BIOLOGY 2B03 Chapter Notes - Chapter 3: Transmission Electron Microscopy, Bright-Field Microscopy, Differential Interference Contrast Microscopy
17 Jun 2018
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BIOLOGY 2B03 - Module 3 Lecture II
Visualizing the Structure of the Cell
If there is smaller than a mm, we would need to use a microscope
Light Microscope: Uses light to illuminate the object that we are looking at
● Allows us to visualize most prokaryotic cells and organelles inside eukaryotic
prokaryotic
● Objects less than 100 nm in size, can not be seen under a light microscope
● Conventional and fluorescent
Electron Microscope: uses a beam of electrons to illuminate the object.
● Allow us to visualize small bacteria, viruses, we could even visualize protein
complexes, lipids or some small molecules
● Transmission and scanning
Resolution or the Limit of Resolution
Our ability to see small objects using a microscope is limited by the wavelength of the
source of illumination
Resolution (D): is a measure of distance: the smallest distance between two ojects at
which the objects still appear distant
● The smaller value = the better resolution; able to see more details
● D = (0.61λ) / (Numerical aperture)
○ Numerical aperture (NA): is a measure of how light is bent as it passes
from the glass of the microscope
■ Closer to 1 the better resolution
● It is better to minimize refraction of the illuminating light
○ Smaller wavelength meaning that a smaller value of D; better resolution
Improving Resolution
Visual Light: wavelength is between 400-700 nm
Sample Calculation
● NA = 0.94, illuminating light λ = 450nm, D=292 nm
● Meaning that if two objects are closer together than 292 nm, they cannot be
distinguished under the microscope
● Also be the limit on the size of objects
● Typically bright field microscopy has a resolving power of about 0.2 um and can magnify
images about 1000x
● Electron microscopy uses a beam of electrons, shorter λ; D is between 0.1 to 10 nm,
UV Light: wavelength is between 200-300 nm
● We will reduce D giving better resolution
Brightfield Microscopy
Light microscopy: samples may be live or fixed, and stained or unstained
find more resources at oneclass.com
find more resources at oneclass.com
● ex) a simple blood smear stained with a whole cell dye
○ Able to distinguish between the biconcave shape of typical red blood cell
and sickled red blood cells (affected by sickle cell anemia)
● The sample is magnified 535X the length of scale bar represents 25 µm
● Therefore we can calculate the diameter of a typical red blood cell to be 8 µm
It is common to embed samples in paraffin to slice thin sections for observation under the
microscope
● ex) section of a small intestine of a mouse
● The surface cells are lined up adjacent to one
another in an even row
● Blue stained = nuclei of the cells, magenta
stained = goblet cells (GC)
○ GC stain particularly strongly with PAS
stain because they are rich in
polysaccharides and glycogen
● The outer surface of microvilli (MV) covering the
columnar epithelial cells
● BUT, we will need a electron microscope to
improve the resolution enough to see these
structures well
Nomarski and Phase Contrast Microscopy
Also known as differential interference contrast or DIC
● Are the complementary techniques capable of producing high
contrast images of unstained and in fixed, transparent biological
specimens
● Both rely on enhancing in density of different regions of the
specimen; each accomplish this in different ways
○ Phase Contrast: tends to favour clease visualization of
internal cellular structures
■ There is a halo created around the external surface
of the cell that obscures visualization
○ DIC: provides clearer, sharper images of the edges and
surfaces
■ Much clearer view of periphery of the cell
Live Imaging: Nomarski/DIC Microscopy
One clear advantage of both techniques: live specimens can be visualized
● Able to look at the dynamics of a living syste,
● Cells may able to migrate along the surface of the underlying media
Global Fluorescence - What Does it Tell Us?
Bright foci distributed in heterogeneous pattern across the earth
● The lights work as marker for human settlements and also for what people are
doing
● Dense light: lights are very closely clustered and this tells us where cities are;
concentrated wealth where people have easy access to electricity
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
If there is smaller than a mm, we would need to use a microscope. Light microscope: uses light to illuminate the object that we are looking at. Allows us to visualize most prokaryotic cells and organelles inside eukaryotic prokaryotic. Objects less than 100 nm in size, can not be seen under a light microscope. Electron microscope: uses a beam of electrons to illuminate the object. Allow us to visualize small bacteria, viruses, we could even visualize protein complexes, lipids or some small molecules. Our ability to see small objects using a microscope is limited by the wavelength of the source of illumination. Resolution (d): is a measure of distance: the smallest distance between two ojects at which the objects still appear distant. The smaller value = the better resolution; able to see more details. Numerical aperture (na): is a measure of how light is bent as it passes from the glass of the microscope.
