Autore Bruno Pacifici
Il microscopio
Introduzione al microscopio
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Typically, a person would think that a
microscope is an instrument design to magnify small things. But, this really
isn't the case. For example, you can go out to any hobby or toy store and for
$49.95 buy an instrument capable of magnifying objects to 1200 times. And that
includes a zoom lens and light source. Most student & research microscopes
magnify no more than 1000 times and cost starting at around $1500.00, with
research microscopes going into the tens of thousands of dollars. Is the
academic community being taken for a ride? No. The $49.95 microscope only gives
you an image that is a soft blur at 1000 times, whereas the research
microscopes image is crystal sharp. This is called resolution,
the ability to see fine details. Once you can resolve fine details then you can
magnify them. Every optical system has a finite resolution, if you magnify
objects beyond the resolution the result will be empty magnification. So, the actual purpose of a
microscope is to see small things clearly.
Another,
desirable attribute of a microscope is depth
of field, which is the range of depth that a specimen is in acceptable
focus. A microscope that has a thin depth of field will have to be continuously
focused up and down to view a thick specimen.
A
third feature that a microscope has its mechanism for contrast
formation. Contrast is the ratio between the dark and the light. Typically,
most microscopes use absorption contrast, that is the specimen is subjected to
stains in order to be seen. This is called bright field microscopy.
There are other types of microscope that use more exotic means to generate
contrast, such as phase contrast, dark field, differential interference
contrast.
The fourth desirable feature is a strong illumination source. The
higher a microscope magnifies the more light will be required. Also, there will
be more optical trade off leeway when there is more light present. The
illumination source should also be at a wavelength (color) that will facilitate
the interaction with the specimen. All microscopes fall into either of two
categories based on how the specimen is illuminated. In the typical compound
microscope the light passes through the specimen and is collected by the image
forming optics. This is called diascopic illumination. Dissecting
(stereo) microscopes generally use episcopic illumination for use with
opaque specimen. The light is reflected onto the specimen and then into the
objective lens.
The
four attributes of an optical system trade off with each other. Resolution and
brightness is antagonistic towards contrast and depth of field. For example,
you can not have maximum resolution and maximum contrast simultaneously.
Theoretically speaking, if you had an infinite resolving system there would be
no contrast to discern the image. It is up to the microscopist to decide which
attribute is needed to view a particular specimen. All of which are controlled
be the iris diaphragm.
Obbiettivi
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The
objective lens is the lens that is closest to the object or specimen. It is
essentially the information-gathering lens of an optical system. Therefore it is
regard as the most important lens of the microscope. There are many different
types of objective lenses. The most common and inexpensive is the achromat.
This lens is usually found on student microscopes. It is corrected for
spherical aberration for only green light. Chromatic aberration is corrected in
only two colors. The apochromat objective is far superior and generally
very expensive. Chromatic aberration is corrected for all three colors and it
spherically corrected for two colors. These objectives quite often will require
a special compensating eyepiece. Semiapochromat objectives have
correction in between the apochromat and achromat. Flat field or plano
objectives compensate for curvature of field and are excellent for histology
work. The flat field objectives can be optically constructed to be also an
achromat, semiapochromat or apochromat. In the latter case the lens would be
called a plano apochromat which is generally regarded as the finest lens
available. The price of a single plano apochromat will run into the many
thousands of dollars.
Each objective has information critical for the maximum resolution
possible written on the side of the barrel. Generally the magnification is
print in the largest text with the manufacturer type designation. The second
value is the numerical aperture. Beneath that, in a smaller font the tube
length and the cover glass thickness is given. Any special information will
also be added such as if it is an oil lens, infinity etc. The tube length
usually 160 refers to the distance between the objective and the eyepiece in
millimeters. It must be maintain if the aberrations are to be corrected. You
can recognize a superior microscope if when adjusting the interpupillary
distance you can see the eyepiece extend. This happens to maintain the proper
tube length. The coverslip thickness usually around .17mm is also critical.
