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Microscopes

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Microscopes
CHAPTER 26 | VISION AND OPTICAL INSTRUMENTS
One theory that takes various factors into account was advanced by Edwin Land (1909 – 1991), the creative founder of the Polaroid Corporation.
Land proposed, based partly on his many elegant experiments, that the three types of cones are organized into systems called retinexes. Each
retinex forms an image that is compared with the others, and the eye-brain system thus can compare a candle-illuminated white table cloth with its
generally reddish surroundings and determine that it is actually white. This retinex theory of color vision is an example of modified theories of color
vision that attempt to account for its subtleties. One striking experiment performed by Land demonstrates that some type of image comparison may
produce color vision. Two pictures are taken of a scene on black-and-white film, one using a red filter, the other a blue filter. Resulting black-and-white
slides are then projected and superimposed on a screen, producing a black-and-white image, as expected. Then a red filter is placed in front of the
slide taken with a red filter, and the images are again superimposed on a screen. You would expect an image in various shades of pink, but instead,
the image appears to humans in full color with all the hues of the original scene. This implies that color vision can be induced by comparison of the
black-and-white and red images. Color vision is not completely understood or explained, and the retinex theory is not totally accepted. It is apparent
that color vision is much subtler than what a first look might imply.
PhET Explorations: Color Vision
Make a whole rainbow by mixing red, green, and blue light. Change the wavelength of a monochromatic beam or filter white light. View the light
as a solid beam, or see the individual photons.
Figure 26.14 Color Vision (http://cnx.org/content/m42487/1.4/color-vision_en.jar)
26.4 Microscopes
Although the eye is marvelous in its ability to see objects large and small, it obviously has limitations to the smallest details it can detect. Human
desire to see beyond what is possible with the naked eye led to the use of optical instruments. In this section we will examine microscopes,
instruments for enlarging the detail that we cannot see with the unaided eye. The microscope is a multiple-element system having more than a single
lens or mirror. (See Figure 26.15) A microscope can be made from two convex lenses. The image formed by the first element becomes the object for
the second element. The second element forms its own image, which is the object for the third element, and so on. Ray tracing helps to visualize the
image formed. If the device is composed of thin lenses and mirrors that obey the thin lens equations, then it is not difficult to describe their behavior
numerically.
Figure 26.15 Multiple lenses and mirrors are used in this microscope. (credit: U.S. Navy photo by Tom Watanabe)
Microscopes were first developed in the early 1600s by eyeglass makers in The Netherlands and Denmark. The simplest compound microscope is
constructed from two convex lenses as shown schematically in Figure 26.16. The first lens is called the objective lens, and has typical magnification
values from 5× to 100× . In standard microscopes, the objectives are mounted such that when you switch between objectives, the sample remains
in focus. Objectives arranged in this way are described as parfocal. The second, the eyepiece, also referred to as the ocular, has several lenses
which slide inside a cylindrical barrel. The focusing ability is provided by the movement of both the objective lens and the eyepiece. The purpose of a
microscope is to magnify small objects, and both lenses contribute to the final magnification. Additionally, the final enlarged image is produced in a
location far enough from the observer to be easily viewed, since the eye cannot focus on objects or images that are too close.
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CHAPTER 26 | VISION AND OPTICAL INSTRUMENTS
Figure 26.16 A compound microscope composed of two lenses, an objective and an eyepiece. The objective forms a case 1 image that is larger than the object. This first
image is the object for the eyepiece. The eyepiece forms a case 2 final image that is further magnified.
To see how the microscope in Figure 26.16 forms an image, we consider its two lenses in succession. The object is slightly farther away from the
objective lens than its focal length f o , producing a case 1 image that is larger than the object. This first image is the object for the second lens, or
eyepiece. The eyepiece is intentionally located so it can further magnify the image. The eyepiece is placed so that the first image is closer to it than
its focal length f e . Thus the eyepiece acts as a magnifying glass, and the final image is made even larger. The final image remains inverted, but it is
farther from the observer, making it easy to view (the eye is most relaxed when viewing distant objects and normally cannot focus closer than 25 cm).
