Possible ways to improve the resolution of an optical microscope. Microscope resolution and magnification. Rules for working with an immersion lens
Methodical instructions
To study objects that are small and indistinguishable with the naked eye, special optical instruments are used - microscopes. Depending on the purpose, they are distinguished: simplified, working, research and universal. According to the light source used, microscopes are divided into: light, luminescent, ultraviolet, electronic, neutron, scanning, tunnel. The design of any of the listed microscopes includes mechanical and optical parts. The mechanical part is used to create observation conditions - to place the object, focus the image, the optical part - to obtain an enlarged image.
Light microscope device
A microscope is called a light microscope because it provides the ability to study an object in transmitted light in a bright field of view. The general view of the Biomed-2 microscope is shown in (Fig. Appearance of Biomed 2).
|
|
The mechanical part of the microscope consists of a microscope base, a movable stage and a revolving device.
Focusing on the object is carried out by moving the stage by rotating the coarse and fine adjustment knobs.
The coarse focusing range of the microscope is 40 mm.
The condenser is mounted on a bracket and is positioned between the stage and the collector lens. Its movement is made by turning the condenser height adjustment knob. Its general view is shown in (Fig. ???) A two-lens condenser with an aperture of 1.25 provides illumination of the fields on the object when working with lenses with magnification from 4 to 100 times.
The subject table is mounted on a bracket. Coordinate movement of the stage, possibly by rotating the handles. The object is fastened to the table by the specimen holders. The holders can be moved relative to each other.
The coordinates of the object and the amount of movement are measured on scales with a graduation of 1 mm and verniers with a graduation of 0.1 mm. The range of movement of the object in the longitudinal direction is 60 mm, in the transverse direction - 40 mm. Condenser
Condenser
The microscope is equipped with a condenser attachment unit with the possibility of centering and focusing movements.
A universal condenser installed in a holder is used as a base in the microscope; when using immersion oil, the numerical aperture is 1.25.
When adjusting the illumination, a smooth change in the numerical aperture of the beam of rays illuminating the preparation is carried out using an aperture diaphragm.
The condenser is installed in the condenser holder in a fixed position and secured with a locking screw.
Condenser centering screws are used during the illumination adjustment process to move the condenser in a plane perpendicular to the optical axis of the microscope while centering the image of the field diaphragm relative to the edges of the field of view.
The up and down condenser handle, located on the left side of the condenser holder bracket, is used when adjusting the lighting to focus on the image of the field diaphragm.
The filters are installed in a rotating ring located at the bottom of the condenser.
Optical part of the microscope
Consists of lighting and observation systems. The lighting system evenly illuminates the field of view. The observation system is designed to enlarge the image of the observed object.
Lighting system
Located under the stage. It consists of a collector lens installed in a housing, which is screwed into the opening of the microscope base and a cartridge with a lamp installed in it. The lamp holder is installed inside the microscope base. The microscope illuminator is powered from the AC mains through a three-pin power cord, which is connected with a plug to the mains. The illumination lamp is switched on by a switch located at the base of the microscope.
Observing system
Consists of objectives, monocular attachment and eyepieces.
Lenses
Objectives are the most important, most valuable and fragile part of a microscope. Magnification, resolution and image quality depend on them. They are a system of mutually centered lenses enclosed in a metal frame. There is a thread on the upper end of the barrel, by means of which the lens is attached to the socket of the revolver. The front (closest to the object) lens in the lens is called the frontal lens, the only one in the lens that produces magnification. All other objective lenses are called correction lenses and are used to eliminate the imperfections of the optical image.
When a beam of light rays with different wavelengths passes through the lenses, a rainbow coloration of the image occurs - chromatic aberration. Unequal refraction of rays on the curved surface of the lens leads to spherical aberration due to uneven refraction of the central and peripheral rays. As a result, the dot image appears as a blurred circle.
Objectives included in the microscope set are designed for optical tube length 160 mm, height 45 mm and cover glass thickness mm.
Objectives with a magnification of more than 10X are equipped with spring-loaded frames that protect the specimen and the front lenses of the objectives from damage when focusing on the surface of the specimen.
