UNITS OF MEASUREMENT
1mm = 1000µm
1µm = 10-3mm (convert mm to µm by multiplying by 1000 = 3 zeros)
1nm = 10-6mm (convert nm to mm by dividing by 1000000 = six zeros)
1000nm = 1µm
0.001µm = 1 nm
Protozoa are fairly large single-celled animals.
Bacteria are so small, they are measured in µm.
Viruses are even smaller, so they are measured in nm.
MICROSCOPY: THE INSTRUMENTS
VOCABULARY
Immersion oil: keeps light from bending and allows lens to be refracted.
Resolution: ability of two lenses to distinguish two points.
Parfocal: focused in all lenses.
Depth of field: how much of the background is in focus at the same time that the
foreground is in focus.
Refractive Index: a measure of the light-bending ability of a medium
Numerical aperture: numerical aperture increases as depth of field decreases.
Resolution power: limits the useful magnification of the microscope resolving power.
RESOLUTION: the ability of the lenses to distinguish two points. A microscope with a
resolving power of 0.4 nm can distinguish between two points greater than or equal to
0.4nm. Shorter wavelengths of light provide greater resolution. When you go to the eye
doctor, you look at the chart (Snellen chart) and read it from 20 feet away. If you can read
what a normal sighted person can read from 20 feet away, it is called 20/20 vision. If you
can’t read it well, your eyesight has less resolution than normal.
RESOLVING POWER: the distance between two closely adjacent objects where the
objects still appear separate and distinct. The shorter the distance (the smaller the
number), the better the resolving power (the sharper the image). To calculate the
resolving power (to see how close two objects can be so you can still see them):
D = distance (smaller number is better)
0.61 = a constant number, does not change
NAobj = numerical aperture of the objective (larger number causes D to decrease,
which is better)
λ = (lambda): the wavelength (nm) of the light going through the microscope.
Convert lambda’s nm to mm by moving the decimal place six places to the left.
D = 0.61 λ / NAobj
PRISM: a triangular device that breaks up light into its various wavelengths so you can
see all the colors of the rainbow (the visible spectrum). The wavelengths of the visible
spectrum range from 350-700nm. The visible spectrum of colors starts with violet
(350nm), and goes on to indigo, blue, green (550nm), yellow, orange, red (700nm). These
colors are picked up with special photoreceptor cells in the human retina called cones,
and the information is transmitted to the brain where we perceive the color.
Sample Problem
When we want to observe the color green (550nm) under an oil-immersion objective lens
of a microscope, where the NAobj is 1.25, what is the resolving power (in microns)?
Solution
* Convert 550 nm to mm by moving the decimal place six places to the left:
550nm = 0.000550 mm
D = (0.61)(0.000550mm) / 1.25
D = 0.0002684mm  convert this to microns (µm) by multiplying by 1000
D = 0.2684 µm  round off to the nearest 1/100 (two decimal places to the right)
D = 0.27 µm
Sample Problem
The NAobj for the high-dry (400x) lens is 0.65
What is the resolving power (D) of this objective when viewing a wavelength of 550nm?
Solution
D = (0.61)(0.000550) / 0.65
D = 5.1615 mm (Now multiply by 1000 to convert to microns)
D = 0.52 µm
Therefore, we can see an organism such as E. coli, which is 2µm long and 1µm wide
because it is larger than the resolving power. However, we could not see Haemophilus
influenza, which is 0.2µm long because it is smaller than our resolving power.
Therefore, the resolving power limits the useful magnification of the microscope.
Resolution determines the magnification.
REFRACTION
Refraction is the bending of light caused by the surrounding medium. When you stick a
pencil in a glass of water and look at it from the side, the pencil appears to be bent. That
is because the water is refracting the light. We need to take that into consideration when
viewing things under oil magnification with the microscope.
N = Refraction Index of the medium surrounding the lens
Air: N= 1
Glass: N = 1.5
Immersion Oil: N = 1.51 (about the same as glass)
TYPES OF MICROSCOPES
SIMPLE MICROSCOPE: Has only one lens, like an ocular (eyepiece)
COMPOUND MICROSCOPE: More than one lens, like an ocular and an objective. An
example is the Brightfield microscope.
There are two main types of compound microscopes: Light Microscopes and Electron
Microscopes.
