MICROSCOPY

MICROSCOPE

An optical instrument that uses a lens or a combination of lenses to produce magnified images of small objects ,especially of objects too small to be seen by the unaided eye.

History of microscopes
Egyptian artifacts include rock crystals in the form of convex lenses (2600 B.C.) The Greeks and Romans continued with these types of lenses up to the end of the Roman Empire (~31 B.C.) knew and practiced the art of glass blowing . Observed that objects placed in a bulb filled with water appeared magnified .

History of microscopes
Roger Bacon (1214-1292), a monk in Oxford, wrote on the topic of Nature often referenced the concepts and use of lenses. Indeed ,Bacon knew of lenses and their abilities. However the type of lenses he talked about would be laid on books or held in the hand just above the printed text.

History of microscopes
Based on the letters of William Boreal ( the Dutch envoy to the Court of France) the father and son team of Hans and Zacharias Jansen are the inventors of the microscope. At least they are the first to have any documentation that substantiates such a claim. Their microscope design was somewhat limited. It could only be used for opaque objects Had a magnification of about 20X

History of microscopes
1665 - Robert Hooke (the Secretary of the Royal Society) publishes "Microgaphia" a folio of thirty-eight copper-plate illustrations of objects drawn with the aid of his microscope. First to describe and coin the phrase "cell" when observing a slice of cork (bark from an oak tree) using a microscope power of 30X.

History of microscopes
1673 - Antony van Leeuwenhoek a tradesman of Delft, Holland with no formal training made some of the most important discoveries in biology. He discovered bacteria, free-living and parasitic microscopic protists, sperm cells, blood cells and more. All of this from a very simple device that could magnify up to 300X.

History of microscopes
1823 - Achromatic lenses introduced now provide resolution of 1 micron or 1/1000 mm. 1839 - Theodor Schwann and Matthias Schleiden formally propose the "Cell Theory." 1840 - Donne' publishes the first micrographs in France. 1880 - Microscope lamp with filters. August Kohler had worked out light source and condenser position to obtain the best image projection.

History of microscopes
1880-1900 - Louis Pasteur and Robert Koch both become engaged in microscopy and the study of bacteria. 1904 - The first commercial UV microscope by Zeiss. The resolution based on Abbe's formula is twice that of a visible light microscope. 1930 - Fritz Zernike discovered he could view unstained cells using the phase angle of rays. It took until 1941 to bring a commercial microscope to market.

History of microscopes
1931 - Max Knoll and Ernst Ruska construct the first electron microscope. 1933 - Ruska builds the first electron microscope that exceeds the resolution of the light microscope. It has an accelerating voltage of 75 kV. 1934 - First electron micrograph of a biological sample. long-leaved sundew fixed with osmium. 1937 - First scanning electron microscope is built. 1939 - Siemens supplies the first commercially available electron microscope.

ELECTRON MICROSCOPE

History of microscopes
1983 - Scanning laser confocal microscope is commercially available 1982 - Scanning Probe Microscope is invented and works by measuring current. 1986 - Atomic Force Microscope is invented and measures force instead of current.

CONCEPTS IN LIGHT
Light can behave as a particle and a wave. AMPLITUDE: Strength of the energy or brightness of the light. WAVELENGTH: Distance between the apex of one wave and the next. Measured in nanometers. It determines color.Wavelength of visible light is 400-800nm. FREQUENCY: The number of waves per second . INTERFERENCE: is the mutual influence of two waves on each other, whereby the resulting crests may be either enhanced or flattened (enhancement of amplitude, reduction of amplitude).

CONCEPTS IN LIGHT
RETARDATION:ray at 90 degrees. The higher the density of the medium,the greater the retardation.

CONCEPTS IN LIGHT
DIFFRACTION: is the partial deflection of a ray of light at the corners of opaque objects.

CONCEPTS IN LIGHT REFRACTION: ray at any other angle than 90 degrees. Deviation in direction and retardation

CONCEPTS IN LIGHT

RETARDATION AND REFRACTION ARE GOVERNED BY:

ANGLE OF INCIDENCE. REFRACTIVE INDEX. CURVATURE OF LENS.

