FUNDAMENTALS OF MICROBIOLOGY - E. Yu. Tyumentseva - 2015

TOPIC 1. MICROSCOPE DESIGN. TYPES OF MICROSCOPY

Objective: to study the design of a light biological microscope and master the techniques of operating it. To get acquainted with various types of Cell/15.html">Microscopy.

Materials, Reagents, equipment: microscope; bacteriological loops; Glass slides.

1.1. Microscope Design

A microscope (from Greek micros, meaning small, and scopio, meaning to look) is an optical instrument consisting of two parts: mechanical (auxiliary) and optical (primary).

1. Optical part: eyepiece, objective lens, Abbe condenser, illumination system (mirror).

2. Mechanical part: stand, base, stage, body tube holder, coarse adjustment knob, fine adjustment knob (Fig. 1).

Class="center">Fig. 1. Microscope design: 1 - base; 2 - illuminator; 3 - light filter; 4 - Abbe condenser; 5 - stage; 6 - objective lenses; 7 - revolving nosepiece;

8 - monocular HEAD; 9 - eyepiece; 10 - stand; 11 - measuring vernier; 12 - stop screw; 13 - specimen holder; 14 - coarse adjustment knob;

15 - fine adjustment knob; 16 - condenser adjustment knob

Mechanical Part of the Microscope.

The stand features a horseshoe-shaped base and an arched Column (body tube holder). Attached to it are the gear mechanism housing and a system of gear wheels for regulating THE POSITION OF the body tube. This system is set in motion by rotating the coarse and fine adjustment knobs.

The coarse adjustment knob (rack and pinion, macro-screw) is used for the preliminary, approximate focusing on the object under observation.

The fine adjustment knob (micro-screw) is used for precise final focusing. One full turn of the fine adjustment knob moves the tube by 0.1 mm (100 µm).

Rotating the knobs clockwise lowers the tube toward the specimen, while counterclockwise rotation raises it away from the specimen.

The stage serves to hold the specimen slide for study. The stage can be rotated and moved in mutually perpendicular planes using adjustment knobs. A round aperture is located in the center of the stage to illuminate the specimen from below with light rays directed by the microscope mirror. Two stage clips—spring-loaded metal plates designed to secure the specimen—are mounted onto the stage.

If it is necessary to examine The surface of a specimen thoroughly without omissions (which is important when counting), or if a specific area of the specimen needs to be re-examined during work, a mechanical stage is placed on the microscope stage. It features a system of vernier scales that allow coordinates to be assigned to any point of the object under study. To do this, when mounting the mechanical stage, its center of rotation and the optical axis of the microscope system must be aligned with the centering plate of the mechanical stage (hence, a stage equipped with a mechanical stage is sometimes referred to as a mechanical cross-stage).

The body tube is the housing that encloses The Optical System elements of the microscope. A nosepiece (objective turret) with sockets for objective lenses is attached to the lower part of the body tube. Modern microscope models feature an inclined body tube with an arched holder, which ensures a horizontal position of the stage.

The optical part of the microscope consists of the primary optical assembly (objective and eyepiece) and the auxiliary illumination system (mirror and condenser). All PARTS OF THE optical system are strictly centered relative to one another.

In many modern microscopes, the mirror and condenser are replaced by an adjustable light source built directly into the instrument.

The illumination system is located beneath the stage. The mirror reflects incident light into the condenser. One side of the mirror is flat, while the other is concave. When working with the condenser, only the flat mirror must be used. The concave mirror is used when working without a condenser with low-magnification objectives. The condenser (from Latin con-denso, meaning to condense or thicken), consisting of 2–3 short-focus lenses, collects the rays coming from the mirror and directs them onto the object. The condenser is essential primarily when working with an immersion system. The condenser lenses are mounted in a metal housing connected to a gear mechanism that allows the condenser to be moved up and down via a special knob. To adjust the illumination intensity, the condenser is equipped with an iris Diaphragm consisting of curved steel blades.

Stained specimens are best viewed with the diaphragm almost fully open, whereas unstained ones are viewed with a narrowed diaphragm aperture.

A ring-shaped holder for light filters is located beneath the condenser (blue and white frosted glass plates are usually supplied with the microscope). When working with an artificial light source, the filters create the impression of daylight illumination, which makes microscopy less fatiguing for the eyes.

The objective (from Latin objectum meaning object) is the most vital component of a microscope. It is a multi-lens, short-focus system whose quality largely determines the image of the object. The outer lens, facing the specimen with its flat side, is called the frontal (or front) lens. It is this lens that provides the magnification. The remaining lenses in the objective system primarily serve to correct optical aberrations that occur during specimen observation.

One such flaw is spherical aberration. It arises from the tendency of lenses to refract peripheral and central light rays unevenly. Peripheral rays are typically refracted more strongly than central ones, causing them to intersect at a point closer to the lens. As a result, the image of a point appears as a blurred spot.

Chromatic aberration occurs when a beam of light with varying wavelengths passes through a lens. Because different wavelengths are refracted to varying degrees, the rays fail to converge at a single point. Short-wavelength blue-violet rays are refracted more strongly than longer-wavelength red rays. Consequently, a colorless object acquires spurious coloration.

