Sb95668

of the studied light (so that the limit for red light comes much earlier than the limit for blue light) and on the diameter of the telescope mirror. This means that a telescope with a certain mirror diameter can theoretically resolve up to a certain limit at a certain wavelength. For conventional telescopes on Earth, the diffraction limit is not relevant for telescopes bigger than about 10 cm. Instead, the seeing, or blur caused by the atmosphere, sets the resolution limit. But in space, or if adaptive optics are used, then reaching the diffraction limit is sometimes possible. At this point, if greater resolution is needed at that wavelength, a wider mirror has to be built or aperture synthesis performed using an array of nearby telescopes.

In recent years, a number of technologies to overcome the distortions caused by atmosphere on ground-based telescopes have been developed, with good results, such as adaptive optics, speckle imaging and optical interferometry.

3. Microscopes

The word microscope origins from Greek μικροζ – micros – small and σκοπεω – scopos – to look. These are various devices, pro viding imaging with

significant magnification of small-size objects and details, which cannot be seen by the naked eye. The simples single-component microscopy device is known since antique times as a magnifying glass. In 16-17th centuries on its basis was developed the two-component system, known as the optical microscope, light microscope or simply microscope. We shall discuss here these two devices in more details. There also exist several other types of microscopes, having either optical nature – like confocal and optical near-field microscopes, or non-optical one, like various kinds of electron microscopes, atomic force and tunnel microscopes.

3.1. Magnifying glass

The simplest type of microscopy device, in its simplest kind comprised by just one converging lens, is known as the magnifying glass. Ray tracing in such glass is shown in the Fig.3.1. The object to be observed is placed in the focal plane of the converging lens (more accurately speak-

ing, nearby the focal plane at the a little bit

Fig.3.1. Magnifying glass

smaller distance), and hence the ob-

server’s eye gets the parallel beam from each point to the object. So the magnify-

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ing glass constructs the virtual magnified image of the object, positioned at the plane of the most convenient observation by the human eye – approximately at 250 mm from the eye.

a b

Fig. 3.2. Visible magnification – (a) observation b y naked eye and (b) with magnifying glass

The visible magnification of the magnifying glass is determined as the ratio of tangent of object vision angle through the glass to that by naked eye (Fig.3.2). For the naked eye this tangent is equal to tgw = y/250, while for the case when the object is placed nearby the focal; plane of the converging glass it is equal to tgw' = y/f’ . Hence one can evaluate the visible magnification of the magnifying glass as follows:

In such a system the diameter of the human eye pupil is usually much smaller than the lens diameter, and hence it serves both the aperture of the system and

its exit pupil.

D' = Deye. (3.2)

In Fig.3.3 is shown the magnifying glass with di-

ameter Dmg. The pupil of the observer’s eye with diameter Deye is positioned at the distance S from the glass. Hence the field of system vision in the image space 2w' is determined by the ray, going through the upper boundaries of the lens and of the pupil:

One can see from (3.4) that the larger field of vision can be obtained when eye is positioned closed to the magnifying glass. Medium magnification glasses (up to 7x) are usually made on the single lens base. The stronger magnification devices (from 7x to 15x) usually comprise two-three lenses. One can use the magnifying glasses for observing the details with the transverse size of down to 0.01 mm. The field of such glass vision can be as high as 20°.

3.2. Optical microscope

The microscope provides imaging of the small-size objects and details with the higher magnification and resolution, than the ones, provided by the magnifying glass. Its optical scheme comprises two separate components – the objective (objective lens) and the eyepiece (ocular). First of them constructs the magnified real image of the object (Fig.3.4) in the forward focal plane of the eyepiece. The eyepiece action is very similar to that of the magnifying glass – it produces the magnified virtual image of the object at the distance of the best vision (250 mm). The plane of object positioning is known as the general forward focal plane of the overall microscope.

Fig. 3.4. Optical scheme of the microscope

The microscope objective lens can be characterized by its linear magnifica-

of the lens and forward focus of the eyepiece. The latter is also known as the optical interval of the microscope or the microscope tubus length.

