Microscopy

Microscopy is the technical field of using microscopes to view samples and objects that cannot be seen with the unaided eye (objects that are not within the resolution range of the normal eye).

Magnification An object can be focussed generally no closer than 250 mm from the eye. This is considered to be the normal viewing distance for 1x magnification. Younger people may be able to focus as close as 125 mm so they can magnify as much as 2x because the image covers a larger part of the retina. As regard microscopy, a typical animal cell is 1020 m in diameter, which is about one fifth the size of the smallest particle visible to the naked eye.

Resolving power This shows the sizes of various cellular and subcellular structures and the ranges of size that different types of microscopes can visualize.

The compound Microscope Be able to draw!!

1. The specimen is mounted on a transparent glass slide and positioned on the movable stage of the microscope. 2. Light from a bright source is focused by the condenser lenses onto the specimen. 3. The objective lenses pick up the light transmitted by the specimen and focuses it on the focal plane of the objective lens, creating a magnified image of the specimen. 4. Usually the image is again magnified by the ocular lens, or eyepiece, which projects it onto the plane of the human eye or onto a piece of photographic film or a video camera.

There are 3 different types of lenses: Ocular, Objective and Condenser.

Resolution The resolution is the most important property of any microscope. It is defined as the ability to distinguish between two very closely positioned objects. Resolution depends on both: 1. The wavelength of the light The limit of resolution of the light microscope is set by the wavelength of visible light. This ranges from about 0.4 m (for violet) to 0.7 m (for deep red). 2. The numerical aperture of the lens system used The numerical aperture of a microscope objective is a measure of its ability to gather light and resolve fine specimen detail at a fixed object distance.

Numerical Aperture The numerical aperture (NA) of an optical system is a dimensionless number that characterizes the range of angles over which the system can accept or emit light. Numerical aperture is commonly used in microscopy to describe the acceptance cone of an objective and hence its light-gathering ability and resolution. NB the following: The higher the NA the greater the resolution. The wider the angle that the lens is capable of receiving light at, the greater its resolving power. The higher the NA, the shorter the working distance.

Under the best conditions, with violet light (wavelength = 0.4 m) and a numerical aperture of 1.4, a limit of resolution of just under 0.2 m can theoretically be obtained in the light microscope. In practical terms, bacteria and mitochondria, which are about 500 nm (0.5 m) wide, are generally the smallest objects whose shape can be clearly discerned in the light microscope; details smaller than this merge into one and are obscured/blurry.

Visualising fixed (i.e., dead) cells/tissues under the light microscope by: (i) Fixing (ii) Sectioning (iii) Staining

(i) Fixing Fixing cells means to Immobilise kill and preserve the cells/tissues. The aim is to rapidly fix structures in place so that they can be seen clearly under the microscope. This method makes cells permeable to staining reagents. Some fixative agents used in the past are ethanol, methanol or acetone. Current fixatives involve the use of reactive aldehydes, particularly formaldehyde and glutaraldehyde, which cross-link adjacent protein molecules so that they are stabilised and locked in position.

(ii) Embedding/Sectioning Embedding Cells are embedded using waxes or resins, usually paraffin or plastic. The cells can also be frozen. This avoids alteration of the cells in undesirable ways i.e., the denaturation of enzymes by fixatives such as formaldehyde. Sectioning The tissues are usually cut into very thin slices, or sections, (typically 110 m thick) with a microtome. These sections are then laid flat on the surface of a glass microscope slide.

