Microscopy PDF

Microscopy in relation to anatomical studies Basic Principles of Microscopy  Microscopy is the use of a microscopes to view samples & objects that cannot be seen with the naked eye Microscope – an instrument that gives an enlarged image of an object that is minute...

Microscopy in relation to anatomical studies Basic Principles of Microscopy  Microscopy is the use of a microscopes to view samples & objects that cannot be seen with the naked eye Microscope – an instrument that gives an enlarged image of an object that is minute or not visible with the unaided eye  Compound light microscope an instrument that uses visible light to produce a magnified image of an object that is seen by the eye. – two lenses: objective lens and eyepiece (or ocular), work together to produce the final magnification of the image  Four parameters in microscopy: Magnification – the enlargement of the appearance of an object – total magnification = magnification of the objective x magnification of the ocular(eyepiece) ex. 10x eyepiece used with a 40X objective lens, will produce a magnification of 400X the eye can now view the specimen at a magnification 400 times greater 1000 millimeters (mm) = 1 meter (m) 1000 micrometers (μm or mcm) = 1 millimeter (mm) 1000 nanometers (nm) = 1 micrometer (mcm)  Four parameters in microscopy: Resolution – the smallest distance between two points visible to the eye Resolving power – the ability to distinguish two points as separate – human eye can resolve about 150 µm between two points – light microscope has a resolving power of 0.2 µm  Four parameters in microscopy: Illumination – for visualization of the specimen – transmitted light or reflected light may be used in light microscopy – source of light can be white light or uv light  Four parameters in microscopy: Contrast – refers to the ability to distinguish an object from the surrounding medium – dyes and stains such as carbol fuchsin, methylene blue and safranin are used to increase contrast of the specimen  10x 40x 100x 0.25 0.65 1.25 10.6 mm 0.6 mm 0.13 mm Numerical aperture (N.A.) of a microscope objective is a measure of its ability to gather light and resolve fine specimen detail at a fixed object distance. Working distance is a linear measurement of the distance between the tip of the objective lens and the specimen.. Refractive index - measures the light bending of a medium Light may bend in air so much that it misses the small high-magnification lens. Immersion oil is used to keep light from bending. In light microscopy, oil immersion is a technique used to increase the resolving power of a microscope. Oil has high refractive index, thereby increasing the numerical aperture of the objective lens.  Working Principle of Compound Microscope Compound microscopes have a combination of lenses that enhances both magnifying power as well as the resolving power. – The specimen to be examined is mounted Primary image of on a glass slide and positioned on the stage the specimen between the condenser lens and objective lens. – A beam of visible light from the base is focused by a condenser lens onto the specimen. – The objective lens picks up the light transmitted by the specimen and create a magnified image of the specimen called primary image inside the body tube. This image is again magnified by the ocular lens or eye piece.  Working Principle of Compound Microscope When higher magnification is required, the nose piece is rotated after low power focusing to bring the objective of higher power (generally 40X) in line with the illuminated part of the slide. For very high magnification (e.g. for observing bacterial cell), oil immersion objective lens (usually 100X) is employed. The common light microscope is also called bright field microscope because the image is produced against a brightly illuminated field. – The image appears darker because the specimen or object is denser and somewhat opaque than the surroundings. Adjusting the Condenser Position and Aperture Iris Diaphragm The condenser is usually used in the highest position. Brightness of the observed field of view may be improved by lowering the condenser slightly. 1. Rotate the condenser height adjustment knob 1 to move the condenser to the highest position. 2. The aperture iris diaphragm ring 2 has an objective magnification scale (4X, 10X, 40X, 100X). Rotate the ring so that the magnification of the objective in use faces frontward. How To Track a Microscopic Image The image observed through the microscope moves in directions opposite to the actual up-down and left-right movements of the specimen.  Orientation of the specimen Images viewed through a microscope would appear upside down and inverted. – ex. wet mount of small letter e (which image is correct?) Answer: Microscope A and D Eyepiece (10x) Binocular observation tube Revolving nosepiece Pre-focusing knob Objectives (4x, 10x, 40x, 100x) Microscope frame Condenser (iris diaphragm ring) Coarse adjustment knob Filter holder or illuminator Fine adjustment knob Base Main switch: power on/off Light intensity adjustment knob  Binocular tube Diopter Interpupillary distance adjustment adjustment ring Observation tube clamping knob Specimen holder OLYMPUS CX21 Stage Specimen holder Y axis feed knob Stage aperture Specimen holder X axis feed knob  Eyepiece or ocular Diopter correction ring Flexible cord for top light Eyepiece tube housing Focus knob Objective (1x, 3x) Lamp cover clamp screw Incident light bulb with lamp cover Switch for incident and Stage clip transmitted light  Diopter correction ring Flexible cord for top light Slide block clamp screw Objective turret Pillar Working Distance: 55 mm Stage plate Cord Base and transmitted light bulb  Eyepiece/ocular Eyepiece tube housing Ojective turret Head locking screw Focus knob Objective (2x, 4x); Working distance: 55 mm Pillar incident light bulb Power switch: incident Transmitted light bulb and transmitted light Light intensity adjustment Locking socket set screw