Microscopy and Its Types
Microscopy and Its Types
▶PROPERTIES OF MICROSCOPES
- Magnification- the power of the microscope to. enlarge the image of an object
- Resolution- the power of the microscope to show detail clearly manage plane.
- It uses light to create specimen image. lenses are glass or plastic High magnification and resolution (2,000X) Path of light in an ordinary light compound microscope
- The ordinary microscope is called a bright- field microscope because it forms a dark image against a brighter background.
- Simple Microscope Has one lens. Ex: Magnifying glass, Leeuwenhoek’s microscope .
- Compound Microscope Has 2 or more lenses (more lenses create better image and better resolution) Ex: Compound microscope.
- The lens closer to eye is called the eyepiece, and the lens closer to the object being viewed is called the objective.
- The object, supported by a glass slide under the objective lens, is illuminated by light beneath it.
- In some microscopes, a third lens, called a condenser is located between the object and the light source and it serves to focus the light on the object.
- The limit of the resolving power of the best compound light microscope is about 0.2 um.
- OCULAR (eyepieces)- part you look through. contains lenses that contribute to total magnification power of 10x (magnifies 10 times)
- BODY TUBE– Hollow tube that keeps the lenses of the ocular and objectives at a set distance
- NOSEPIECE: holds objectives.
- OBJECTIVES-contain lenses that contribute to total magnification 3-4 Objectives present (4X 10X, 40X,100x)
- The Total magnification ocular power X objective power 10 40- 400 times for 40X objective.
- ARM-supports body tube.
- BASE: supports entire microscope Stage: tray-like structure that supports specimen/slide over stage.
- OPENING STAGE CLIPS- keep specimen/slide tight.
- AGAINST STAGE OPENING- allows light to pass through/around specimen.
- SUBSTAGE CONDENSER- It is mounted within or beneath the stage and focuses a cone of light on the slide.
- DIAPHRAGM-controls amount of light that reaches your eye Light source which provides light to create the image.
- The minimum distance (d) between two objects that reveals them as separate entities is given by the Abbé equation.
- In which lambda (2) is the wavelength of Light used to illuminate the specimen and n sine is the numerical aperture (NA).
- Resolution (d) = wavelength (2)/NA (n= refractive index, 0 angle of 0.5 n sin 6 aperture of lens As d becomes smaller, the resolution increases, and finer detail can be discerned in a specimen.
- The NA is a measure of the ability of a lens to collect light from the specimen.
- The working distance of an objective is the distance between the front surface of the lens and the surface of the cover glass (if one is used) or the specimen when it is in sharp focus.
- Objectives with large numerical apertures and great resolving power have short working distances
- The resolution of a microscope depends upon the numerical aperture of its condenser as well as that of the objective.
▶LIMIT OF RESOLUTION
- The maximum theoretical resolving power of a microscope with an oil immersion objective (numerical aperture of 1.25) and blue-green light is approximately 0.2 micron.
- There are two basic types of compound light microscope:
▶AN UPRIGHT LIGHT MICROSCOPE
- The light source is below the condenser lens in the upright microscope and the objectives are above the specimen stage.
- This is the most commonly used format for viewing specimens.
▶AN INVERTED MICROSCOPE
- The light source and the condenser lens are above the specimen stage, and the objective lenses are beneath it.
- The condenser and light source can often be swung out of the light path to perform manipulation of the specimen directly on the stage.
- As example the microinjection of macromolecules into tissue culture cells, for in vitro fertilization of eggs or for viewing developing embryos over time.
▶TYPES OF MICROSCOPY
▶DARK FIELD MICROSCOPY
- A hollow cone of light is focused on the specimen in such a way that unreflected and unrefracted rays do not enter the objective.
- Only light that has been reflected or refracted by the specimen forms an image.
- The image appears bright on a dark background.
- Best suited for viewing Living, unstained cells and organisms. Determination of motility in cultures.
▶PHASE CONTRAST MICROSCOPY
- The method images differences in the refractive index of cellular structures.
- Light that passes through thicker parts of the cell is held up relative to the light that passes through thinner parts of the cytoplasm.
- It requires a specialized phase condenser and phase objective Each phase setting of the condenser lens is matched with the phase setting of the objective lens.
- The light source : electron gun- When a high voltage of between 40-100 kV is passed between the cathode and the anode, a tungsten filament emits electrons.
- The negatively charged electrons pass through a hole in the anode forming an electron beam.
- The beam of electrons passes through a stack of electromagnetic lenses (the column).
- Focussing of the electron beam is achieved by changing the voltage across the electromagnetic lenses.
- When the electron beam passes through the specimen some of the electrons are scattered while others are focussed by the projector lens onto a phosphorescent screen or recorded using photographic film or a digital camera.
- There are two different types of electron microscope – The transmission electron microscope (TEM) and the scanning electron microscope (SEM).
- TEM: electrons that pass through the specimen are imaged. Phase contrast Image is formed by the interference between electrons that passed through the sample and ones that did not.
- SEM: Electrons that are reflected back from the specimen (secondary electrons) are collected, and the surfaces of specimens are imaged.