(iii) Staining: revealing the structure Cells are tiny, colourless and translucent. Therefore we need to stain them in order to see them clearly under the microscope. Chemical stains H & E staining Haematoxylin (Purple) binds to basic amino acids (lysine and arginine) on many different kinds of proteins. Eosin (red) binds to acidic molecules (such as DNA, and aspartate and glutamate side chains). The only limitation of H& E staining is that they lack sensitivity. Fluorescent stains can be used when we want to stain specific proteins e.g. to distinguish between resting platelets and active platelets. This is called Immunofluorescent microscopy. Fixed cells or tissues are permeabilised and stained with fluorescently-labelled antibodies. Using antibodies to localise proteins (in fixed cells) Staining with antibodies can be direct (primary antibodies) or indirect (secondary antibodies). Some markers for secondary antibodies include; Enzymes- e.g., horse radish peroxidase. This enzyme will catalyse a colour producing reaction. Fluorophores e.g., FITC, Rhoda mine, Texas red. Colloidal gold spheres- for immunogold electron microscopy.

Immunohistochemistry (in fixed cells) Samples are sections of tissue, where each cell is surrounded by tissue architecture and other cells normally found in the intact tissue. This is used in diagnostics- clinical biopsies and the diagnosis of tumour samples. Fluorescent dyes (Fluorophores) A fluorophore is a chemical that is said to be fluorescent if it absorbs light at one wavelength (the excitation wavelength) and emits light (fluoresces) at a specific and longer wavelength. The emitted light is of longer wavelength (and hence of lower energy) than the light absorbed. For example: DAPI absorbs UV/emits bright blue (used routinely to stain DNA). Cy3, absorbs green and emits orange light. Fluorescein (FITC) absorbs blue/green and emits green light. Cy5 absorbs red and emits infrared (far red) light. Fluorescence Microscopy FM is similar to a light microscope except two filters and dichroic mirror are used. Only fluorescent light emitted by the sample is used to form an image; light of the exciting wavelength induces the fluorescence but is then not allowed to pass the filters placed between the objective lens and the eye/camera.

Visualising living cells and tissues

Living cells are dynamic cells and are in constant motion: trafficking From one organelle to another In and out of the cell. Degradation/replacement Introducing membrane impermeant molecules into living cells Microinjection Purified protein coupled to a fluorescent dye Antibodies Disadvantage: only one cell at a time Partial disruption of the cell membrane Electroporation Pores stay open for mins/hrs. Brief but large electric shock. Creates large transient holes in the plasma membrane Without disruption of intracellular membranes Low concentration of a detergent Imaging dynamic processes in living cells Optical enhancement of light microscopy Fluorescent tags/Fluorescent microscopy Attached to molecules to be imaged which range from small inorganic ions to large macromolecules Confocal microscopy: Produces sharper images and allows 3D reconstruction of cells/tissues

Optical Enhancement of microscopy for live material No need to Stain cells!! Normal Bright field Light microscopy (in comparison) Images appear dark, background is white; specimens need either natural pigments or stained. Not good for live material Darkfield The cell appears as a bright object against a dark background. The illuminating rays of light are directed from the side so that only scattered light enters the microscope lenses. Phase Contrast/Differential-Interference-Contrast (DIC) Images appear bright, background is dark; measures variations in light intensity via density of specimen

Phase-Contrast and DIC Microscopy This method offers detailed views of transparent, live, unstained cells and tissues. The phase of the light is altered by its passage through the cell, and small phase differences can be made visible by exploiting interference effects using a phase-contrast or a differential-interferencecontrast (DIC) microscope. For example: light passing through the nucleus, is retarded and its phase shifted relative to light passing through the cytoplasm. This can be converted into differences of light and dark in the final image. Time-lapse microscopy Here the same cell is photographed at regular intervals over periods of several hours. This method of microscopy is used to observe cell movement such as mitosis or cell migration. Fluorescent microscopy in living cells Fluorescence-microscopy techniques are also useful living cells Confocal microscopy generally always used. Confocal Microscopy This is microscopy in a Conjugate focal plane i.e., same plane imaging. There are different types of confocal microscopes. Generally used with fluorescence optics. The main difference is that it gets rid of out of focus light. This results in a sharper image. Confocal uses a laser beam as a pinpoint source of illumination to produce an incredibly clear/sharp image of one point. It can also be used to build a two-dimensional Optical section. Data is collected from each point in the plane of focus and a composite image is computer generated. Three-dimensional reconstruction of confocal images This method records individual fluorescent images of planes at different depths of the sample and combines the stack of images into one three-dimensional image. Applications of fluorescent microscopy in living cells - in real time - fixing over time Cell Motility Assays Monitoring Cytoskeleton Dynamics Embryo Development- mitosis Intracellular Ion Signalling Using dyes whose fluorescence is proportional to the concentration of Ca2+ or H+ ions, fluorescence microscopy can measure the local concentration of Ca2+ ions and intracellular pH in living cells. GFP expression Fluorescent Resonance Energy Transfer (FRET)