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The real image, produced by the lens in the forward focal plane of the eyepiece, acts as the object for the latter. The eyepiece action is identical to that of the magnifying glass, providing the visible magnification of:

The total magnification of the microscope is equal to the product of objective and eyepiece magnifications:

Similarly to the magnifying glass, one can determine the general magnification of the microscope in terms of its general focal length or vice versa:

G = 250 (3.8) fm¢

Fig.3.5. Microscope objective lenses (left) and eyepieces (right)

Typical microscope lenses and eyepieces are shown in the Fig.3.5. The overall design of optical microscope is shown in the Fig.3.6. It comprises various mechanical adjustment components and devices for object illumination. Majority of microscopes have an opportunity of fast interchange of lenses and eyepieces, providing various magnification.

Each objective lens consists of six or more pieces of glass that combine to produce a clear image of an object. The six or more lenses in the objective lens are needed to provide corrections that produce image clarity. The interaction of light with the glass in a lens produce aberrations that result in a loss in image quality because light waves will be bent, or refracted, differently in different portions of a lens, and different colors of light will be refracted to different extents by the glass. Spatial aberrations (e.g., spherical aberration) can be corrected by using lenses

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with different curvature on their surfaces, and chromatic (i.e., color) aberrations can be minimized by using multiple kinds of glass in combination. These corrections increase the cost of the lens to the extent that an apochromatic objective lens exhibiting full color correction and extremely high N.A. can cost several thousand dollars. This objective lens is about the size of your thumb.

Fig.3.6. Optical miscroscope outlook

The objective lenses in most microscopes are achromats, and best suited for imaging with green light. Green filters narrow the bandwidth of the light, and make achromat objectives reasonably effective for most routine uses. The achromat lenses are not suitable for critical high-resolution imaging with white light, because red and blue light do not focus in the same plane as green light. Chromatic aberrations will degrade resolution in color images obtained with achromatic ob-

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jectives. Color photomicrography aimed at the highest level of resolution and image clarity should be performed with totally corrected apochromatic objective lenses. Fluorite lenses, offer intermediate levels of correction, better than achromats but not as good as apochromats. Fluorite lenses are well suited for fluorescence microscopy because of their high transmittance of shorter wavelength light.

The oculars in most microscopes are designed to work optimally with the objective lenses from the same manufacturer. Each manufacturer makes some of the color and spatial corrections in the objective and the remainder of the corrections in the ocular. Mixing brands will usually result in a degraded image. In addition, when you look into a microscope, the magnified and corrected image you see through the oculars is actually a virtual image (as opposed to a real image). The ocular, designed to provide a corrected virtual image when viewed by eye, is not suitable for the generation of photographic or video images through the microscope. For photography or video microscopy it is necessary to use a projection lens that generates a corrected real image. Many of the newer microscopes provide total image corrections in the objective lens, thus obviating many of the concerns about matching glass components from the same manufacturer. Nevertheless, it is a good practice not to mix parts from one manufacturer with those of another, because unintended image degradation can result.

An essential factor in producing a good image with the light microscope is obtaining adequate levels of light in the specimen, or object plane. It is not only necessary to obtain bright light around the object, but for optimal imaging, the light should be uniform across the field of view. The best way to illuminate the specimen involves the use of yet another lens system, known as a condenser. The front element of the condenser is usually a large, flattened lens that sits directly beneath the specimen. Its placement on a movable rack provides you with the means to focus the light beam coming past the object and maximize the intensity and control the uniformity of illumination. Two apertures in the illumination system allow you to regulate the diameter of the illumination beam by closing or opening iris diaphragms. One of these diaphragms, housed within the brightfield condenser and known as the condenser diaphragm, allows you to increase contrast, but at the cost of worsening resolution. The second of these diaphragms, known as the field aperture diaphragm, does not affect resolution as dramatically and is regularly adjusted for optimal illumination.