Since each lens produces a magnification that multiplies the height of the image, it is apparent that the overall magnification m is the product of the
individual magnifications:
m = m om e,
(26.12)
m o is the magnification of the objective and m e is the magnification of the eyepiece. This equation can be generalized for any combination
of thin lenses and mirrors that obey the thin lens equations.
where
Overall Magnification
The overall magnification of a multiple-element system is the product of the individual magnifications of its elements.
Example 26.5 Microscope Magnification
Calculate the magnification of an object placed 6.20 mm from a compound microscope that has a 6.00 mm focal length objective and a 50.0 mm
focal length eyepiece. The objective and eyepiece are separated by 23.0 cm.
Strategy and Concept
This situation is similar to that shown in Figure 26.16. To find the overall magnification, we must find the magnification of the objective, then the
magnification of the eyepiece. This involves using the thin lens equation.
Solution
The magnification of the objective lens is given as
mo = –
where
di
,
do
d o and d i are the object and image distances, respectively, for the objective lens as labeled in Figure 26.16. The object distance is
given to be
d o = 6.20 mm , but the image distance d i is not known. Isolating d i , we have
1 = 1 − 1,
fo do
di
where
(26.13)
(26.14)
f o is the focal length of the objective lens. Substituting known values gives
We invert this to find
1 =
1
1
−
= 0.00538
mm .
d i 6.00 mm 6.20 mm
(26.15)
d i = 186 mm.
(26.16)
di :
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CHAPTER 26 | VISION AND OPTICAL INSTRUMENTS
Substituting this into the expression for
m o gives
mo = −
di
= − 186 mm = −30.0.
do
6.20 mm
(26.17)
Now we must find the magnification of the eyepiece, which is given by
me = −
where
d i′
,
d o′
(26.18)
d i′ and d o′ are the image and object distances for the eyepiece (see Figure 26.16). The object distance is the distance of the first
image from the eyepiece. Since the first image is 186 mm to the right of the objective and the eyepiece is 230 mm to the right of the objective,
the object distance is d o′ = 230 mm − 186 mm = 44.0 mm . This places the first image closer to the eyepiece than its focal length, so that
the eyepiece will form a case 2 image as shown in the figure. We still need to find the location of the final image
magnification. This is done as before to obtain a value for
1 / d i′ :
d i′ in order to find the
1 = 1 − 1 =
1
1
−
= − 0.00273
mm .
f e d o′ 50.0 mm 44.0 mm
d i′
(26.19)
d i′ = − mm = −367 mm.
0.00273
(26.20)
Inverting gives
The eyepiece’s magnification is thus
me = −
d i′
= − −367 mm = 8.33.
44.0 mm
d o′
(26.21)
So the overall magnification is
m = m om e = ( − 30.0)(8.33) = −250.
(26.22)
Discussion
Both the objective and the eyepiece contribute to the overall magnification, which is large and negative, consistent with Figure 26.16, where the
image is seen to be large and inverted. In this case, the image is virtual and inverted, which cannot happen for a single element (case 2 and
case 3 images for single elements are virtual and upright). The final image is 367 mm (0.367 m) to the left of the eyepiece. Had the eyepiece
been placed farther from the objective, it could have formed a case 1 image to the right. Such an image could be projected on a screen, but it
would be behind the head of the person in the figure and not appropriate for direct viewing. The procedure used to solve this example is
applicable in any multiple-element system. Each element is treated in turn, with each forming an image that becomes the object for the next
element. The process is not more difficult than for single lenses or mirrors, only lengthier.
Normal optical microscopes can magnify up to
1500× with a theoretical resolution of – 0.2 µm . The lenses can be quite complicated and are
composed of multiple elements to reduce aberrations. Microscope objective lenses are particularly important as they primarily gather light from the
specimen. Three parameters describe microscope objectives: the numerical aperture (NA) , the magnification (m) , and the working distance. The
NA is related to the light gathering ability of a lens and is obtained using the angle of acceptance
on the specimen (see Figure 26.17(a)) and is given by
NA = n sin α,
θ formed by the maximum cone of rays focusing
(26.23)
n is the refractive index of the medium between the lens and the specimen and α = θ / 2 . As the angle of acceptance given by θ
increases, NA becomes larger and more light is gathered from a smaller focal region giving higher resolution. A 0.75NA objective gives more
detail than a 0.10NA objective.
where
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CHAPTER 26 | VISION AND OPTICAL INSTRUMENTS
Figure 26.17 (a) The numerical aperture
acceptance θ . (b) Here,
entering the lens.
