Microscopy Microscopy Microscope is used to view objects

Microscopy

Microscopy • Microscope is used to view objects or specimens that are too small to be seen with just the human eye. • Microscopy is a technical field that involves the use of Microscopical components such as microscopes or microscope objectives to obtain greater detail of examined samples. • Definition-A microscope is a high precision optical instrument that uses a lens or a combination of lenses to produce highly magnified images of small specimens or objects especially when they are too small to be seen by the naked (unaided) eye. • A light source is used (either by mirrors or lamps) to make it easier to see the subject matter.

TERMS AND DEFINITIONS Principle Microscopy is to get a magnified image, in which structures may be resolved which could not be resolved with the help of an unaided eye. Magnification • It is the ratio of the size of an object seen under microscope to the actual size observed with unaided eye. • The total magnification of microscope is calculated by multiplying the magnifying power of the objective lens by that of eye piece. Resolving power • It is the ability to differentiate two close points as separate. • The resolving power of human eye is 0. 25 mm • The light microscope can separate dots that are 0. 25µm apart. • The electron microscope can separate dots that are 0. 5 nm apart.

TERMS AND DEFINITIONS Limit of resolution • It is the minimum distance between two points to identify them separately. • It is calculated by Abbé equation. Working distance • It is the distance between the objective and the objective slide. • The working distance decreases with increasing magnification.

TERMS AND DEFINITIONS Numerical aperture(NA) The numerical aperture of a lens is the ratio of the diameter of the lens to its focal length. NA can be decreased by decreasing the amount of light that passes through a lens. Diameter of the lens

Imaging Techniques Technique Image Formed By Optical Microscopy Light Rays Confocal Microscopy Coherent Light Source (Laser) Transmission Electron Microscopy (TEM) Electrons Scanning Electron Microscopy (SEM) Electrons Atomic Force & Scanning Tunneling Microscopies (AFM/STM) Molecular Mechanical Probes

History of microscope • • In 1590 F. H Janssen & Z. Janssen constructed the first simple compound light microscope -10 x to 30 x. In 1665 Robert Hooke developed a first laboratory compound microscope. Later, Kepler and Galileo developed a modern class room microscope. In 1672 Anton Von Leeuwenhoek developed a first simple microscope with a magnification of 200 x – 300 x. In 1674, Anton was the first to see and describe bacteria, yeast, plants, and life in a drop of water- He is called as Father of microscopy. The term microscope was coined by Faber in 1623. In the early 1930’s the first electron beam microscopes were developed which were a breakthrough in technology as they increased the magnification from about 1000 x or so up to 250, 000 x or more.

Compound Microscope • Common type of microscope. • High power microscope- The magnification (power) 40 x to 1000 x. • Compound refers to the fact that in order to enlarge an image - a single light path passes through a series of lenses in a line where each lens magnifies the image over the previous one. • In the standard form – 2 lenses • an objective lens (closest to the object or specimen) • an eyepiece lens (closest to the observers’ eye) • Uses light to illuminate the specimen • The objective lens usually consists of three or four lenses. • The most used light method is trans-illumination. • At 400 x much detail can be seen at the cellular level of biological specimens. • Applications: Learn about cells and microorganisms in both medical and science field.

Objective lenses • One of the most important parts of a compound microscope, as they are the lenses closest to the specimen. • A standard microscope has three, four, or five objective lenses that range in power from 4 X to 100 X. • Objectives vary in power from 1 x to 160 x in compound microscopes but the most common power range is from 4 x to 100 x. • Most compound microscopes have three or four (occasionally five) objectives usually of 4 x, 10 x, 40 x, and 100 x (oil immersion) which revolve on a nosepiece (turret) to give different magnifying powers.

Numerical Aperture • NA of a microscope objective is a measure of its ability to gather light • The more light (higher NA) the better the resolving power of the lens – Better resolution • The N. A. will be marked on the objective and the typical N. A. for the following are; • 4 x=0. 10, • 10 x=0. 25 • 40 x=0. 65 • 100 x=1. 25.

Ocular Lens or Eye piece • The eyepiece consists of a series of lenses mounted in a tube (barrel) at the upper end of the microscope. • Its basic function is to look at the focused, magnified image projected by the objective lens and magnify that image a second time before your eye looks at the image of the specimen. • The eyepieces are usually 10 x but also come in 5 x, 12. 5 x, 15 x, and 20 x. The “x” refers to the amount of magnification (power) that this lens adds as a multiplier to the magnification of the objective. • For special applications, eyepieces can have scales, pointers, crosshairs, markers, etc. on them. • The eyepoint is the location (or position) of the eye from the eyepiece which allows for the best possible viewing of the image.

Other parts • Diopter Adjustment: Useful as a means to change focus on one eyepiece so as to correct for any difference in vision between your two eyes. • Body tube (Head): The body tube connects the eyepiece to the objective lenses. • Arm: The arm connects the body tube to the base of the microscope. • Coarse adjustment: Brings the specimen into general focus. • Fine adjustment: Fine tunes the focus and increases the detail of the specimen. • Nosepiece: A rotating turret that houses the objective lenses. The viewer spins the nosepiece to select different objective lenses. • Specimen or slide: The specimen is the object being examined. Most specimens are mounted on slides, flat rectangles of thin glass.

• Stage: The flat platform where the slide is placed. • Stage clips: Metal clips that hold the slide in place. • Stage height adjustment (Stage Control): These knobs move the stage left and right or up and down. • Aperture: The hole in the middle of the stage that allows light from the illuminator to reach the specimen. • On/off switch: This switch on the base of the microscope turns the illuminator off and on. • Illumination: The light source for a microscope. Illumination is the application of light onto an object or specimen in a microscope. • Diaphragm: Adjusts the amount of light that reaches the specimen. • Condenser: Gathers and focuses light from the illuminator onto the specimen being viewed. • Base: The base supports the microscope and it’s where illuminator is located.