The human eye can perceive changes in light amplitude (intensity). Unstained biological specimens, such as living cells, are essentially transparent to our

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eyes, but they interact with light in a fairly uniform way, by retarding (slowing) the passage of a light beam by approximately 1/4 of a wavelength. By slowing a light beam this much relative to another light beam that had passed though the surrounding medium, the biological specimen alters the phase of the beams. Intensity (amplitude) is additive and light rays that are 1/2 out of phase are perceived as darkness. Zernicke realized that if he could retard the light passing through biological specimens without affecting the light passing through the surrounding medium, he could generate changes in amplitude within living cells. The phase contrast microscope was invented by Zernicke in the 1930's as a means to generate contrast in biological specimens, changing these invisible phase differences into visible amplitude differences.

Zernicke employed an optical trick to separate the light beams interacting with the specimen from those that do not encounter the specimen. To separate the beams of light from each other, he placed a transparent ring (known as an annulus) in an opaque disk and inserted this disk into the optical path of the microscope, within the condenser. He placed a complementary ring inside the objective lens. Nearly all of the light that passes through the sample but misses the specimen then passes through the objective lens through this ring. Most of the light that passes through the specimen is scattered and some of it enters the objective lens in such a way that it will not pass through the objective lens ring, but will pass this plane in some other location. He designed the glass plate holding the ring so that all light missing the ring would encounter an additional 1/4 of retardation relative to the beams of light that had not interacted with the specimen, placing the light rays that had interacted with the specimen out of phase with rays that had not interacted with the specimen by 1/2. He found that a reduction in intensity of the light that had not passed through the specimen would create a grey background and increase contrast even more, with some parts of the specimen darker and other parts of the specimen brighter than the background.

The operation of any microscope in the phase contrast mode requires that you first set up proper brightfield illumination, with a centered field iris diaphragm whose edges are in focus in the specimen plane. Next, rotate the condenser turret cylinder until the number on the condenser turret matches the number engraved on the objective lens. Under this condition, the condenser annulus is matched to the phase ring present in the objective. Next, remove one of the oculars and insert the Bertrand focusing telescope into the ocular hole. This lens enables you to see the rear focal plane of the objective lens, the plane where the ring resides. You will see

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a bright circle of light (the condenser annulus) and a dark ring (present within the objective). The dark ring is stationary, but the bright annulus is not. You may need to align the annulus with the ring so that the two are superimposed. On the back side of your condenser, you will find two adjustment screws that permit this alignment to be performed. When the ring and the annulus are aligned, place the ocular back into the microscope. The difference between phase contrast and brightfield for the observation of living cells is significant.

In certain classes of atoms and molecules, electrons absorb light, become energized, and then rapidly lose this energy in the form of heat and light emission. If the electron keeps its spin, the electron is said to enter a singlet state, and the kind of light that is emitted as the electron returns to ground state is called fluorescence. If the electron changes its spin when excited, it enters the triplet state, and the kind of light that is emitted as the electron returns to ground state is known as phosphorescence. Phosphorescence is much longer-lived than fluorescence. Both fluorescence and phosphorescence emissions are of particular wavelengths for specific excited electrons. Both types of emission are dependent on specific wavelengths of excitation light, and for both types of emission, the energy of excitation is greater than the energy of emission. Described another way, of excitation light is shorter than of emission light. In biology, we can utilize fluorescence in localization reactions, to identify particular molecules in complex mixtures or in cells. Fluorescence has the advantage of providing a very high signal-to-noise ratio, which enables us to distinguish spatial distributions of rare molecules. To utilize fluorescence, we need to label the specimen (a cell, a tissue, or a gel) with a suitable molecule (a fluorochrome) whose distribution will become evident after illumination. The fluorescence microscope is ideally suited for the detection of particular fluorochromes in cells and tissues.

All microscopes operate in the same general fashion. Light beams pass through a condenser lens system and provide illumination of an object at many points simultaneously. For incident light fluorescence microscopy, the objective lens also acts as a condenser for the excitation light beam. In its interaction with the object, some of this light is absorbed, some of this light is scattered, some of this light is reflected, and some of this light is slowed or retarded (relative to a beam of light that does not pass through the object). A portion of the light that has interacted with the object then passes through the imaging lens system of the microscope where it provides us with visual or pictorial image information about the object. Like the process of illumination, the process of image generation operates

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in a parallel fashion, where large numbers of light beams contribute to the image simultaneously. Resolution is limited by the closeness of overlapping points of brightness or darkness. In a practical sense, the limit of resolution is 0.18-0.2 µm with the best available objective lenses and a good specimen.