CONCEPTS IN LIGHT

TOTAL INTERNAL REFLECTION

IMAGE FORMATION

REAL IMAGE.

VIRTUAL IMAGE.

COMPOUND MICROSCOPE

IMAGE QUALITY
1.ACHROMATIC LENSES

2.APOCHROMATIC LENSES

1.FLUORITE

IMAGE QUALITY

COMPONENTS OF A MICROSCOPE
Light source Condensers Object stage Objectives Body tube Eyepiece

Light source
Bright,glare free Sunlight Oil lamps to low-voltage electric lamps Tungsten-halogen bulb

Light source

Condensers
To focus or concentrate the available light into the plane of the object.

Iris diaphragm
Closed too much contrasty and refractile Left wide open - glare

Swing top lens

Used with higher power objectives with smaller diameter lenses

Illumination
CRITICAL: the light source is focused by the sub stage condenser in the same plane as the object, when the object is in focus.the image of the filament causes uneven illumination.

Illumination
Kohler: the image of the light source is focused by the lamp collector or field lens in the focal plane of the sub stage condenser. {on the aperture diaphragm}

Kohler illumination

Conjugate planes in the path of the illuminating light rays The lamp filament. The condenser aperture diaphragm (at the front focal plane of the condenser). The back focal plane of the objective. The eye point (also called the Ramsden disk) of the eyepiece, which is located approximately one-half inch (one centimeter) above the top lens of the eyepiece, at the point where the observer places the front of the eye during observation.

The conjugate planes in the image-forming light path The field diaphragm. The focused specimen. The intermediate image plane (i.e., the plane of the fixed diaphragm of the eyepiece). The retina of the eye or the film plane of the camera.

OBJECT STAGE
Rigid platform with aperture Supports the glass slide Sturdy Perpendicular to the path of light. Provided with a mechanical stage.

Objective lenses
To collect the maximum amount of light possible from the object ,unite it and form a high quality magnified real image some distance above

specifications

Power of resolution
Ability to tell two points apart. Depends on numerical aperture and the wavelength of light used. NA = n X sin u n = refractive index of the medium between the cover glass over the object and the front lens of the objective. u = angle between the optical axis of the lens and the outermost ray which can enter the front lens. NA = 0.95 Water and oil immersion objectives = 1.20 and 1.30 respectively

Numerical aperture

Mathematical expression of the light-gathering ability of the lens.

Numerical aperture

Power of resolution
RESOLUTION = 0.61 / NA.
MAGNIFICATION : 1:1 ,100: 1. TUBE LENGTH OF 160mm OR 170 mm INFINITY CORRECTED OBJECTIVES. TYPES OF OBJECTIVES: 1.ACHROMAT 2.APOCHROMAT 3.PLAN-APOCHROMAT PHOTOMICROGRAPHY 4.PLAN-ACHROMAT CYTOLOGICAL SCREENING

BODY TUBE

BODY TUBE
MONOCULAR BINOCULAR COMBINED PHOTO-BINOCULAR

Intermediate optics- rotating turret. Infinity-corrected= additional tube lens.

BODY TUBE

EYEPIECE
To magnify the image formed by the objective within the body tube and present the eye with a virtual image apparently in the plane of the object being observed.

EYEPIECES
HUYGHENS {1690}. Peri-planatic and wide field types For holding measuring graticules and photographic formats. High focal point spectacle wearers.

EYEPIECES

EYEPIECES

Total magnification
Product of magnification values of objective and eyepiece. Optical tube length/ focal length of objective X magnification of eyepiece. Empty magnification

USE OF MICROSCOPE
CLEAN AND FREE FROM DUST USE OF OIL IMMERSION OBJECTIVE SWABS OF COTTON WOOL OR POLYSTYRENE

SETTING UP OF MICROSCOPE
CENTERING THE LAMP ADJUSTING THE CONDENSER INSERTING THE SLIDE