History ofmicroscope

Figure 2. 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.

The magnifying power MP of a telescope is the (apparent) increase in the size of an object relative to its size when viewed with the unaided eye.  MP is equal to the magnitude of the angular magnification mθ = -f1/f2.  The ratio of the diameter of the entrance pupil to the exit pupil is also equal to MP.  The magnifying power should be chosen so that the diameter of the exit pupil is approximately equal to the diameter of the pupil of the eye.

A basic refracting telescope is an optical instrument that has two optical elements, an objective and an eyepiece.  We have two thin lenses in air.  The objective is a large lens that collects light from a distant object and creates an image in the focal plane, which is a faithful representation of the object.  The eyepiece is a sophisticated magnifying glass through which we view this image.

Microscope

where λ is the wavelength of the light and D the diameter of the objective.  For the human eye and visible light D = 0.8 cm and λ = 500 nm, therefore θmin = 7.62*10-5 rad = 4.37*10-3 degree.  Earth-bound telescopes rarely reach their theoretical resolution limit because of the blurring of the images due to atmospheric turbulence.

The resolution depends on the wavelength of the light entering the objective as well as on the diameter of the objective.  The bigger the diameter of the objective the smaller the resolving power.  For a telescope the theoretical intrinsic minimum angular separation is given by

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 6 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.

As the 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 greater 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 4 shows the angle used in calculating the NA of an optical fiber.

A Keplerian telescope has a converging lens eyepiece and a Galilean telescope has a diverging lens eyepiece.  The distance between the image and the eyepiece is the sum of the focal lengths of the two lenses. (Remember that for a diverging lens the focal length is negative.)  A telescope by itself is not an image forming system.  The eye of the observer or the camera attached to the telescope forms the image.

This situation is similar to that shown in Figure 2. 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.

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 2. 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.

MicroscopeParts

We use a telescope to gather light and to increase the angle that a distant object subtends at the eye.  If the eye is relaxed for distant viewing, the telescope simply produces an angular magnification.  An incident (approximately) parallel beam from a distant source point, which makes an angle θ with respect to the optical axis, emerges as a parallel beam which makes a larger angle θ' with respect to the axis.

To see how the microscope in Figure 2 forms an image, we consider its two lenses in succession. The object is slightly farther away from the objective lens than its focal length fo, 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 fe. 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 = mome, where mo is the magnification of the objective and me 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.

Figure 7. An electron microscope has the capability to image individual atoms on a material. The microscope uses vacuum technology, sophisticated detectors and state of the art image processing software. (credit: Dave Pape)

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 1.) 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.

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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 θ formed by the maximum cone of rays focusing on the specimen (see Figure 3a) and is given by NA = n sin α, where n is the refractive index of the medium between the lens and the specimen and [latex]\alpha=\frac{\theta}{2}\\[/latex]. 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 0.75 NA objective gives more detail than a 0.10 NA objective.

[latex]\displaystyle\frac{1}{d_{\text{i}}\prime}=\frac{1}{f_{\text{e}}}-\frac{1}{d_{\text{o}}\prime}=\frac{1}{50.0\text{ mm}}-\frac{1}{44.0\text{ mm}}=\frac{0.00273}{\text{mm}}\\[/latex]

Figure 6. 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.

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.

Compoundmicroscope

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.

Figure 5. 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.

Electronmicroscope

The image as viewed through the Keplerian telescope is inverted, and the image formed by the objective lens is in the second focal plane of that lens which is also the first focal plane of the eyepiece lens.  The image formed by the eyepiece is at infinity.  The telescope is not an image forming system until we add another optical system, such as the lens of an eye or a camera.