A colored ring can be applied to the lens barrel in accordance with the magnification, as well as:
- numerical aperture;
- optical length of the tube 160;
- cover glass thickness 0.17, 0 or - ";
- type of immersion - oil OIL (MI) or water VI;
Objectives marked with 0.17 are designed to study specimens with only 0.17 mm cover slips. Objectives marked 0 are designed for examining specimens without cover slips only. Objectives of low magnification (2.5 - 10), as well as immersion objectives can be used in the study of preparations both with a cover slip and without a cover slip. These lenses are marked with a -.
Eyepieces
The microscope eyepiece consists of two lenses: an eye (upper) and a collective (lower). There is a diaphragm between the lenses. The diaphragm delays the side beams and transmits those close to the optical axis, which enhances the contrast of the image. The purpose of the eyepiece is to magnify the image provided by the lens. The eyepieces have their own magnification of x5, x10, x12.5, x16 and x20 as indicated on the rim.
The choice of eyepieces depends on the set of lenses used. When working with objectives with achromats, achrostigmata and achrofluars, it is advisable to use eyepieces with a linear field of view of no more than 20 mm, with planachromats and planapochromats - eyepieces with a linear field of view of 20; 22 and 26.5 mm.
Additionally, the microscope can be equipped with an eyepiece WF10 / 22 with a scale; scale division value 0.1 mm.
Microscopes characteristics
Microscope magnification
The main characteristics of a microscope are magnification and resolution. The total magnification that a microscope gives is defined as the product of the objective magnification times the eyepiece magnification. However, the magnification does not characterize the quality of the image; it can be clear and unclear. The clarity of the resulting image is characterized by the resolution of the microscope, i.e. the smallest size of objects or their details that can be seen with this device.
The total magnification G of the microscope during visual observation is determined by the formula: G = βok × βok, where:
βob - lens magnification (marked on the lens); βok - eyepiece magnification (marked on the eyepiece).
The diameter of the field observed in the object, Dob mm, is determined by the formula: Dock = Dock × βob. Dock - the diameter of the ocular field of view (marked on the eyepiece) mm. The calculated values of the magnification of the microscope and the diameter of the observed field on the object are given in Table 3.
| Lens magnification | Microscope magnification and observed field at an object with an eyepiece: |
|||||
|---|---|---|---|---|---|---|
| 5/26* | 10/22 | 15/16* | ||||
| G | Dob, mm | G | Dob, mm | G | Dob, mm | |
| 4 | 20 | 4,0 | 50 | 4,5 | 64 | 3,75 |
| 10 | 50 | 2,0 | 100 | 1,8 | 160 | 1,5 |
| 20 | 100 | 1,0 | 200 | 0,9 | 320 | 0,75 |
| 40 | 200 | 0,5 | 420 | 0,45 | 640 | 0,38 |
| 100 | 500 | 0,2 | 1000 | 0,18 | 1600 | 0,15 |
- By additional order
Microscope resolution
The resolution of the microscope is determined by the minimum (resolving) distance between two points (or two thinnest strokes), visible separately, and is calculated by the formula
D = λ / (A1 + A2), where d is the minimum (permissive) distance between two points (strokes); λ is the wavelength of the used light; A1 and A2 are the numerical aperture of the objective (indicated on its barrel) and the condenser.
You can increase the resolution (i.e., reduce the absolute value of d, since these are reciprocal values) in the following ways: illuminate the object with light with a shorter wavelength λ (for example, ultraviolet or short wavelengths), use lenses with a larger A1 aperture, or increase the aperture condenser A2.
Lens working distance
The microscopes are equipped with four detachable objectives with their own magnifications of 4 ×, 10 ×, 40 × and 100 ×, indicated on a metal mount. The lens magnification depends on the curvature of the main front lens: the greater the curvature, the shorter the focal length and the greater the magnification. This must be remembered during microscopy - the greater the magnification of the objective, the shorter the free working distance and the lower it should be lowered over the plane of the specimen.
Immersion
All lenses are divided into dry and immersion, or submersible. Dry is a lens that has air between its front lens and the drug in question. In this case, due to the difference in the refractive index of glass (1.52) and air (1.0), part of the light rays is deflected and does not fall into the eye of the observer. Dry lenses usually have long focal lengths and give low (10x) or medium (40x) magnifications.