This corresponds to a cover glass of No. 1.5. The more sophisticated objectives
even have a coverglass compensation control that you dial in the thickness of
the coverglass.
Condensatori
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The
substage condenser of a microscope is design to focus the light onto the specimen
(il campione). In addition it must also fill the numerical aperture of the
objective. Like objective lenses there are several different types. The most
common being the Abbe condenser. This type is not corrected for optical
aberrations. The achromatic condenser is corrected for both spherical
and chromatic aberrations. Both types of condenser have their numerical
aperture printed on the side. This needs to be of equal or greater value then
that of the objective N.A., otherwise, the full resolution of the objective
will not be utilized. Most substage condensers can use immersion oil like that
of the objectives to achieve their full N. A. This is not recommended unless
you are doing very demanding photomicroscopy work.
Diaframma a iride
The iris diaphragm is the most important single control on the
microscope. There is a misconception that it is used to regulate the amount of
light. The light intensity control is the sole means to adjust the brightness. The
iris diaphragm is the resolution verses contrast control. It does this by
varying the size of the numerical aperture of the objective lens. Usually,
lenses such as those found on cameras have the iris diaphragm built in the
objective lens. In a microscope objective the iris diaphragm would have to be
very small, which would be difficult to manufacture. So the optical engineers
put the iris diaphragm at the optical equivalent of being in the objective
lens, in the condenser assembly. This is one of the reasons why the condenser
lens has to be set at the correct distance to the objective. In addition the
iris diaphragm controls the depth of field.
Oculare
The eyepiece is basically a
projection lens system. There are three types generally used in light
microscopy. The most common is the Huygenian type. This eyepiece is used
with low and medium magnification and is designed to project the image into a
human eye. Some of these eyepiece will have a long eyepoint, the spot there
your eye should be, so you can focus with your glasses on. If you suffer from
stigmatism you should ware your glasses while using the microscope. If you are
near or far sighted then you can adjust the eyepiece for your personal
correction using the diopter corrector and leave your glasses off. The second
type of eyepiece is the compensating eyepiece and is generally used with
apochromate or flat field objectives. These provide superior image quality. The
third type is the photo eyepiece. These are designed to project a
corrected image onto film plane in a camera. These are generally considered the
finest of eyepieces. All eyepieces will have a relative magnification written
on the side of the barrel. They range in magnification from 2.5X to 15X with
the lower magnifications used with the photo eyepiece.
Apertura limitante il campo
The
field limiting aperture is used to determine the correct position and center of
the condenser lens. It is used in conjunction with the condenser centering
knobs to place the illumination in the center. It also helps in reducing the
amount of optical flare.
La risoluzione
Resolution
is the ability to discern fine details. Typically, for image system it is
express as a linear dimension. Such as the resolution of a typical electron
microscope is about 0.2nm. This means that objects separated by more than 0.2nm
will be resolved as being separate. Lord Rayleigh in 1896 first
described resolution as a function of the airy disc.
Airy disc of two point light sources.
If
you have a point light source on one side of a lens the opposite side will show
an image of the light. The image will have the appearance of a larger diameter
then the source. This is a result of the diffraction of light from the edge of
the lens. Notice how there are discrete bands of decreasing intensity radiating
out from the center of the spot. Rayleigh showed how the fundamental resolution
is when two light sources must be separated by at least the distance of the
first band.
light distribution of a
cross section of respective airy disc.
Ernst Abbe was able to
derive an expression for resolution by optical geometry. The Abbe equation is base
on the size of the lens that will capture the light.
= refractive index of the medium.
=wavelength of the light.
=half the acceptance angle of the lens.
The
resolution will be expressed in the same units as the wavelength of the light.
Alpha is one half the acceptance angle of the lens and n is the index of
refraction of the medium between the specimen and the lens.
