a molecule remains in an excited state prior to returning to the ground state. The lifetimes of many fluorophores are altered by the presence of ions such as Ca 2+, Mg 2+, Cl or K+. This allows the researcher to follow environmentally induced changes. An advantage of lifetime imaging is that the absolute values of lifetimes are independent of the probe concentration, photobleaching, light scattering and the amount of excitation intensity. Fluorescence lifetime imaging (FLIM) thus offers several opportunities to study dynamic events of living cells.
5. FLAP (Fluorescence Localization After Photobleaching) The molecule to be located has 2 fluorophores, one to be bleached, and the other to act as a reference label. One can then track the distribution of the molecule after it is bleached. The FLAP signal is obtained by subtracting the bleached signal from the unbleached one, allowing the tracking of the labelled molecule.
Most confocal microscopes generate a single image in 0.1 1 sec. For many dynamic processes this rate may be too slow, particularly if 3D stacks of images are required. Two commonly used designs that can capture images at high speed are : (a) the Nipkow disk confocal microscope, and (b) a confocal microscope that uses an acousto-optic deflector (AOD) for steering the excitation light.
Acousto-Optic Deflector (AOD) Speeding up the image acquisition rate can be achieved by making the excitation light beam scan more quickly across the specimen. An AOD is a device that deflects light by creating a diffraction grating out of a crystal using sound waves. The major disadvantage of AODs is that they are wavelength sensitive. That is, different wavelengths experience different degrees of deflection.
Nipkow Disk Instead of scanning a single point across the specimen the Nipkow disk microscope builds an image by passing light through a spinning mask of pinholes, thereby simultaneously illuminating many discrete points. The disadvantage of the Nipkow disk microscope is that only a small fraction (1%) of the illuminating light makes it through the pinholes to the specimen.
Provides excellent optical sectioning. It addresses a fundamental drawback of confocal laser scanning microscopy: that the beam also excites the specimen above and below the focal-plane In confocal microscopy, a single highenergy photon excites a fluorophore molecule while it takes two lowerenergy photons absorbed simultaneously to achieve the same result in two-photon microscopy.
Review of Confocal Microscopy Denis Semwogerere and Eric R. Weeks Emory University, Atlanta, Georgia, U.S.A. Yvona Ward Cell and Cancer Biology Branch Helix Systems, NIH, U.S.A Michael Hooker and Michael Chua Microscopy Facility, Cell and Molecular Physiology University of North Carolina, U.S.A Microscopy and Imaging Facility School of Biochemistry and Immunology Trinity College, London, U.K. Lecture in Pricipals of Confocal Microscopy Bartek Rajwa, PhD Purdue University, West Lafayette, Indiana, U.S.A.