Introduction to Microscopy

Magnification

Process of enlarging the apparent size (not physical size) of something

Resolution

The level of detail accuracy observed in the specimen

Aberrations

Distortion of an image from the ideal optical image.

Pincushion distortion. Barrel distortion. Chromatic aberrations. Astigmatism.

Resolving power

The smallest detail that a microscope can resolve when imaging a specimen

Microscopy

1. Light: Compound microscope (light/photons pas through specimen to form a 2D image). Phase Contrast. Differential Interference Contrast. Digital. Fluorescence. Confocal.

2. Electron: Transmission (electrons transmitted through specimen 2D image formed). Scanning (electrons scan surface of the specimen by deflection, 3D image formed).

Optical principals of Microscopy Light

Elements to form an image: specimen, source of illumination and system of lenses to focus illumination and form image.

1. Source of illumination: visible light (^ ~400-700nm).

2. System of lenses: series of glass lenses (focus protons)

3. Image capture: directly through the eyepiece or detector- photographic film, electronic camera.

Optical Principals of Microscopy Electron

1. Source of illumination: beam of electrons (tungsten filament). Wavelength size limits how small an object may be seen in a microscope.

2. System of lenses: series of electromagnetic (focus electrons).

3. Image capture: electrons beam focused on fluorescent screen or photographic paper/film or digitally using a detector.

History

1. Antonie van Leeuwenhoek (1632-1723): father of microbiology discovered protozoa, spermatozoa and bacterial

2. Ernst Abbe (1876) analyzed the effects of diffraction on inage formation in microscope and showed how to optimize design.

3. Carl Zeiss (1886) made a series of lenses to Abbe design that enabled microscopists to resolve structures at theoretical limits of visible light.

Diffraction

When a beam of light/waves is spread out after passing through a narrow aperture or across an edge

Convex lens

Converges rays of light that are traveling parallel to its principal axis

Van Leeuwenhoek

Very simple device using only one convex lens mounted in a tiny hole in the brass plate making up the body of the instrument. The specimen was mounted on the sharp point in front of the lens and its position and focus coukf be adjusted by turning the two screws. The entire instrument was only 3-4 inches long and had to be held up closse to the eye, Required good lighting and patience with a magnification of 300x (only one lenses, convex lens gave optical aberrations, light wavelength which effects the resolution). Modern light microscopes have 2 lens (objective- corrects aberrations. Ocular- clarifies the details the 1st lens collected by further magnification). Development of electron Microscopy and other optional techniques further advanced modern cellular biology cell structure and function.

Light

Waves of the electromagnetic field which carry electromagnetic radiant energy, include radio waves, microwaves, infrared, light, ultraviolet, X-rays, and gamma rays. Light (electromagnetic radiation in the electromagnetic spectrum that can be perceived by the human eye). Has wavelengths in the range of 400-700nm between infrared (longer wavelength) and ultraviolet (shorter wavelengths).

Photons

Massless elementary particles that propagate visible light. Always move at speed of light in a vacuum releases energy.

Electrons absorb or release energy, when electrons absorbs a photon, energy can free the electron to move around or the electron can release the energy as another photon.

Exhibit wave-particle duality = properties of both wave and particles.

Nature of light

Wave motion of a rope held between two people analogous to waveform of photons and electrons, can be used to illustrate the effect of the size of an object on its ability to disturb wave motion.

The need

To create a magnified image of an object. To measure length, angles and area in objects. Visualize fine details of structured that the naked eye is unable to see.

The eye

Image to be seen must be presented in colours of the visible spectrum (400-700nm) and/or varying degrees of light intensity. Can sense differences in brightness/intensity ranging from black to white and grays (radiation around 550nm-green). Receptors for sensing colour and distinguishing levels of intensity are located on retina. Iris, curved cornea and lens are the mechanisms for admitting light and focusing it on retina. Resolve 2 details in the object lying 0.1mm (100microm) apart at thr point of nearest distance of distinct vision (~250mm from the eye). This is determined by spacing between receptors in retina eye (~3microm)

Micrometer

Size of cells and organelles are one millionth of a meter (10^-6) .

Nanometer: size of a molecule and subcellular structures (10^-9) therefore 1000nm = 1microm

Different microscopes

Allow viewing of details at different wavelengths.

1. Light: visualize fine details of structures that the naked eye is unable to see.

2. Electron: work on shorter wavelengths than light mics, up to 100000x magnification, detailed ultrastructure of ribosomes, cell membrane, microtubules, microfilaments, macromolecules, organelle features and cytoplasmic inclusion can be identified.

Wave motion

a rope held between 2 people is comparable to waveform of electrons and photons can be used to illustrate the effect of the size on an object on its ability to disturb wave motion. a) Moving a slack rope up and down rhythmically will generate a waveform which is characteristic to wavelength. b) When thrown against a rope, an object with a diameter comparable to the wavelength of the rope will disturb the motion of the rope. c) An object with a diameter significantly less than the wavelength of the rope will probably cause little or no disturbance of the rope because with its smaller diameter it is not likely to strike the rope when tossed towards it. d) If the rope is moved more rapidly the wavelength will be reduced substantially. e) A softball can now disturb the motion of the rope because its diameter is comparable to the wavelength of the rope.