Microscopy Basics Quiz

Microscopy and Micrometry

• A person is paid based on time or value
• Microscopy is the technical field using microscopes or microscope objectives to examine samples in greater detail
• Objectives include Light, Fluorescent, phase contrast, and electron microscopy
• Microscopic principle is to magnify an image that could not be resolved with the naked eye
• Magnification is the ratio of the size observed under a microscope to the actual size of the object under a naked eye
• Total magnification is the result of multiplying the objective lens and eyepiece magnification
• Maximum magnification doesn't equal maximum resolution
• Resolving power: The ability to differentiate two close points as separate
• Human eye resolving power is 0.25 mm
• Light microscope resolving power is 0.25µm
• Electron microscope resolving power is 0.5nm
• Limit of resolution is the minimum distance between two points distinguishable as separate and is calculated by the Abbé equation
• Resolving power (R.P.) = Wavelength of light in nm / (2 × Numerical aperture of objective lens)
• Numerical Aperture (NA): The ratio of the diameter of the lens to its focal length
• NA is an estimate of how much light from the sample is collected by the objective
• NA = n sin α (n = refractive index, α = angle of incident illumination)
• The higher the NA the better the resolution of a specimen is
• d = 0.5 λ/n sin θ (d = resolution, λ = wavelength of light used)
• Numerical aperture, not magnification determines resolution
• Imaging techniques include Optical Microscopy, Confocal Microscopy, Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), and Atomic Force & Scanning Tunneling Microscopies (AFM/STM). Their respective image formation methods are: Light Rays, Coherent light source (laser), Electrons, Electrons, and Molecular Mechanical Probes, respectively.
• A microscope is a high-precision optical instrument using a lens or a combination of lenses for highly magnified images of small specimens that cannot be seen by the naked eye.
• A light source (mirrors or lamps) is used to illuminate the specimen.
• Microscopes are used to view specimens too small to be seen with the naked eye.

History

• In 1590, F.H Janssen and Z.Janssen made the first simple compound light microscope (10x to 30x)
• In 1665, Robert Hooke developed the first laboratory compound microscope
• Later scientists (Kepler and Galileo) developed advanced classroom microscopes
• In 1672, Anton Von Leeuwenhoek built a simple microscope with a magnification of 200x to 300x
• Anton Von Leeuwenhoek observed bacteria, yeast, plants, and life in a water drop in 1674
• In 1623, the term "microscope" was coined by Faber
• Electron microscopes were developed in the early 1930s, dramatically increasing magnification from about 1000x to 250,000x or more

Light Microscopy

• Visible light is used in light microscopy
• Glass lenses are used to focus light
• A significant advantage is that light microscopy can often be used on living cells
• Allows watching processes like cell migration or division
• Types of light microscopes include Brightfield, Phase Contrast, Dark-Field, and Fluorescence

Contrasting Techniques

• Differential interference contrast (DIC) introduces contrast to images of specimens with lack thereof when using a brightfield microscopy
• Brightfield microscopy: Light transmitted through the sample is absorbed by it.

Compound Microscope

• A common type of microscope with high magnification (40x to 1000x)
• Compound microscopes use multiple lenses in a line for magnification.
• Standard microscopes have objective lens and eyepiece lens.
• Objective lenses typically have 3, 4 or 5 lenses rangings from 4x to 100x (including oil immersion)
• Usually have 40x, 10x, 40x, and 100x (oil immersion) objective lenses
• The lenses revolve on a nosepiece to change the magnification

Anatomy of a Microscope

• Components include the ocular lens, diopter adjustment, nose piece, mechanical stage, condenser, illumination, brightness adjustment, base, objective lens, frame (arm), stage control, coarse adjustment, fine adjustment, and light switch.

Parts of a Compound Microscope

• Mechanical parts include base/metal stand, pillars, inclination joint, curved arm, body tube, draw tube, coarse adjustment, fine adjustment, stage, mechanical stage (slide mover), revolving nosepiece
• Optical parts include light source, diaphragm, condenser, objectives, and eyepiece

Objective Lenses

• Critical components of a compound microscope
• Vary in power from 1x to 160x
• Standard microscopes usually have 4x, 10x, 40x, and 100x (oil immersion) objectives

Numerical Aperture

• Measurement of a microscope objective's ability to gather light
• Higher NA leads to better resolution
• Typical NA values for objective lenses include 4x (0.10), 10x (0.25), 40x (0.65), and 100x (1.25).

Ocular Lens (Eye Piece)

• Consists of a series of lenses that magnify the image projected by the objective lens.
• Common magnification is 10x.
• Includes features like scales, pointers, crosshairs, and markers for special applications

Nosepiece

• Holds the objective lenses
• Rotates to allow for changing magnifications

Arm

• Supports the upper parts of the microscope and is used to carry the microscope (using two hands)

Base

• Supports the entire microscope, used to carry (with two hands)

Stage

• Supports the microscope slide

Stage Clip

• Holds the slide in place
• Typically, a pair for holding specimens

Light Source

• Provides illumination for viewing specimens
• Can be a mirror or an illuminator

Diaphragm

• Controls the amount of light entering the lenses
• Adjustable for different thicknesses of specimens for optimal illumination

Coarse Adjustment Knob

• Used to rapidly adjust focus, mainly for low-power objectives (4x)

Fine Adjustment Knob

• Used for precise focusing, usually for medium and high-power objectives (10x and 40x).

Phase Contrast

• A technique for enhancing contrast in specimens with low inherent contrast.
• Used widely in tissue culture and timelapse microscopy

Applications of Phase Contrast

• Determining morphologies of living cells (plant and animal)
• Studying microbial motility and structures
• Detecting microbial elements like bacterial endospores

Fluorescence

• Uses fluorescent dyes to highlight specific structures
• Visualization of bacterial agents like Mycobacterium tuberculosis and other agents
• Identify antibodies against pathogens and microorganisms
• Differentiation between dead and live bacteria
• Used in identifying and observing microorganisms labeled with fluorophores in ecological studies

Darkfield

• Illuminating rays of light are directed through the sample from the side
• Light scattered by specimens is enhanced
• Visualization of unstained/colourless specimens, like diatoms

Electron Microscopy (general)

• Uses electrons instead of visible light
• Electromagnets act as lenses
• Entire system operates in a vacuum
• Includes transmission and scanning electron microscopy

Transmission electron microscopy (TEM)

• Used for viewing interior structures of specimens
• Thin sections are needed, as well as negative stains, for specimen preparation.

Scanning electron microscopy (SEM)

• Visualizes external features of organisms or cells
• Coating of specimens with heavy metals improves contrast.

Electron Micrographs

• Typically, black and white images
• Often have false color added to enhance visual appeal.