The numerical aperture (N.A.) is basically a value that describes the quality
of a lens. It is
derived from the size of the lens, its working distance and the index of
refraction. All quality objective lens will state the numerical aperture on the
side of the barrel. A good rule of thumb is that the effective magnification of
an objective is its numerical aperture times 1000. So a 40 x objective that has
a N.A. of 0.65 has an effective magnification of 650 times. If you magnify
beyond this you will only get empty magnification. You can calculate the
theoretical resolution of any optical system using Abbe's equation. To
calculate the resolution of the objective above multiple the wavelength of
green light (0.5 micrometers) times the constant .61 divided by the N.A. The
result will be 0.47 micrometers. In another example you can calculate the
resolution of a pair of 8 x 20 binoculars. The number 8 is the magnification
and the number 20 is the diameter of the objective lens. Assume you were
looking at a specimen 100 ft away the alpha would be 0.0188 degrees. Plugging
in abbe's equation the result for red light (650 nm) is 1.2 mm. Remember, this
is a theoretical value with is the best possible resolution possible. The
practical resolution will always be less due to optical
aberrations.
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Achromate
objectives |
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Magnification |
N.A. |
Theorectical
Resolution (micrometers) |
Practical
Resolution (micrometers) |
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4X |
0.10 |
3.05 |
3.40 |
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10X |
0.25 |
1.22 |
1.30 |
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40X |
0.65 |
0.47 |
0.52 |
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100X |
1.30 |
0.24 |
0.26 |
La profonditą di campo
Depth
of field is the area in front of and behind the specimen that will be in
acceptable focus. For example, when you take a photograph of a close up of a
person the background will often be out of focus. Below are photos of a
histology specimen that were taken under high power at different heights.
Notice
how whole nuclei in one photo are completely absent in the other. The histology
section was cut from a microtome at about 20 microns thick. The optical section
from the depth of focus of the microscope is much, much thinner (<1
micrometer). This occurs in all optical devices and is dependent on a number of
parameters. The single most influential is the numerical aperture. The diagram
shows a lens a its full aperture opening.
On
the right side, the focus point is a vertical line representing the specimen
plane. The horizontal line shows the range of acceptable focus. The criteria
for acceptable focus is ultimately dependent on the circle of minimum
confusion, the summation of all the optical aberrations. However, in a
practical sense the acceptable focus is dependent on effective magnification.
The higher you magnify an object the more critical the focus.
In
the second diagram, the numerical aperture of the lens is stopped down by an
aperture. This decreases the angle of acceptance. Since, the rays of light are
now at a shallower angle the range of focus is increased. The focal length of a
lens is also a factor in controlling depth of field. Since the angle of acceptance
is dependent on the focal length, which in turn determines the numerical
aperture.
The
diagram illustrates how a lens with a short focal length will have a very tight
depth of field. While a lens with a long focal length will be much deeper.
Finally it turns out that the wavelength of the light is also a factor. So,
large lens with short focal length and high magnifications will have a very
short depth of field. Small lens with long focal length and low magnifications
will be much better.
Depth of field
The
depth of field deals with the focus plane of the specimen. On the other side of
the lens is the focus plane of the image. The range of acceptable focus for the
image is called depth of focus. It is essential the same as depth of field but
for one important difference, that being magnification. With higher
magnification depth of field becomes shorter, however higher magnification
increase the depth of focus for the image. This is because the magnification is
done with a projection lens. An example is when a slide projector is moved
further away from the screen the magnification increases. In addition, so does
the focal length affecting the angle of acceptance and ultimately the depth of
focus.
Depth of focus
The
table shows optical data for typical student light microscope. Notice that the
practical depth of field is much better than what the equation would predict.
This is because the manufacturer has stop down the iris diaphragm to get
superior results. Also notice that at the low magnification the difference
between the practical and the theoretical is much greater. This is a function
as to how small and round the iris diaphragm can be made.