Light Microscopy
Light microscopes have an RP of about 0.25 µm, about the size of the very largest viruses
and the very smallest cells. They extend the resolving power of the human eye by about
1000x. One major advantage of light microscopes is their low cost and relative ease of
use. Another is that it is possible to view living tissue in some microscopes, especially
with phase optics. For many applications, light microscopy is the preferred tool, such as
in screening tissues for signs of cancer or other pathology. A disadvantage of light
microscopes is that they do not have good depth of field; when you look at the
foreground, the background is out of focus.
Light microscopes can have a variety of configurations. Some popular types are:
Dissecting
Brightfield
Darkfield
Phase-contrast
Differential Interference contrast
Fluorescence
DISSECTING MICROSCOPE
This type of microscope is for viewing in 3D objects that are very large (visible to the
naked eye) but in greater detail than the naked eye can see. It shows a view like a
powerful magnifying glass. It typically magnifies 20x to 40x. It requires an external light
source (plug in a lamp and face it to the scope).
BRIGHTFIELD ILLUMINATION
Most student microscopes (including the ones we use in lab) are Brightfield microscopes,
requiring that objects to be viewed either have natural contrast or be stained to provide
sufficient contrast for viewing. Some stains, called vital stains, can be used on living
tissue; most require the sample to be dried and fixed.
In general, the Brightfield microscope is not very good for looking at live cells (without
stain) because the contrast is not good; cells look white on a white background. You can
attempt to look at live cells with this microscope by opening up the iris all the way to let
all the light in to improve contrast. This microscope is very good for looking at stained
cells. When you apply stain to cells it kills them, but the contrast is improved and you can
see them well. This type of light microscope produces the most realistic images of a cell,
compared to other light microscopes.
DARKFIELD ILLUMINATION
This has limited applications, but it is good for looking at live cells because the cells look
white against a dark background. You can even see the cilia and flagella.
PHASE CONTRAST MICROSCOPY
This is the most sensitive type of microscopy. Live cells are visible because contrast is
increased. You can even see cilia and flagella.
DIFFERENTIAL INTERFERENCE CONTRAST
This shows depth in 3D. It has two beams of light and a prism. By moving the prism, you
see different colors. This is a good way to view live cells; it has the best resolution of the
light microscopes. To get better resolution, you need to use electron microscopy.
FLUORESCENCE MICROSCOPY
In this technique, ultraviolet light (invisible) is used. Cells are stained with a fluorescent
dye call fluorochromes, which energizes electrons to create visible light. You can see the
light given off by cells. It is not possible to see live cells with this. It is often used for
viewing syphilis and TB.
ELECTRON MICROSCOPY
Electron microscopes use a beam of electrons rather than light to illuminate an object.
Magnification of up to a million-fold is possible. Creating an electron beam requires very
high voltages, typically 100,000 volts, and the electron beam works well only in a
vacuum, so air must be removed prior to specimen examination. Electrons are not easily
scattered by the light atoms of common cellular materials: C, H, O, and N. To improve
electron scattering and increase contrast, heavy metals are used, either as coatings over
exposed surfaces or as stains applied to tissues.
The primary advantage of all electron microscopes is the added resolving power and the
extraordinary clarity of the images.
Disadvantages include: the cost and complexity of the equipment; the need to isolate
them from vibrations (vibrations from a passing truck can be easily visible when viewing
an object at 1,000,000x) the inability to study living tissue; the very thin samples
required; and the need for heavy metals to obtain good contrast, with possible damage or
distortion of the sample.
The two major forms of electron microscope, TEM and SEM, are very different. One
views surfaces or "outsides," the other views "insides." Together, they reveal an
extraordinary world of very small objects.
Transmission Electron Microscope (TEM)
Transmission electron microscopes (the most common types of electron microscopes)
transmit a beam of electrons through the sample. To achieve this, samples must be very
small and very thin. Therefore, the specimen is sliced thin using an instrument called an
ultramicrotome. Most samples are mounted on tiny circular grids with a very fine mesh
screen. The grid is coated with a thin plastic layer, and sample is either adsorbed directly
(as in a virus sample) or prepared as an ultra thin section of tissue that has been fixed,
stained, dehydrated, and embedded in resin to form a hard block of plastic. This gives a
view of sections of the specimen’s cells, revealing the structures of its organelles.
This type of microscope uses electromagnetic lenses and creates a flat image, and since
there is no light, there is no color. Electrons float better in a vacuum, so it has a vacuum
pump. Staining is done with heavy metal salts instead of dyes. Areas with low levels of
impregnation of these metal salts produce bright regions on the screen, and heavily
impregnated areas produce dark ones (the electrons can't get through). This improves
how thing look with this microscope.