Immersion, or submersible, are called such objectives, between the front lens of which and the drug is placed a liquid medium with a refractive index close to the refractive index of glass. Cedar nut oil is usually used as an immersion medium. You can also use water, glycerin, transparent oils, monobromnaphthalene, etc. In this case, a homogeneous (homogeneous) medium (drug glass - oil - objective glass) with the same refractive index is established between the frontal objective lens and the preparation. Due to this, all rays, without refracting or changing direction, fall into the lens, creating the conditions for the best illumination of the drug. The value (n) of the refractive index is 1.33 for water, 1.515 for cedar oil, 1.6 for monobromonaphthalene.
Microscopy technique
The microscope is connected to the electrical network using a power cable. Using a revolver, a lens with a magnification of × 10 is installed in the course of the rays. A slight stop and the click of the revolver spring indicate that the lens is mounted on the optical axis. Use the coarse focusing knob to lower the lens at a distance of 0.5 - 1.0 cm from the stage.
Rules for working with dry lenses.
The prepared preparation is placed on a stage and secured with a clamp. Using a dry lens with a magnification of × 10, several fields of view are viewed. Move the stage with the side screws. The area of the preparation required for the study is set in the center of the field of view. Raise the tube and rotate the revolver to move the lens with a magnification of × 40, observing from the side, again lower the tube with the lens with the macrometric screw until it almost touches the preparation. Look through the eyepiece, very slowly raise the tube until the contours of the image appear. Precise focusing is carried out using a micrometric screw, rotating it in one direction or the other, but not more than one full turn. If resistance is felt during the rotation of the micrometer screw, it means that its stroke has been passed to the end. In this case, turn the screw one or two full turns in the opposite direction, again find the image using the macrometric screw and proceed to work with the micrometric screw.
It is useful to train yourself to keep both eyes open during microscopy and to use them alternately, as this will result in less eye fatigue.
When changing lenses, remember that the resolution of the microscope depends on the ratio of the lens aperture to the condenser. The numerical aperture of a lens with a magnification of × 40 is 0.65, that of a non-immersed condenser is 0.95. It is practically possible to bring them into compliance with the following technique: after focusing the preparation with the objective, remove the eyepiece and, looking into the tube, cover the iris diaphragm of the condenser until its edges become visible at the border of the uniformly illuminated rear objective lens. At this point, the numerical apertures of the condenser and the lens will be approximately equal.
Rules for working with an immersion lens.
A small drop of immersion oil is applied to the preparation (preferably fixed and colored). The revolver is turned and an immersion objective with a magnification of 100 × is installed along the central optical axis. The condenser is lifted up to the stop. The iris diaphragm of the condenser is fully opened. Looking from the side, the tube is lowered with a macrometric screw until the objective is immersed in oil, almost until the lens touches the specimen slide. This must be done very carefully so that the front lens does not move or get damaged. They look through the eyepiece, very slowly rotate the macrometric screw towards themselves and, without removing the lens from the oil, raise the tube until the contours of the object appear. It should be remembered that the free working distance in the immersion lens is 0.1 - 0.15 mm. Then, precise focusing is performed with a macroscopic screw. Several visual fields are examined in the preparation by moving the table with side screws. At the end of work with the immersion lens, raise the tube, remove the preparation and carefully wipe the front lens of the objective, first with a dry soft cotton cloth, then with the same cloth, but slightly moistened with pure gasoline. Do not leave oil on the surface of the lens, as it promotes dust settling and can damage the microscope optics over time. The drug is freed from oil first with a piece of filter paper, then the glass is treated with gasoline or xylene.
Increase A microscope is defined as the product of the objective magnification times the eyepiece magnification. Typical research microscopes have an eyepiece magnification of 10, and an objective magnification of 10, 45 and 100. Accordingly, the magnification of such a microscope ranges from 100 to 1000. Some of the microscopes have magnifications of up to 2000. An even higher magnification does not make sense, since this the resolution does not improve. On the contrary, the image quality deteriorates.
Microscope Magnification Formula
Image quality is determined resolution of the microscope, i.e. the minimum distance at which the microscope optics can distinguish two closely spaced points separately. the resolution depends on the numerical aperture of the objective, the condenser and the wavelength of the light that illuminates the preparation. Numerical aperture (opening) depends on the angular aperture and refractive index of the medium located between the front lens of the objective and condenser and the drug.