Fluorescence resonance energy transfer (FRET). FRET is used to monitor protein interactions. Two molecules of interest are each labelled with a different fluorochrome, chosen so that the emission spectrum of one fluorochrome overlaps with the absorption spectrum of the other. If the two proteins bind so as to bring their fluorochromes into very close proximity (closer than about 2 nm), the energy of the absorbed light can be transferred directly from one fluorochrome to the other. When the complex is illuminated at the excitation wavelength of the first fluorochrome, light is emitted at the emission wavelength of the second.

Electron Microscopy
Electron Microscopy is a very useful way of imaging cells that are fixed or dead. The atoms appear black because they are electron dense. White on the image represents the spaces between them. EM uses a beam of electron radiation to produce images. The best resolution is in the range of 0.1-0.2nm which is nearly 1000 times greater than light microscopy. EM involves the expensive chemical preparation of specimens and the use of very sophisticated equipment. There are two types of EM; Transmission Electron Microscopy (TEM) Scanning Electron Microscopy (SEM)

An Electron Microscope is similar to a much larger upside-down light microscope. The source of illumination is the electron gun. This is a filament or tungsten cathode that is heated and emits electrons. The Cathode voltage ranges from 50,000 100,000 volts. The magnetic coil puts the electrons back on course so that we can view the on the photographic plate.

Transmission Electron Microscopy (TEM) TEM is what is usually considered electron microscopy. The highest resolution TEM can reach is 0.1nm which allows extremely detailed images to be produced. Because of this, TEM machines are very expensive.

Fixing of Specimens for the EM Specimens for EM are generally fixed, sectioned, and dehydrated, and then stained with electrondense heavy metals such as Glutaraldehyde (which covalently cross links protein molecules to neighbours) and Osmium tetroxide. Preparing sections Dehydration/Embedding Permeate tissue with a monomeric resin that polymerises to form a solid block of plastic. Sometimes Supercool (rapid) first before dehydration. This is where water becomes a glass called vitreous ice.

Sectioning Like microtome for light microscopy but the blade is different. Ultra-thin sections of 50-100 nm thickness are cut. These thin sections, free of water and other volatile solvents, are placed on a small circular metal grid for viewing in the microscope.

Staining Staining can take place before or after sectioning. The tissues are stained/impregnated with electron-dense material to make cells visible or else the image will all just appear bright. Areas with reduced electron flux: electrons passing through are scattered by structures stained with electron dense material. These areas appear dark as they are not focused by the electromagnetic lenses. Areas with normal electron flux: Other electrons pass through the non-stained sections and appear bright as these electrons are focused by the electromagnetic lenses to form an image. Electron-dense material: Usually with salts of heavy metals, e.g., gold, osmium, uranium or lead. Osmium tetroxide preferentially stains membranes. Stain all proteins in a cell with electron-dense gold particles coated with protein A, a bacterial protein that binds protein molecules nonspecifically.

Immunogold staining and Immunogold Electron Microscopy Immunogold EM is used to localise proteins in EM. It involves the use of Antibodies and the usual procedure is Indirect antibody binding. Usual protocol: Thin section Primary antibody Secondary antibody/colloidal gold used for particles seen as black dot as they are electron dense.