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Achromate
objectives |
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Magnification |
N.A. |
DF
theorectical (micrometers) |
DF
Practical (micrometers) |
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4X |
0.1 |
50.0 |
172.5 |
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10X |
0.25 |
8.0 |
27.6 |
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40X |
0.65 |
1.2 |
3.0 |
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100X |
1.30 |
0.3 |
0.7 |
Contrasto
Contrast is number of shades found
in an image. A high contrast picture will have only two shades, black and white.
The more shades you have, the less contrast, but it should be understood that
you also have more information. This is called dynamic range. Color is also
considered a form of contrast. As an example, the more colors and shades a
computer picture has the more memory it will take. Optically speaking, contrast
is necessary since it is possible to generate an image of high resolution but
it is the contrast that lets you see it. In standard bright field microscopy
contrast and resolution are mutually exclusive. The result is that if you have
high contrast you will have poor resolution. It wasn't until the twenty century
that optical instruments were able to have both high resolution and contrast.
With respect to microscopy there are several mechanism that can form contrast.
Absorption
contrast is the contrast that is involved in normal human vision and bright
field microscopy. The light is literally absorbed by pigments in the specimen.
The result is less light is transmitted to the eye so the specimen appears
dark. If the pigments absorb only a specific wavelength of light the specimen
will appear the complimentary color.
Diffraction
contrast is when light hitting the edge of the specimen bends and will
diffracted out of the optical path. This is the mechanism used for dark field
and stop contrast microscopy. Interference contrast uses constructive and
destructive wave interference. It requires the splitting of light waves to
create a reference and analytic waves. The analytic wave passes through the specimen
and will be retarded relative to the density of the specimen. The two waves
will then be brought together where they can interfere with each other
producing contrast. This is the basis of phase contrast and differential
interference contrast microscopy. Which are highly desirable for biologist
since they do not erode resolution and do not require staining of the specimen.
Scatting is a form of contrast generation typically found in electron
microscopy.
Le aberrazioni
Aberrations are optical imperfections which impair the theoretical
resolution of a lens.
There are many different types of aberration of that only the more significant
to microscope operation will be discussed here. Chromatic aberration is the
inability of a lens to focus different colors of light to the same spot. The
shorter the wavelength of light the more it will be refracted by an optical
surface. As a result blue light has a shorter focal length then red light.
The
diagram shows a lens focusing a white light point source. The point at where
the green light is focused the red and blue light will be a blur. A classic example of this is in
a beginning microbiology class. Students will be asked to identify a bacteria
as being either gram positive (blue) or negative (pink). Since most student
microscope use achromat objectives and are not set up properly, the colors of
light will focus at different levels. The effect is as you pass through
focus the specimen turns from gram positive to negative. The student then
reports the specimen as being gram variable.
Spherical aberration result when the edges of a lens refract light more
than the center.
The diagram shows the effect of this. The area at which most of the rays focus
together the image will form a disc. This is called the circle of minimum
confusion. If you were to view the image, of a point source, it will have a
diffuse halo around it. It is possible to add in a compensating lens to correct
the effect, however it is generally only effective for a particular wavelength
(color) of light. The optical complexity goes up as you try to compensate for
more colors.
Curvature of field is another aberration caused by the fact that a lens
focuses not on a flat surface but on that of a sphere. As the object moves off the
optical axis the focal distance to the lens is farther. This will impart a
magnification error on the image. The resulting image will have either a pin
cushion or barrel distortion effect. This aberration as with all the others can
be minimized by the use of compensating lenses.
There is an intimate relationship
between the amount of aberration a lens has to its numerical aperture.
Typically, the optical aberration increases at cube power of the numerical aperture.
So if you were to increase the diameter of a lens, the theoretical resolution
would increase, while the aberration would erode the image quality. This is
dependent on the quality of the lens. A high quality lens allows you to use
the full numerical aperture. Achromat lenses allow you to use about 70% of
the numerical aperture. Apochromats yield 95% to 100% of its numerical
aperture.