Introduction to Microscopy

• Microscopy encompasses the use of microscopes to observe small structures and details.

Learning Outcomes

• Ability to convert between different lengths within the metric system.
• Knowledge of the history and development of early microscopes.
• Understanding of the three elements of good microscopy.
• Differentiation of the principles behind light and electron microscopy.
• Enumeration and description of different types of microscopes.

Microbial Dimensions

• Microbes are too small to be seen without magnification.
• There are two size categories of organisms: macroscopic and microscopic.
• Macroscopic organism dimensions are measured in centimeters or meters.
• Microscopic organism dimensions are measured in millimeters, micrometers (µm), or nanometers (nm).
• The Metric system is used in Microbiology because of its divisibility by 10.
• Obsolete terms micron (µ) and millimicron (mµ) have been replaced by micrometer (µm) and nanometer (nm).
• Smallest bacteria measure 200 nm
• Protozoa and algae size range from 3 to 4 mm and are visible to the naked eye.
• Red blood cells measure 7µm.
• Viruses range in size from 20 to 800 nm.

Metric Units

• Metric units of length, include kilometers, meters, decimeters, centimeters, milimeters, micrometers, nanometers, picometers.
• 1 kilometer (km) is 1000 meters.
• 1 decimeter (dm) is 0.1 meters.
• 1 centimeter (cm) is 0.01 meters.
• 1 millimeter (mm) is 0.001 meters.
• 1 micrometer (μm) is 0.000001 meters.
• 1 nanometer (nm) is 0.000000001 meters.
• 1 picometer (pm) is 0.000000000001 meters.
• Relative size of microbes varies with viruses being the smallest in diameter ranging from 0.01-0.3 μm.
• Cocci(spherical bacteria) have an average diameter of 1 μm.
• Bacilli (rod-shaped bacteria) have an average width x length of 1 x 3 μm.
• Fungal Yeasts have a diameter of 3-5 μm.
• Septate hyphae have a width of 2-15 μm
• Pond water protozoa sizes vary with some being visible with the unaided human eye.

Defining the Microscope

• "Microscope" is derived from the Latin word "micro" (small) and the Greek word "skopos" (to look at).
• Instruments such as telescopes, binoculars, magnifying glasses, and microscopes are all types of optical instruments.
• Microscopes are optical instruments that can magnify organisms up to a hundred or even a thousand-fold.

History of Microscopes

• Spectacles were first made in Italy during the 14th century.
• The first microscope was created in 1590 by Hans and Zacharias Janssen, but its magnification was limited (x3-x9).
• In 1667, Robert Hooke published "Micrographia," detailing his studies using the microscope.
• Anton van Leeuwenhoek made the first observations of bacteria using a single-lens microscope around 1675.
• Around the 18th century, technology improvements made microscopy more popular, including discovering that combining two types of glass reduced the chromatic effect.
• In 1830 Joseph Jackson Lister discovered that using weak lenses together at various distances provided clear magnification.
• Ernst Abbe developed a mathematical theory in 1878 linking resolution to light wavelength.
• Richard Zsigmondy invented the ultramicroscope in 1903, enabling observation of specimens smaller than the wavelength of light.
• Fritx Zernike invented phase-contrast microscopy in 1932 to study biological materials.
• Ernst Ruska developed the electron microscope in 1938, enhancing resolution using electrons.
• Gerd Binnig and Heinrich Rohrer invented the scanning tunneling microscope in 1981, which enabled 3D specimen imaging.

Simple vs. Compound Microscopes

• A simple microscope contains only one magnifying lens.
• Magnifying glasses are simple microscopes and magnify objects X3-X20 their original size.
• A compound microscope uses multiple magnifying lenses.
• Compound light microscopes usually magnify objects about 1,000 times.
• Photographs taken through compound microscopes are called photomicrographs.
• Compound microscopes use visible light, often from a built-in bulb.

Microscope Design

• Early microscopes used a simple magnifying lens and limited parts.
• Later compound microscopes included a second magnifying lens.
• The base of a compound microscope contains a lamp for specimen illumination.
• A condenser focuses light onto the specimen.

Principle of Light Microscopy

• Light microscopy uses visible light to observe specimens.
• Compound light microscopy uses multiple lenses and visible light.
• Modern compound light microscopes have two lens systems: ocular lens and objectives.

Properties of Light Microscopy

• Magnification, resolution, and contrast are three key properties of an effective microscope.

Magnification

• Clear glass spheres can magnify objects and magnification results from visible light waves interacting with the curvature of a lens.
• Refraction is the bending of light as it passes through a medium, such as a lens.
• The greater the difference between two substances, the greater the refraction.
• Image formation occurs when light passes through a spherical lens and magnification depends on lens size and curvature.
• Magnification refers to the ability to enlarge an image.
• Compound light microscopes use multiple lenses to achieve magnification.
• Total magnification is calculated by multiplying the magnifying powers of each individual lens.
• Total magnification = Power of objective x Power of ocular.
• The first lens in a microscope (closest to the specimen) is called an objective lens.
• The Objective Lens forms a real image of the specimen and typically magnifies it in the range of 4x-100x .
• The second lens (closest to the eye) is called an ocular lens or eyepiece.
• The Ocular Lens/Eyepiece forms a virtual image typically magnifying 10x-20x.

Ocular and Objective Lenses

• The ocular lens is closest to the eye forming the virtual image.
• The objective lens is closest to the specimen forming the real image.