α
(NA)
of a microscope objective lens refers to the light-gathering ability of the lens and is calculated using half the angle of
is half the acceptance angle for light rays from a specimen entering a camera lens, and
D
is the diameter of the aperture that controls the light
While the numerical aperture can be used to compare resolutions of various objectives, it does not indicate how far the lens could be from the
specimen. This is specified by the “working distance,” which is the distance (in mm usually) from the front lens element of the objective to the
specimen, or cover glass. The higher the NA the closer the lens will be to the specimen and the more chances there are of breaking the cover slip
and damaging both the specimen and the lens. The focal length of an objective lens is different than the working distance. This is because objective
lenses are made of a combination of lenses and the focal length is measured from inside the barrel. The working distance is a parameter that
microscopists can use more readily as it is measured from the outermost lens. The working distance decreases as the NA and magnification both
increase.
f / # in general is called the f -number and is used to denote the light per unit area reaching the image plane. In photography, an image
of an object at infinity is formed at the focal point and the f -number is given by the ratio of the focal length f of the lens and the diameter D of the
aperture controlling the light into the lens (see Figure 26.17(b)). If the acceptance angle is small the NA of the lens can also be used as given
The term
below.
f /# =
f
≈ 1 .
D 2NA
(26.24)
f -number decreases, the camera is able to gather light from a larger angle, giving wide-angle photography. As usual there is a trade-off. A
f / # means less light reaches the image plane. A setting of f / 16 usually allows one to take pictures in bright sunlight as the aperture
diameter is small. In optical fibers, light needs to be focused into the fiber. Figure 26.18 shows the angle used in calculating the NA of an optical
As the
greater
fiber.
Figure 26.18 Light rays enter an optical fiber. The numerical aperture of the optical fiber can be determined by using the angle
α max.
Can the NA be larger than 1.00? The answer is ‘yes’ if we use immersion lenses in which a medium such as oil, glycerine or water is placed
between the objective and the microscope cover slip. This minimizes the mismatch in refractive indices as light rays go through different media,
generally providing a greater light-gathering ability and an increase in resolution. Figure 26.19 shows light rays when using air and immersion lenses.
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CHAPTER 26 | VISION AND OPTICAL INSTRUMENTS
Figure 26.19 Light rays from a specimen entering the objective. Paths for immersion medium of air (a), water (b)
(n = 1.33) , and oil (c) (n = 1.51)
are shown. The
water and oil immersions allow more rays to enter the objective, increasing the resolution.
When using a microscope we do not see the entire extent of the sample. Depending on the eyepiece and objective lens we see a restricted region
which we say is the field of view. The objective is then manipulated in two-dimensions above the sample to view other regions of the sample.
Electronic scanning of either the objective or the sample is used in scanning microscopy. The image formed at each point during the scanning is
combined using a computer to generate an image of a larger region of the sample at a selected magnification.
When using a microscope, we rely on gathering light to form an image. Hence most specimens need to be illuminated, particularly at higher
magnifications, when observing details that are so small that they reflect only small amounts of light. To make such objects easily visible, the intensity
of light falling on them needs to be increased. Special illuminating systems called condensers are used for this purpose. The type of condenser that is
suitable for an application depends on how the specimen is examined, whether by transmission, scattering or reflecting. See Figure 26.20 for an
example of each. White light sources are common and lasers are often used. Laser light illumination tends to be quite intense and it is important to
ensure that the light does not result in the degradation of the specimen.
Figure 26.20 Illumination of a specimen in a microscope. (a) Transmitted light from a condenser lens. (b) Transmitted light from a mirror condenser. (c) Dark field illumination
by scattering (the illuminating beam misses the objective lens). (d) High magnification illumination with reflected light – normally laser light.
We normally associate microscopes with visible light but x ray and electron microscopes provide greater resolution. The focusing and basic physics is
the same as that just described, even though the lenses require different technology. The electron microscope requires vacuum chambers so that the
electrons can proceed unheeded. Magnifications of 50 million times provide the ability to determine positions of individual atoms within materials. An
electron microscope is shown in Figure 26.21. We do not use our eyes to form images; rather images are recorded electronically and displayed on
computers. In fact observing and saving images formed by optical microscopes on computers is now done routinely. Video recordings of what occurs
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