In addition to the resolution of the system, the numerical aperture characterizes the lens aperture: the light intensity per unit image area is approximately equal to the square of NA. The NA is about 0.95 for a good lens. The microscope is usually sized to have a total magnification of about 1000 NA.
Resolution limit- the smallest dist. Between two closely spaced points of an object that can be smoothed through a microscope (perceived as two points).
Aperture (Latin apertura - hole) in optics is a characteristic of an optical device that describes its ability to collect light and resist diffraction blurring of image details. Depending on the type of optical system, this characteristic can be linear or angular. As a rule, among the details of an optical device, the so-called aperture diaphragm is specially distinguished, which most of all limits the diameters of the light beams passing through the optical instrument. Often, the role of such an aperture diaphragm is played by a frame or, simply, the edges of one of the optical elements (lenses, mirrors, prisms).
Angle aperture - the angle between the extreme rays of the conical light beam at the entrance (exit) of the optical system.
Numerical Aperture - is equal to the product of the refractive index of the medium between the object and the lens by the sine of the aperture angle. It is this value that most fully determines at the same time the luminosity, the resolution of the microscope objective. To increase the numerical aperture of objectives in microscopy, the space between the objective and the cover glass is filled with an immersion liquid.
Corner The lens aperture is the maximum angle (AOB) at which rays passing through the specimen can enter the lens. Numerical aperture objective is equal to the product of the sine of half the angular aperture and the refractive index of the medium between the slide and the front lens of the objective. N.A. = n sinα where, N.A. - numerical aperture; n is the refractive index of the medium between the preparation and the objective; sinα is the sine of the angle α equal to half of the angle AOB in the diagram.
Thus, the aperture of dry systems (between the frontal lens of the objective and the drug-air) cannot be more than 1 (usually not more than 0.95). The medium placed between the drug and the lens is called immersion liquid or immersion, and a lens designed to work with immersion liquid is called immersion. By immersion with a higher refractive index than air, it is possible to increase the numerical aperture of the objective lens and hence the resolution.
Numerical aperture lenses are always engraved on their frames.
The resolution of the microscope also depends on the condenser aperture. If we consider the condenser aperture equal to the lens aperture, then the resolution formula has the form R = λ / 2NA, where R is the resolution limit; λ is the wavelength; N.A is the numerical aperture. It can be seen from this formula that when observed in visible light (green part of the spectrum - λ = 550nm), the resolution (resolution limit) of the microscope cannot be> 0.2μm
Immersion (from Latin immersio - immersion) - a liquid that fills the space between the object of observation and a special immersion objective (condenser and slide). Three types of immersion fluids are mainly used: oil immersion (MI / Oil), water immersion (VI / W) and glycerol immersion (GI / Glyc), the latter being mainly used in ultraviolet microscopy.
Immersion is used in cases where it is required to increase the resolution of the microscope or its application requires the technological process of microscopy. This happens:
1.increasing visibility by increasing the difference between the refractive index of the medium and the object;
2. an increase in the depth of the viewed layer, which depends on the refractive index of the medium.
In addition, the immersion liquid can reduce the amount of stray light by eliminating glare from the object. This eliminates the inevitable loss of light when it enters the lens.
Refraction of light - a change in the direction of light rays in a medium with a refractive index n changing in space. Usually, the term “R. with." use when describing the distribution of optical. radiation in inhomogeneous media with smoothly varying n from point to point (the trajectories of light rays in such media are smoothly curved lines). A sharp change in the direction of the rays at the interface between two homogeneous media with different n is usually called. refraction of light. Atm. optics, spectacle optics traditionally use the term "refraction". Since the atmosphere is an inhomogeneous medium, then, as a result of R. s. there is a shift in the apparent position of the celestial bodies relative to the true one, which must be taken into account in astronomy. R. s. in the atmosphere should be taken into account when geodesic. measurements. R. s. is the cause of mirages. R.'s phenomenon with. allows you to visualize optical. inhomogeneities in solid, liquid and gaseous media.
Refractometer and I ( from lat. refractus - refracted and Greek. metreo - I measure) is a method for studying substances based on the determination of the index (coefficient) of refraction (refraction) and some of its functions. Refractometry (refractometric method) is used for identification of chemical compounds, quantitative and structural analysis, determination of physical and chemical parameters of substances.
