Review of the Microscope

Ch. 1 Biology

Microscopy

Things are not all the same size, are they?

Figure shows the resolution of the naked eye, optical (light) microscopy and electron microscopy.

History of Microscopy

• Founding Fathers Of Microscopy:Hans and Zacharias Janssen, ~1590, Dutch Eyeglass Makers, Inventors Credit for the first microscope is usually given to Zacharias Janssen, pictured at the left, in Middleburg, Holland, around the year 1595. Since Zacharias was very young at that time, it's possible that his father Hans made the first one, but young Zach took over the production.

• The first compound microscopes produced by the Janssen's was simply a tube with lenses at each end. The magnification of these early scopes ranged from 3X to 9X, depending on the size of the diaphragm openings.

History of Microscopy

• Anton van Leeuwenhoek, 1632-1723, Wine Assayer, Surveyor, Cloth Merchant, Minor Public Official, and Inventor Leeuwenhoek was a man with many talents, his most important attributes were creativity, power of observation, and ingenuity. Leeuwenhoek was a common man without any fortune or formal education, so he had to work for a living. Leeuwenhoek made simple (one lens) microscopes. He was not the first person to build a microscope, but the microscopes that he did build were the best ones for that time period. Leeuwenhoek was the first person to describe bacteria (from teeth scrapings), protozoans (from pond water), helped to prove the theory of blood circulation. He gained much of his inspiration form reading Hooke's Micrographia.

• A simple microscope has only one lens.

Microscopy: The Instruments

Figure 1.2b

Discovery of Microorganisms

• Antony van Leeuwenhoek (1632-1723)– first person to

observe and describe micro-organisms accurately

Figure 1.1b

History of Microscopy• Robert Hooke, 1635-1703, English Chemist, Mathematician,

Physicist, and Inventor Hooke's remarkable engineering abilities enabled him to invent and improve many mechanical devices, including timepieces (for which he invented the spiral spring), the quadrant, and the Gregorian telescope. Perhaps even more intriguing than his actual inventions are the devices he designed but never built: he anticipated the invention of the steam engine, and as early as 1684 he described a working telegraph system.

• Hooke balanced his inventions with more pure research. Hooke improved on early compound microscopes around 1660. In Micrographia (1665), he coined the word cell to describe the features of plant tissue (cork from the bark of an oak tree) he was able to discover under the microscope. He put his extensive mathematical knowledge in formulating the theory of planetary movement, which provided a basis for Sir Isaac Newton's theories of gravitation. In 1667 he discovered the role of oxygenation in the respiratory system.

Figure 2.3

The Light Microscope

• many types– bright-field microscope– dark-field microscope– phase-contrast microscope– fluorescence microscopes

• are compound microscopes– image formed by action of 2 lenses

• In a compound microscope the image from the objective lens is magnified again by the ocular lens.

• Total magnification =objective lens ocular lens

Microscopy: The Instruments

Figure 3.1b

•working distance— distance between the front surface of lens and surface of cover glass or specimen

Figure 2.2

The Light Microscope

Microscope Resolution

• ability of a lens to separate or distinguish small objects that are close together

• wavelength of light used is major factor in resolutionshorter wavelength greater resolution

How do we use the microscope correctly?

Using the Microscope

The Bright-Field Microscope

• produces a dark image against a brighter background

• has several objective lenses– parfocal microscopes remain in

focus when objectives are changed

• total magnification– product of the magnifications of

the ocular lens and the objective lens

The Dark-Field Microscope

• produces a bright image of the object against a dark background

• used to observe living, unstained preparations

Figure 2.7b

The Phase-Contrast Microscope

• enhances the contrast between intracellular structures having slight differences in refractive index

• excellent way to observe living cells

The intact chloroplasts and broken chloroplasts can be differentiated by phase contrast microscopy:

The Differential Interference Contrast Microscope

• creates image by detecting differences in refractive indices and thickness of different parts of specimen

• excellent way to observe living cells

• Accentuates diffraction of the light that passes through a specimen; uses two beams of light.

Differential Interference Contrast Microscopy

Figure 3.5

• Uses UV light.

• Fluorescent substances absorb UV light and emit visible light.

• Cells may be stained with fluorescent dyes (fluorochromes).

Fluorescence Microscopy

Figure 3.6b

Fluorescence Microscopy Is Widely Used to Visualize Specific Cellular Structures and to Localize Proteins

A Multiprobe Fluorescent Micrograph of a Mitotic Cell

Microtubules: green DNA: blue centromeres; red

Newer Techniques in Microscopy

• confocal microscopy and scanning probe microscopy

• have extremely high resolution

• can be used to observe individual atoms

Figure 2.20

Confocal Microscopy

• confocal scanning laser microscope

• laser beam used to illuminate spots on specimen

• computer compiles images created from each point to generate a 3-dimensional image

Figure 2.29

Scanning Probe Microscopy

• scanning tunneling microscope– steady current (tunneling

current) maintained between microscope probe and specimen

– up and down movement of probe as it maintains current is detected and used to create image of surface of specimen

Scanning Probe Microscopy

• atomic force microscope– sharp probe moves over

surface of specimen at constant distance

– up and down movement of probe as it maintains constant distance is detected and used to create image

Electron Microscopy

• beams of electrons are used to produce images

• wavelength of electron beam is much shorter than light, resulting in much higher resolution

Figure 2.20

The Scanning Electron Microscope

• uses electrons reflected from the surface of a specimen to create image

• produces a 3-dimensional image of specimen’s surface features

Scanning Electron Microscopy (SEM) Visualizes Surface Features

Specimens are coated with metals to deflect electrons from a beam scanned across the sample.

SEM of Stereocilia Projecting from a Cochlear (inner ear) Hair Cell

Fly head

Ebola virus

The Transmission Electron Microscope

• electrons scatter when they pass through thin sections of a specimen

• transmitted electrons (those that do not scatter) are used to produce image

• denser regions in specimen, scatter more electrons and appear darker

In Transmission Electron Microscopy, Magnetic Lenses Replace Glass As a Means of Focusing Electromagnetic Waves

The shorter wavelength of the accelerated electrons permits much higher resolution.

TEMs Must be Carefully Interpreted

Imagine all the misleading interpretations of this branched mitochondrion possible from viewing a single section.

A TEM of an Onion Root Tip Cell

Comparison of Microscopic Techniques for Visualizing a Living, Unstained Cell

Differential-interference-contrast (Nomarski)

Dark field

Comparison of Microscopic Techniques for Visualizing a Living, Unstained Cell

Bright field

Phase-contrast

Micro$cope$

• Costs

• $150 - $10,000 (Compound)

• $100-$1500 (Dissection or Stereoscope)

• $20,000-100,000 (Confocal Microscope)

• more than $50.000 (Scanning Electron Microscope(SEM)Transmission Electron Microscope (TEMMicroscope)