Magnifying Power

• Set power of the ocular lens remains constant.
• Total magnification levels of light microscope range from 40X- 1000X.
• The 4X objective is rarely used.
• Start by observing specimen using the 10X objective.
• High power objectives are used to view algae or protozoa and oil immersion objectives for bacteria.
• Optimum specimen observation requires properly focused light.
• Condenser focus the light into the specimen.
• As Magnification increases so should the light, done through three methods:
• Opening the iris diaphragm.
• Opening the field diaphragm.
• Increasing light intensity using the rheostat knob clockwise.

Resolution

• Resolution allows the lenses to distinguish fine details and distinguish points at a specific distance.
• Optical systems need sufficient resolution in order to distinguish or separate from one another.
• The human eye can resolve 0.2 mm.
• Microorganisms cannot be seen with the naked eye.
• Compound Microscopes can resolve 0.2μm.
• Resolution is determined by the objective lens characteristics and the illuminating light wavelength.
• Optical microscopes, utilize light to work.
• Light sources release colored wavelength.

Wavelength and Resolution

• Shorter wavelengths offer better resolution as they must pass between the objects.
• Microscopes may use blue filters to limit the longer wavelengths.
• Clarity depends on resolving power.

Oil Immersion Lens

• Maximum resolution is achieved by placing a drop of oil on the slide when using the oil immersion (100x) objective lens.
• Immersion oil has the same refractive index as glass to become part of the glass optics of the microscope.
• Oil prevents light scattering, increasing numerical aperture and resolution.
• Oil immersion resolves objects of 0.2µ in diameter and at least 0.2 μ apart.

Contrast

• Contrast is the difference in intensities between an image and its adjacent background.
• The Degree of contrast between a magnified image is determined by its refractive index.
• A higher refractive index, means a sharper contrast.
• Too much light can reduce the contrast.
• Lack of contrast can be overcome using adding stains.

Types of Light Microscopes

• Variations of Light Microscopy:
• Bright-field.
• Dark-field.
• Phase-contrast.
• Interference.
• Other Types of Microscopes:
• Fluorescence utilizes ultraviolet radiation.
• Confocal utilizes laser beams.

Brightfield Microscopy

• Use a series of lenses and visible light to illuminate.
• Microscopic observation appears dark against a bright field of view.
• Magnification is 1,000 -1,500x.
• Primarily used for bacteria and fungi visualization.
• Cells often need staining to be observed to create contrast.
• Objects cannot be visualized below 0.2 μm.

Darkfield Microscopy

• Uses reflect light instead of transmitted light, with a special opaque disc.
• Examine live and transparent microorganisms which cannot be stained or distorted due to staining.
• The specimen appear bright against a dark background.
• It is used to view spirochetes (treponema, leptospira, borrelia).

Phase-Contrast Microscopy

• Based on differences in the refractive indices and light waves passing through transparent objects.
• One set of rays comes direct from the light other is reflected away from the specimen.
• Diffracted rays are extracted from the direct rays.
• The 2 sets brought together on the oculars to form image.
• Diffraction is the scattering of light as it touches a specimen's edge.
• Uses include special condenser containing a annulur/ring-shaped diaphragm.
• Diaphragm allows light to travel in circle shape.
• Diffraction plate exists inside the objective lens.
• Produces high-contrast for transparent objects such as tissue slices.

Interference Microscope

• Utilizes 2 beams of light.
• Produces higher resolution.
• Uses the differences in refractive indices to produce the image.
• Prisms split light beam creating different colors.
• Used for viewing living specimens when standard staining is not ideal.
• Provides 3 dimensional image of cell.

Fluorescence Microscopy

• It uses fluorescence, substances that absorbs short wavelengths of lights and gives off longer wavelength of light.
• Some specimens fluoresce naturally if do not then stain.
• Fluorochromes are a group of fluorescent dyes that are examined under a fluorescence scope source.
• Used for structural visualization of a specimen, genetic material, viability of cell population and detects antigens and antibodies.

Confocal Microscopy

• Also known as CLSM(confocal laser scanning microscope), LCSM (laser confocal scanning microscope).
• Light microscopy used to reconstruct 3 dimension images.
• Specimens stained with fluorochromes, instead one plane of small specimen is illuminated with short wavelength light.
• The laser then scans object together with computer to produce 3D image.
• Studies the cell physiology.

Electron Microscopy

• Utilizes beams of electrons instead of light through magnets.
• Resolves shorter wavelengths of electron at 100,000x vs visible light.
• Modern microscopes magnify up to 2 million times.
• Images are always black and white.
• Uses electromagnetic lenses instead of glass lens.
• Visualizes cells and viruses.

Types of Electron Microscopy

• TEM (Transmission Electron Microscopy).

• Uses a finely focused beam electrons that that passes through a small part of a prepared, ultrathin specimen.

• Specimen put on mesh made with copper.

• Resolution of 0.2 nm.

• Magnifies approximately 1 to 10 million times.

• Saved as digital image onto viewing screen.

• SEM (Scanning Electron Microscopy).

• Relies on interaction at surface.

• Uses primary beams to knock out electrons and create secondary electrons.

• Secondary electrons are transmitted to electron which makes image onto viewing screen.

• Studies surface structure of intact cells and viruses.

• Can magnify up to 500,000 times.

• Resolution of 20nm (nanometers).