The refractive index, n, is the ratio of the speeds of light in adjoining media. For liquids and solids, n is usually determined relative to air, and for gases - relative to vacuum. The values of n depend on the wavelength l of the light and the temperature, which are indicated in subscripts and superscripts, respectively. Refractometry methods are divided into two large groups: objective and subjective. Despite the undeniable advantage of objective methods, each objective study, as a rule, ends with subjective adjustments. Objective methods. There are two subgroups of objective refractometry methods:
1. Objective in relation to the patient and subjective in relation to the doctor. An example is skiascopy, the objective data of which can be obtained through the physician's subjective assessment of the skiascopic reflex of the subject. 2. Objective in relation to both the investigator and the investigator, realized with the help of a refractometric automaton.
Light polarization- physical optical characteristic radiation, describing the transverse anisotropy of light waves, i.e., the nonequivalence of dec. directions in the plane perpendicular to the light beam. Creatures. value for P.'s understanding of the page. had its manifestation in effects light interference and, in particular, the fact that two light beams with mutually perpendicular polarization planes do not directly interfere. P. s. found naturals. explanation in e-magn. the theory of light, developed in 1865-73 by J.C. Maxwell, later in quantum electrodynamics.
The term polarization of waves was introduced by Malus in relation to transverse mechanical waves
For receiving polarized light and its detection there are special physical devices called in the first case polarizers, and in the second, analyzers. They usually work in the same way. There are several ways to obtain and analyze polarized light.
1. Polarization using polaroids. Polaroids are celluloid films coated with the thinnest layer of crystals of nodquinine sulfate. The use of polaroids is currently the most widespread method of polarizing light.
2. Polarization by reflection. If a natural ray of light strikes a black polished surface, the reflected ray is partially polarized. As a polarizer and analyzer can be used mirror or fairly well polished ordinary window glass, blackened on one side with asphalt varnish. The degree of polarization is the greater, the more correct the angle of incidence is maintained. For glass, the angle of incidence is 57 °.
3. Polarization by means of rejection. The light beam is polarized not only upon reflection, but also upon
refraction. In this case, a stack is used as a polarizer and analyzer.
stacked together 10-15 thin glass plates, located to the incident light rays at an angle of 57 °.
Prism nicolas (abbr. nicole) is a polarizing device based on the effects of birefringence and total internal reflection. Nicolas prism is two identical triangular prisms made of Icelandic spar, glued together with a thin layer of Canadian balsam. The prisms are milled so that the end face is beveled at an angle of 68 ° relative to the direction of the transmitted light, and the sides to be glued make a right angle with the ends. In this case, the optical axis of the crystal ( AB) is at an angle of 64 ° with the direction of the light.
The full polarization aperture of the prism is 29 °. A feature of the prism is the change in the direction of the outgoing beam when the prism rotates, due to the refraction of the beveled ends of the prism. The prism cannot be used to polarize ultraviolet radiation, since Canadian balsam absorbs ultraviolet light. Light with arbitrary polarization, passing through the end of the prism, experiences birefringence, splitting into two rays - an ordinary one with a horizontal plane of polarization ( AO) and extraordinary, with a vertical plane of polarization ( AE). After that, the ordinary ray undergoes total internal reflection on the bonding plane and exits through the side surface. The extraordinary comes out unhindered through the opposite end of the prism.
Brewster's law - the law of optics, which expresses the relationship between the refractive index and such an angle at which the light reflected from the interface will be completely polarized in the plane perpendicular to the plane of incidence, and the refracted ray is partially polarized in the plane of incidence, and the polarization of the refracted ray reaches the highest value. It is easy to establish that in this case the reflected and refracted rays are mutually perpendicular. The corresponding angle is called Brewster's corner.
This optical phenomenon is named for the Scottish physicist David Brewster, who discovered it in 1815.
Brewster's law : , where n 12 is the refractive index of the second medium relative to the first, θ Br- angle of incidence (Brewster angle).