Now we must find the magnification of the eyepiece, which is given by [latex]m_{\text{e}}=-\frac{d_{\text{i}}\prime}{d_{\text{o}}\prime}\\[/latex], where di′ and do′ are the image and object distances for the eyepiece (see Figure 2). 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 do′ = 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 di′ in order to find the magnification. This is done as before to obtain a value for [latex]\frac{1}{d_{\text{i}}\prime}\\[/latex]:

A compound microscope uses a simple combination of two converging lenses to produce a very effective magnifier.   A sketch is shown below.  The lens closest to the object is known as the objective, and the second lens is the eyepiece.  The object is placed between fo and 2fo.  An intermediate image is formed by the objective lens near the object focal plane of the eyepiece. The tube length g is the distance between the secondary focal point of the objective and the primary focal point of the eyepiece.  The intermediate image serves as the object for the eyepiece.

Stereomicroscope

A Galilean telescope has an objective lens with f1 = 20 cm and the eyepiece lens with f2 = -5 cm.  The lenses are separated by 15 cm.  Calculate the matrix for this system and find mθ.

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 5 shows light rays when using air and immersion lenses.

How to usemicroscope

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 in a microscope can be made for viewing by many people at later dates. Physics provides the science and tools needed to generate the sequence of time-lapse images of meiosis similar to the sequence sketched in Figure 8.

Both the objective and the eyepiece contribute to the overall magnification, which is large and negative, consistent with Figure 2, 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.

Look through a clear glass or plastic bottle and describe what you see. Now fill the bottle with water and describe what you see. Use the water bottle as a lens to produce the image of a bright object and estimate the focal length of the water bottle lens. How is the focal length a function of the depth of water in the bottle?

[latex]m_{\text{e}}=-\frac{d_{\text{i}}\prime}{d_{\text{o}}\prime}=-\frac{-367\text{ mm}}{44.0\text{ mm}}=8.33\\[/latex].

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.

5. (a) +18.3 cm (on the eyepiece side of the objective lens); (b) −60.0; (c) −11.3 cm (on the objective side of the eyepiece); (d) +6.67; (e) −400

The resolving power or resolution of a telescope is the smallest angular separation between two objects that can be seen.  The smaller the resolving power of the telescope the better is the telescope.  If two objects in the sky have a smaller angular separation than the resolving power of the telescope, the two objects will appear as one.  However, if their angular separation is larger than the resolving power, then they will be resolved and appear as two distinct objects.

Figure 3. (a) The numerical aperture of a microscope objective lens refers to the light-gathering ability of the lens and is calculated using half the angle of acceptance . (b) Here, is half the acceptance angle for light rays from a specimen entering a camera lens, and is the diameter of the aperture that controls the light entering the lens.

Figure 4. Light rays enter an optical fiber. The numerical aperture of the optical fiber can be determined by using the angle αmax.

compound microscope: a microscope constructed from two convex lenses, the first serving as the ocular lens(close to the eye) and the second serving as the objective lens

Figure 8. The image shows a sequence of events that takes place during meiosis. (credit: PatríciaR, Wikimedia Commons; National Center for Biotechnology Information)

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 7.

where do and di are the object and image distances, respectively, for the objective lens as labeled in Figure 2. The object distance is given to be do=6.20 mm, but the image distance di is not known. Isolating di, we have

The term 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 3b). If the acceptance angle is small the NA of the lens can also be used as given below.

Gathering as much light as possible is the principal function of astronomical telescopes.  The larger the area π(D/2)2 of the objective the more light the telescope can collect.  To compare the light gathering power (LGP) of two telescopes we compare the areas of their objectives.  For the eye the objective is just the pupil, with a diameter of only ~0.8 cm.

To have a tube length g an object distance xo satisfying 1/xo = 1/fo - 1/(g + fo) is needed.  If the intermediate image is in the object focal plane of the eyepiece then the linear magnification of the objective is -g/f0 and and the angular magnification of the eyepiece is dv/fe.  The total magnifying power of the microscope is given by MP = -g dv/(fofe) = -(g/fo)MPeyepiece.  Here dv = 25 cm, the near point of the human eye.  The focal lengths of both lenses should be extremely short to generate maximum angular magnification.

numerical aperture: a number or measure that expresses the ability of a lens to resolve fine detail in an object being observed. Derived by mathematical formula NA = n sin α, where n is the refractive index of the medium between the lens and the specimen and [latex]\alpha=\frac{\theta}{2}\\[/latex]