Metal Shadowing

TEM metal shadowing allows the study of surface features/shape at High Resolution Use a thin layer of evaporated metal, e.g., platinum. This gives a 3D effect on the

Freeze-Fracture Microscopy A preparation method particularly useful for examining lipid membranes and their incorporated proteins in "face on" view. The fresh tissue or cell suspension is frozen rapidly (cryofixation), then fractured by simply breaking or by using a microtome while maintained at liquid nitrogen temperature. This method allows us to visualise the interior of cell membranes i.e., distribution of proteins To carry out the technique: Frozen block Cracked with knife Freeze-etching The cold fractured surface (sometimes "etched" by increasing the temperature to about 100 C for several minutes to let some ice sublime) is then shadowed with evaporated platinum or gold at an average angle of 45 in a high vacuum evaporator. The specimen is returned to room temperature and pressure, then the extremely fragile "preshadowed" metal replica of the fracture surface is released from the underlying biological material by careful chemical digestion with acids, hypochlorite solution or SDS detergent. The still-floating replica is thoroughly washed free from residual chemicals, carefully fished up on fine grids, dried then viewed in the TEM. Technique: Frozen block Cracked with knife blade Difference: Freeze-drying then shadowed In this way get 3D organisation of exposed interior structures. Negative Staining Negative staining is used to view: large macromolecular aggregates e.g., viruses or ribosomes, subunit structure of protein filaments Finer detail can be seen than by using shadowing alone. The technique involves: Molecules adsorbed on a thin film of carbon Wash with a heavy-metal salt solution e.g., uranyl acetate. Dry sample a very thin layer of metal salt covers the carbon film except over the adsorbed macromolecule. Electrons pass through the macromolecule and not the surrounding heavy-metal stain

Cryoelectron Microscopy This method is used for UNSTAINED and UNFIXED cells. Ultra-thin (~100nm), unfixed, unstained specimens (virus/purified macromolecule complex) are rapidly frozen to form vitreous ice (hydrated form).

EM Tomography EMT is a widely used imagine and diagnostic technique. The normal EM detector gives a 2D image. However, EM has a large depth of field with all parts in focus so it can take images from many different directions (up to ~10,000) to reconstruct a 3D image. Individually each image can be quite noisy but average of combination will eliminate the noise. EMT is advantageous as we can begin to see the internal atomic arrangements in a protein to rival x-ray crystallography.

Scanning Electron Microscopy A scanning electron microscope (SEM) is a type of electron microscope that images a sample by scanning it with a beam of electrons in a raster scan pattern. The electrons interact with the atoms that make up the sample producing signals that contain information about the sample's surface topography, composition, and other properties such as electrical conductivity. SEM has a resolving power of only about 10nm (much less than TEM) so usually used to study whole cells and tissues.The narrow electron beam scans surface of a metal-coated specimen and excited molecules in the specimen release secondary electrons that bounce back to detector to produce image The result is fluctuations in electron signals which produce a 3D image of the specimens signals. Elevations result in more secondary electrons and depressions result in fewer secondary electrons. Specimens dont need to be sectioned as SEM can look at entire small plant/animal. They are fixed, dried, and coated with a thin layer of heavy metal. Or they can be rapidly frozen, and then transferred to a cooled specimen stage for direct examination in the microscope. SEM is less expensive than TEM. The specimen is scanned by a beam of electrons brought to a focus on the specimen by the electromagnetic coils that act as lenses. Ones with best resolution have a high powered electron gun at source. The quantity of electrons scattered/emitted as the beam bombards each successive point on the surface of the specimen is measured by the detector and is used to control the intensity of successive points in an Image built up on a video screen. The SEM creates striking images of 3D objects with great depth of focus and a resolution between 3 nm and 20 nm depending on the instrument.

Possible Exam Qs: Types of preparative Centrifugation Viewing stained (dyes + fluorescent) specimens under the microscope Viewing living specimens under the microscope Comment on confocal + electron microscopy as biochemical tools.