MOLECULAR CELL BIOLOGY

MICROSCOPY
Cell Diversity in Human Body
About 200 cell types in
human body
Major common features
-plasma membrane
-nucleus
-cytoplasm(containing
organelles and
cytoskeleton)
Six Principal Organelles
Endoplasmic reticulum(rough and smooth)
Golgi Apparatus
Lysosomes
Peroxisomes
Mitochondria
The Three Principal Components
of the Cytoskeleton
Microfilaments
Intermediate Filaments
Microtubules
Three branches of Microscopy
Optical
Electron
Scanning Probe
Optical and Electron microscopy measure refraction, diffraction, and
reflection of the source radiation
Optical uses white light, fluorescent light, or lasers
Electron uses electromagnetic radiation/electron beams
Scanning uses a physical probe to interact with the surface of the
specimen
Imaging Techniques
Technique Image Formed By Lowest Resolvable Unit Approx Lower Limit

1 m
Optical Microscopy Light Rays Microns (m)
(monochromatic light)

Coherent Light Source .1 m

Confocal Microscopy Microns (m)
(Laser) (X-Y Direction)

Transmission
Electron Microscopy (TEM) 2
Electrons Angstroms ()
(high resolution TEM)

Scanning Electron Nanometers (nm) to 10 nm

Electrons (100 )
Microscopy (SEM) Angstroms ()

Atomic Force & Scanning

Tunneling Microscopies
Molecular Mechanical 40
(AFM/STM) Angstroms ()
Probes (theoretical)
Units of Measure
m - Micrometer
1,000,000 micrometers = 1 meter
Strand of hair has a diameter of ~ 20-180 m
106
nm - Nanometer
1,000,000,000 nanometers = 1 meter
109
Wavelength of visible light (400-700 nm)
- Angstrom
10,000,000,000 Angstroms = 1 meter
1010
Used to measure the size of atoms/bond lengths
Length of a C-H bond in methane is ~1 Angstrom
Properties of light

Reflection-
Refraction
Numerical Aperture
Refraction
Change in the direction of a wave
(light) due to a change in speed
The straw in the picture looks bent
because the light is bending as it
moves from the water to the air
Reflection

Reflection is defined as a
change in direction of a wave
at an interface between 2
different media so that the
waveform returns to the
media from which it came
Used in focusing light waves
to increase transmitted light
Numerical Aperture

NA of a microscope objective is
a measure of its ability to gather
light
The more light (higher NA) the
better the resolving power of the
lens
Better resolution
NA = (n)sin()
n = Refractive Index
= the maximum cone of
light than can enter the lens
Usually the NA of an objective
increases with its magnifying
power.
The smallest detail that can be
resolved is proportional to:
/NA
Optical Microscope

1. Ocular lens
2. Objective turret
3. Objective
4. Coarse Adjustment
5. Fine Adjustment
6. Stage
7. Light source
8. Condenser
9. X-Y Control
 The common light microscope used in the laboratory is called a
compound microscope because it contains two types of lenses that
function to magnify an object.
The lens closest to the eye is called the ocular, while the lens closest
to the object is called the objective.
Most microscopes have on their base an apparatus called a
condenser, which condenses light rays to a strong beam.
A diaphragm located on the condenser controls the amount of light
coming through it.
Both coarse and fine adjustments are found on the light microscope
 To magnify an object, light is projected through an opening in the stage,
where it hits the object and then enters the objective.
An image is created, and this image becomes an object for the ocular lens,
which remagnifies the image.
Thus, the total magnification possible with the microscope is the
magnification achieved by the objective multiplied by the magnification
achieved by the ocular lens.

A compound light microscope often contains four objective lenses: the

scanning lens (4X), the lowpower lens (10X), the highpower lens (40 X),
and the oilimmersion lens (100 X).
With an ocular lens that magnifies 10 times, the total magnifications
possible will be 40 X with the scanning lens, 100 X with the lowpower lens,
400 X with the highpower lens, and 1000 X with the oilimmersion lens.
Most microscopes are parfocal.
This term means that the microscope remains in focus when one switches
from one objective to the next objective.
Types of Microscopes
divided the different types of microscopes into three main
categories: Optical Microscopes,
Electron Microscopes, and
Other.
Compound Microscope
The compound microscope consists of two optical components : the objective lens system, which
has a very short focal distance and is placed very close to the object; and the ocular or eyepiece
system, which has a longer focal length, lower magnification; and which further magnifies and
projects the image onto the retina of the eye.
The objective lens projects a real image (the intermediate image) of the object up in the body of
the microscope onto the objective conjugate image plane or primary image plane.
The distance from the back focal plane of the objective to the primary image plane within the
microscope body is referred to as the optical tube length. Depending on the type of microscope
and eyepiece, the primary image plane may be either within the microscope body itself or within
the eyepiece (see Infinity corrected optics).
The objective lens forms a real image in the microscope body that acts as the object for the ocular
lens.
The real image becomes the object for the eye itself and is projected onto the retina.
The ocular in turn acts as a simple magnifier to form a large virtual image at the distance of most
distinct vision (25 cm) from the eye
Total Magnification = Magnification of the Objective Lens X Magnification of the Ocular Lens
Dark Field Microscope
In dark field microscopy , a special condenser is used so that only light
reflected off the specimen enters the objective.
The appearance is of a brightly lit specimen against a dark
background, and often with better resolution than that of the bright
field microscope.
Phase contrast microscopy
uses special optical components to exploit subtle differences in the
refractive indices of water and cytoplasmic components to produce
contrast.
Light waves that are in phase (that is, their peaks and valleys exactly
coincide) reinforce one another and their total intensity increases.

 Phase contrast microscopy takes advantage of fact that structures with

different refractive indexes bend the light differently.
With special phase optics, this difference in the ability to bend light
translates into a difference in contrast.
For example, light rays traveling through cellular material get bent and
retarded.
We probably could not detect this bending in bright field. In phase
contrast, a phase plate slows down the highly refracted light rays and puts
them "out of phase".
This is seen as a difference in contrast between the cell and its background.
The background is generally seen as dark, with the organism in sharp
contrast.
This is extremely useful for microbiologists because we can view
microorganisms without physically or chemically altering the cell. Bacteria
(and other microorganisms) can be viewed alive. Simple wet mounts can
be made so such properties as motility and growth can be observed..
Fluorescence microscopy
uses a fluorescent dye that emits fluorescence when illuminated with
ultraviolet light. In some cases, specimens possess naturally
fluorescing chemicals and no dye is needed.
The Electron Microscope
The electron microscope uses an electron beam to create an image,
with electromagnets acting as lenses.
The limit of resolution is improved by a factor of 1000 (theoretically
down to 0.1 nm, but more realistically down to 2 nm) over the light
microscope.
The transmission electron microscope
produces a two-dimensional image of an
ultrathin section by capturing electrons
that have passed through the specimen.
The degree of interaction between the
electrons and the heavy metal stain
affects the kinetic energy of the electrons,
which are collected by a fluorescent plate.
The light of varying intensity produced is
directly proportional to the electron's
kinetic energy and is used to produce the
image.
The TEM is useful for studying a cell's
interior, its ultrastructure.
A scanning electron microscope
used to make a three-dimensional image of the specimen' surface. In
this technique, a beam of electrons is passed over the stained surface
of the specimen.
Some electrons are reflected (backscatter electrons), whereas other
electrons (secondary electrons) are emitted from the metallic stain.
These electrons are captured' and used to produce the three-
dimensional image.