Advantages of TEM are the ability to view objects at a very high level of resolution, and
to see the insides of common biological materials. Disadvantages include the limit of
only viewing a small sample at one time, and the need for very thin specimens. If the
specimen is thicker than 0.25 µm, too many electrons are adsorbed to obtain a good
image. The TEM and the SEM have the highest resolution of all the microscopes.
Scanning Electron Microscope (SEM)
Scanning electron microscopes work by scanning a beam of electrons across the surface
of an object. Each area so illuminated discharges a small shower of electrons, called
secondary electrons. These are trapped by a detector that converts electrons into light
signals, which in turn are trapped by a photomultiplier and used to drive a cathode-ray
tube (similar to a television tube). The resulting image can be viewed and photographed.
A unique feature of an SEM is its great depth of focus; objects in both foreground and
background appear equally sharp and crisp, giving the best 3D image because it has the
highest resolution. By contrast, light microscopes have a very shallow depth of focus, so
as one moves to high-power lenses, only a small part of the image is in focus at any one
time.
Another advantage of SEM is that sample size can be very large. For example, an entire
insect or small flower can be viewed at one time. The SEM gives the most realistic view
of a specimen.
This provides a view of the outside of the cell only; it cannot penetrate to the inside of
cells like the TEM. It also needs a vacuum, and cells are never alive. Its resolution is 10x
better than a Brightfield microscope.
A special type of SEM is called the Scanning Probe Microscope.
Scanning Probe Microscope
This microscope forms images of surfaces using a physical probe that scans the
specimen. An image of the surface is obtained by mechanically moving the probe in a
raster scan of the specimen, line by line, and recording the probe-surface interaction as a
function of position.
A Raster scan, or raster scanning, is the pattern of image detection and reconstruction in
television, and is the pattern of image storage and transmission used in most computer
image systems. The word raster comes from the Latin word for a rake, as the pattern left
by a rake resembles the parallel lines of a scanning raster.
In a raster scan, an image is cut up into successive samples called pixels, or picture
elements, along scan lines. Each scan line can be transmitted as it is read from the
detector, as in television systems, or can be stored as a row of pixel values in an array in a
computer system. On a television receiver or computer monitor, the scan line is turned
back to a line across an image, in the same order. After each scan line, the position of the
scan line is advanced, typically downward across the image in a process known as
vertical scanning, and a next scan line is detected, transmitted, stored, retrieved, or
displayed.
One advantage of scanning probe microscopy is that the resolution of the microscopes is
not limited by diffraction, but only by the size of the probe-sample interaction volume.
Disadvantages of scanning probe microscopy are that the scanning techniques are
generally slower in acquiring images due to the scanning process, and the maximum
image size is generally smaller. This type also gives a 3D view of the specimen.
COMPARISON OF MICROSCOPES
BRIGHTFIELD
DARKFIELD
PHASECONTRAST
DIFFERENTIAL
INTERFERENCE
CONTRAST
FLUORESCENCE
TRANSMISSION
ELECTRON
SCANNING
ELECTRON
SCANNING
PROBE
Dark objects are visible against a bright background.
Light reflected off the specimen does not enter the objective lens
Not for looking at live cells
Maximum resolution is 0.2µm and maximum magnification is
2000x
Stains are used on specimens
Light objects are visible against dark background
Used for live cells, cilia, flagella
Especially good for spirochetes
Uses special condenser with an opaque disc that eliminates all
light in the center
No staying required
Accentuates diffraction of the light that passes through a
specimen
Good for live cells; good contrast
Most sensitive; cilia shows up
Not three-dimensional
Uses two beams of light
Shows three dimensions
Has a prism to get different colors
Good for live cells (unstained)
Best resolution
Uses ultraviolet light
Stained cells with fluorescent dye; energizes electrons and
creates visible light
No live cells
Quick diagnosis of TB and syphilis
Get flat images
Have vacuum pumps to allow electrons to float better
Stain with heavy metal salts
Shows sections of cell, revealing organelles
Requires an ultramicrotome
Best resolution of all microscopes
Surface view only
Needs a vacuum
No live cells
Three-dimensional view
Physical probe scans the specimen
Raster scan: image is cut up into pixels and transmitted to
computer
Not limited by diffraction
Slower in acquiring images
Maximum image size is smaller