MODULE BIOSL 1: EXPLORE

CONTINUITY AND DIVERSITY OF LIFE

Learning Outcome: BIOSL 1.1 – Analyse cell processes and maintenance
Performance Criteria: PC - 1.1.1 to 1.1.11

MICROSCOPY (PC 1.1.1 – 1.1.2)

 Microscopes and microbiology  linking advance
 The microscope is the microbiologist’s most basic tool.
 Microscopes use lenses to magnify object’s images.
 There are many types of microscopes (look at the next slide) but the most common include
two types:
◦ Light microscopes (5 types)
◦ Electron microscope (2 types)
 Light microscope used to examine cells at relatively low magnifications and electron
microscope used to lock at cells and cell structure at very high magnification.

Principles of Light Microscope

 Compound light microscope uses visible light to illuminate cells.

 Many different types of light microscopy:
 1-Brightfield
 2-Darkfield
 3-Phase-contrast
 4-Differential interference contrast (DIC)
 5-Fluorescence
Bright-field microscope
 Specimens are visualized because of differences in contrast (density) between
specimen and surroundings.
 Contrast differences arise because cells absorb or scatter light to varying degrees.
Two sets of lenses form the image.
 Objective lens and ocular lens (compound)
 Total magnification = objective magnification  ocular magnification
 Maximum magnification is ~2,000
Some Principles of Light Microscope

 Magnification is not the limiting factor in the ability of seeing small things but also, we need
a good resolution which is the ability to distinguish two adjacent objects.
 Magnification can be increased without limit, but resolution cannot because it is the function
of physical properties of light.
 Light microscopy limits resolution is about 0.2µm, electron microscope resolution is greater
than of light microscope.
 Resolution: the ability to distinguish two adjacent objects as separate and distinct
 Resolution is determined by the wavelength of light used and numerical aperture of
lens.
Try Labelling
Phase – Contrast and Dark – Field Microscopy

 Two forms of light microscopy improve image without using staining.

 Phase –contrast microscopy is based on the principle that cells differ in refractive index from
their surroundings.
 Phase – contrast microscopy resulting a dark image on light background.
 The dark-fielded microscopy is a light microscope in which the light reaches the specimen
from the sides only. Thus, the specimens appear light on a dark background.
 The dark-fielded microscopy used in observed microbial motility.

Fluorescence Microscopy

 Used to visualize specimens that fluoresce – emit light of one colour following absorption of
light of another colour.
 Cells fluoresce either:
 Contain naturally fluorescent substances such as chlorophyll.
 Because cells have satin with fluorescent dye.
 DAPI (4`,6-diamidino-2-phenylindole) is widely used fluorescent dye staining cell`s DNA.

Electron Microscopy

 Electron microscopes use electrons instead of photons (visible light) to image cells and
structures.
 Electromagnets function as lenses in EM, whole system operates in a vacuum.
 EM are fitted with cameras to allow a photograph to be taken.
 Two types of electron microscopes:
 Transmission electron microscopes (TEM)
(need thin section), negative stain
 Scanning electron microscopes (SEM)
Coat with heavy metal
 Transmission electron microscopy is used to examine cells and cell structure at very high
magnification and resolution, even enabling one to view structures at the molecular level.
 This is because the wavelength of electrons is much shorter than the wavelength of visible
light and wavelength affects resolution.
 Unlike visible light, electron beams cannot penetrate very well. So, special techniques of thin
sectioning are needed to prepare specimens before observing them.
 To obtain sufficient contrast, the preparation are treated with stains such as osmic acid,
permanganate, uranium, lanthanum; because these substances are composed of atoms of
high atomic weight, they scatter electrons well and thus improve contrast.
 MODULE BIOSL 1: EXPLORE
CONTINUITY AND DIVERSITY OF LIFE
Learning Outcome: BIOSL 1.1 – Analyse cell processes and maintenance
Performance Criteria: PC - 1.1.1 to 1.1.11

MICROSCOPY (PC 1.1.1 – 1.1.2)

 Microscopes and microbiology  linking advance
 The microscope is the microbiologist’s most basic tool.
 Microscopes use lenses to magnify object’s images.
 There are many types of microscopes (look at the next slide) but the most common include
two types:
◦ Light microscopes (5 types)
◦ Electron microscope (2 types)
 Light microscope used to examine cells at relatively low magnifications and electron
microscope used to lock at cells and cell structure at very high magnification.

Principles of Light Microscope

 Compound light microscope uses visible light to illuminate cells.

 Many different types of light microscopy:
 1-Brightfield
 2-Darkfield
 3-Phase-contrast
 4-Differential interference contrast (DIC)
 5-Fluorescence
Bright-field microscope
 Specimens are visualized because of differences in contrast (density) between
specimen and surroundings.
 Contrast differences arise because cells absorb or scatter light to varying degrees.
Two sets of lenses form the image.
 Objective lens and ocular lens (compound)
 Total magnification = objective magnification  ocular magnification
 Maximum magnification is ~2,000
Some Principles of Light Microscope

 Magnification is not the limiting factor in the ability of seeing small things but also, we need
a good resolution which is the ability to distinguish two adjacent objects.
 Magnification can be increased without limit, but resolution cannot because it is the function
of physical properties of light.
 Light microscopy limits resolution is about 0.2µm, electron microscope resolution is greater
than of light microscope.
 Resolution: the ability to distinguish two adjacent objects as separate and distinct
 Resolution is determined by the wavelength of light used and numerical aperture of
lens.
Try Labelling
Phase – Contrast and Dark – Field Microscopy

 Two forms of light microscopy improve image without using staining.

 Phase –contrast microscopy is based on the principle that cells differ in refractive index from
their surroundings.
 Phase – contrast microscopy resulting a dark image on light background.
 The dark-fielded microscopy is a light microscope in which the light reaches the specimen
from the sides only. Thus, the specimens appear light on a dark background.
 The dark-fielded microscopy used in observed microbial motility.

Fluorescence Microscopy

 Used to visualize specimens that fluoresce – emit light of one colour following absorption of
light of another colour.
 Cells fluoresce either:
 Contain naturally fluorescent substances such as chlorophyll.
 Because cells have satin with fluorescent dye.
 DAPI (4`,6-diamidino-2-phenylindole) is widely used fluorescent dye staining cell`s DNA.

Electron Microscopy

 Electron microscopes use electrons instead of photons (visible light) to image cells and
structures.
 Electromagnets function as lenses in EM, whole system operates in a vacuum.
 EM are fitted with cameras to allow a photograph to be taken.
 Two types of electron microscopes:
 Transmission electron microscopes (TEM)
(need thin section), negative stain
 Scanning electron microscopes (SEM)
Coat with heavy metal
 Transmission electron microscopy is used to examine cells and cell structure at very high
magnification and resolution, even enabling one to view structures at the molecular level.
 This is because the wavelength of electrons is much shorter than the wavelength of visible
light and wavelength affects resolution.
 Unlike visible light, electron beams cannot penetrate very well. So, special techniques of thin
sectioning are needed to prepare specimens before observing them.
 To obtain sufficient contrast, the preparation are treated with stains such as osmic acid,
permanganate, uranium, lanthanum; because these substances are composed of atoms of
high atomic weight, they scatter electrons well and thus improve contrast.
 Scanning electron microscopy used to observed external features of an organisms or cell.
 No need for thin sections.
 The specimen is coated with a thin film of a heavy metal such as gold.
 An electron beam then scans back and forth across the specimens. Electrons scattered from
the metal coating are collected and activate a viewing screen to produce an image.