When reflected from one plate at the Brewster angle, the intensity of linearly polarized light is very low (about 4% of the incident beam intensity). Therefore, in order to increase the intensity of the reflected light (or to polarize the light transmitted into the glass in a plane parallel to the plane of incidence), several bonded plates are used, folded into a stack - Stoletov's foot. It is easy to trace what is happening in the drawing. Let a ray of light fall on the top of your foot. The first plate will reflect a fully polarized beam (about 4% of the original intensity), the second plate will also reflect a fully polarized beam (about 3.75% of the original intensity), and so on. In this case, the beam emerging from the bottom of the foot will increasingly polarize in a plane parallel to the plane of incidence as the plates are added. full refraction is essential for radio communications: most whip antennas emit vertically polarized waves. Thus, if a wave hits the interface (land, water, or ionosphere) at the Brewster angle, there will be no reflected wave, and there will be no channel, respectively.
Malus law is the dependence of the intensity of linearly polarized light after its passage through the polarizer on the angle between the polarization planes of the incident light and the polarizer, where I 0 - the intensity of the light incident on the polarizer, I- the intensity of the light coming out of the polarizer. Light with a different (not linear) polarization can be represented as the sum of two linearly polarized components, to each of which the Malus law is applicable. According to Malus's law, the intensity of transmitted light is calculated in all polarizing devices, for example, in polarizing photometers and spectrophotometers. Reflection losses, which depend on and are not taken into account by the Malus law, are determined additionally.
Optically active substances , environments with natural optical activity... O.-a. v. are subdivided into 2 types. Those belonging to the 1st of them are optically active in any state of aggregation (sugars, camphor, tartaric acid), to the 2nd, they are active only in the crystalline phase (quartz, cinnabar). In substances of the 1st type, optical activity is due to the asymmetric structure of their molecules, the 2nd type - the specific orientation of molecules (ions) in the unit cells of the crystal (the asymmetry of the field of forces that bind particles in the crystal lattice). Crystals O.-a. v. always exist in two forms - right and left; in this case, the lattice of the right-hand crystal is mirror-symmetric to the lattice of the left one and cannot be spatially combined with it (the so-called enantiomorphic forms, see Fig. Enantiomorphism). Optical activity of the right and left forms of O. - a. v. Type 2 crystals have different signs (and are equal in absolute value under the same external conditions); therefore, they are called optical antipodes (sometimes this is also the name of type 1 crystals ).
Rotation of the plane of polarization light - united by a common phenomenological. manifestation of a group of effects consisting in turning polarization plane transverse waves as a result of interaction with an anisotropic medium. Naib. the effects associated with V.p. are well-known. light, although similar phenomena are observed in other areas of the spectrum e-magn. waves (in particular, in the microwave range), as well as in acoustics, physics of elementary particles, etc. p.p. is usually due to the difference in coeff. refraction of the medium for two circularly polarized (in the right and left circles) waves (the so-called circular anisotropy) and is described in the general case by a second-rank axial tensor connecting the axial vector of the angle of rotation of the plane of polarization with the polar wave vector. In a medium with only circular anisotropy, a linearly polarized wave can be decomposed into two normal circularly polarized waves of equal amplitude (see Sec. Normal fluctuations), the phase difference between them determines the azimuth of the plane of polarization of the total wave.In homogeneous media with circular anisotropy, the VF angle linearly depends on the path length in the medium. Circular anisotropy can be both natural (spontaneous, inherent in the environment in an unperturbed state), and artificial, induced externally. impact. In the second case, the circular asymmetry can be due to the asymmetry of the disturbing action or the combined symmetry properties of the medium and the disturbance
Angle of rotation. The light beam can be natural and polarized. In a natural ray of light, the oscillations of the vector occur in a disordered manner.
Polarized light beams, in turn, are subdivided into linearly polarized, when the oscillations occur in a straight line perpendicular to the beam; circularly polarized, when the end of the vector describes a circle in a plane perpendicular to the direction of the beam, and elliptically polarized, in which the oscillations are performed along an ellipse.
The plane in which the oscillations occur in a plane-polarized beam is called the oscillation plane.
The plane passing through the direction of the polarized beam and perpendicular to the plane of oscillation is called the plane of polarization.
Light waves with the help of polarizing devices (polaroid, tourmaline plate, nicole, etc.) can be polarized.
Where to get money for business?
Trade tax or "rent tax
Background Open un contour elba