In the incident light fluorescence microscope, a light beam passes through a chromatic beam splitter and then the objective lens to illuminate a specimen. This light beam is used to excite electrons in fluorochrome molecules present in the object. As some of those excited electrons return to their ground state, the emission of light is detectable through the oculars of the microscope, or with a camera or video printer. The image is generated continuously, across the entire field of view. A primary problem with the fluorescence images generated in this way is that out-of- focus fluorescence appears as 'flare' in the object, and reduces the signal substantially. In addition, human eyes are not sufficiently sensitive photodetectors for the lowest levels of fluorescence, and most video-based imaging systems are only slightly better than your eyes. Under conditions where there is sufficient signal for you to easily observe fluorochrome distribution patterns, the excitation light can be of sufficient intensity to photooxidize (i.e., burn) your specimen. Much information can be lost with just a few seconds of exposure to the excitation lamp.

The Confocal Scanning Optical Microscope, an expensive piece of instrumentation that illuminates the object with a small beam of light in a point-by-point (i.e., serial) fashion, eliminates most of the photoxidation problems, permitting the observation of objects for extended periods at very high resolution with little loss of signal. The placement of a small aperture in the beam path generates a small depth of field, and effectively eliminates out of focus information in image formation.

The confocal scanning optical microscope is designed to illuminate an object in a serial fashion, point by point, where a small beam of light (from a LASER) is scanned across the object rapidly in an X-Y raster pattern. The raster pattern can be created in several ways, but in one of the more popular instruments, it occurs as a consequence of the simultaneous rotation and vibration of a polygonal mirror. The vibration is caused by the activity of a servogalvanometer, while the rotation is caused by the activity of a small electric motor. Thus, a bright spot of light scans across an object from top to bottom, line by line. The image is also generated point-by-point. Image formation is translated into intensities at each spot in the X- Y raster by a photomultiplier tube. The intensity information is digitized and stored in a computer. A complex image processing software package permits visualiza-

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tion and manipulation of the images. Resolution is limited by spot size for the LASER and approaches 0.12-0.15 µm for an ideal spec imen and with the best available objective lenses.

The manufacturers of confocal scanning optical microscopes include a pinhole diaphragm at a very special place in the optical path, near to the site of the photomultiplier tube. This pinhole is situated in a plane where the light from the in-focus part of the image converges to a point. Light from object planes above or below that of the focused image do not converge at the spot in the optical path occupied by the pinhole. Because of this design, out of focus image information is darkened to the extent that it is not detectable. The consequence is that all out of focus information is removed from the image and the confocal image is basically an 'optical section' of what could be a relativelythick object. The 'thickness' of the optical section may approach the limit of resolution, but in practice, the resolution in the Z-direction is somewhat greater, approximately 0.4-0.8 µm. The value of optical sectioning is best realized with fluorescence microscopy, where out-of-focus information alters, distorts, or even degrades the image. Because the confocal images are stored in a computer, it is possible to stack them up and generate threedimensional reconstructions. The image processing programs also enable us to rotate these images and observe three-dimensional aspects of cellular structure. It may be clear to you that the computer responsible for these image manipulations must be fast and powerful. The biggest problem is one of image storage, where single images can routinely occupy >1,000,000 bytes of space. In rather short periods of use, it is easy to accumulate sufficient numbers of images to fill the largest of hard disks.

When light passes through an object, it interacts with some or all of the atoms and molecules present in that object. In these interactions, sometimes light of a particular (i.e., color) is absorbed by the atoms or molecules, while sometimes light is scattered. The interaction of light with a translucent object often results in a slight reduction in the velocity of the light beam. The extent of this reduction in velocity can be measured as the refractive index of the object. For certain kinds of objects, especially those with high order in particular axes of the object, such a crystalline or paracrystalline arrays, the interaction with light beams is vastly different, depending on the orientation of the object relative to the impinging light beam. As a result, the refractive indices are measurably different in different axes of the object. Such an object with multiple refractive indices is termed birefringent. Birefringence (multiple refractive indices) results from the alignment of atoms or

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