Objectives that eliminate spherical aberration and partially correct chromatic aberration are known as achromats. They contain up to 6 lenses and correct for the primary spectrum (the yellow-green region), without eliminating the secondary spectrum. The image produced by achromats is uncolored, though its edges may exhibit a red or bluish halo. In modern achromats, this flaw is practically imperceptible. The best material for achromatic lenses is flint glass—older types of glass with a high lead oxide content.

Objectives that eliminate chromatic aberration even for the secondary spectrum are called apochromats. They may consist of anywhere from 1 to 12 lenses. The lenses of apochromats designed to correct secondary spectrum rays are crafted from fluorite, rock salt, alum, and other materials. Apochromats make it possible to eliminate object coloration and yield equally sharp images across rays of different colors. Maximum performance with apochromats is achieved only when they are paired with compensating eyepieces, which counteract the optical flaws of the objectives. In compensating eyepieces, the chromatic error is opposite in sign to that of the objective, resulting in near-complete compensation of the microscope's chromatic aberration.

Planachromats are a specialized type of apochromat featuring a flat field of view. Planachromatic objectives completely eliminate field curvature, which otherwise causes uneven focus across the specimen (with curved fields, only a portion of the field is in focus). Planachromats and planapochromats are widely used in photomicrography.

Objectives are classified as either dry or immersion types. When using dry objectives, air is present between the front lens of the objective and the specimen under study. The optical design of immersion objectives requires them to operate with the front lens submerged in a liquid, homogeneous medium. When using a dry objective, due to the difference in refractive indices between glass (1.52) and air (1.0), some light rays are deflected and fail to reach the observer's eye (Fig. 2).

Fig. 2. Light path in dry and immersion systems: I–V represent light rays

When working with an immersion objective, it is necessary to place cedar wood oil between the coverslip and the objective lenses; its refractive index is very close to that of glass (Table 1).

Table 1. Refractive indices of various compounds

The magnification value of objectives is engraved on their mount (8x, 40x, 90x). In addition, each objective is characterized by a specific working distance in millimeters.

Low-magnification objectives have a greater distance from the front lens to the specimen than high-magnification objectives. For instance, objectives with 8x, 40x, and 90x magnification have working distances of 13.8, 0.6, and 0.12 mm, respectively. Depending on the objective in use, either the coarse or fine adjustment knob is used for focusing. An immersion objective has a working distance of up to 0.12 mm, which is why it is often referred to as "short-sighted."

Low-magnification objectives offer not only longer working distances but also wider fields of view. Therefore, it is recommended to begin examining any specimen using a low-magnification objective.

Objectives are designed to be used with a coverslip 0.17±0.1 mm thick. If the coverslip deviates from this standard, the objective must be adjusted by turning the correction collar found on modern, high-quality objectives. In the absence of such a collar, spherical aberration caused by the coverslip should be compensated for by raising or lowering the microscope tube.

One of the most important characteristics of an objective is its resolving power, which ultimately determines the resolving power of the microscope as a whole. It defines the minimum distance between two points on a specimen at which their images can still be distinguished as separate.

The eyepiece (or ocular, from Latin ocularis meaning ocular) serves as a direct continuation of the human eye's own lenses. The refractive System of the eye can be modeled as a biconvex lens with an average focal length of 15 cm (the distance of most distinct Vision being 25 cm).

An eyepiece consists of two lenses—the eye lens (upper) and the field or collective lens (lower)—enclosed in a metal barrel. The purpose of the field lens is to gather the light rays emerging from the objective so that they pass through the small aperture of the eye lens.

The function of the eyepiece is to provide a direct, virtual, magnified image of the real, inverted, and enlarged image produced by the objective. The eyepiece magnification is engraved on its mount. Working magnifications for eyepieces typically range from 4x to 15x. The intrinsic magnification of an eyepiece is calculated using the formula applied for determining magnifier power:

K = L/F,

where L is the distance of most distinct vision, equal to 25 cm; and F is the focal length of the eyepiece lenses.

Eyepieces come in various types, the choice of which depends on the objective. For low- and medium-power achromatic objectives as well as low-power planachromats, Huygens eyepieces or orthoscopic eyepieces are used; for high-power apochromatic, planachromatic, and achromatic objectives, compensating eyepieces are required.

Huygens eyepieces consist of two planoconvex lenses with their convex surfaces facing the objective. The lower lens typically has a larger diameter and a longer focal length than the upper lens. The focal plane of a Huygens eyepiece lies between the eye lens and the field lens.

During extended microscopy sessions, one should use a dual eyepiece setup known as a binocular head. Binocular heads often feature their own intrinsic magnification (approximately 1.5x) and are equipped with correction lenses. The housing of the head can be adjusted laterally within a range of 55–75 mm to match the interpupillary distance of the observer. The technical specifications of the microscope are presented in Table 2.

Table 2. Technical specifications of the microscope

* Magnification up to 1600x is an optional feature; when ordered, the unit is equipped with a 16x eyepiece

Using a binocular head improves object visibility, reduces image glare, and thus helps protect eyesight.

1.2. Operating the microscope

Basic guidelines for microscope use. Choose a Location for the microscope away from direct sunlight. Working on a dark-surfaced desk causes less eye strain.

It is best to look into the eyepiece with your left eye while keeping your right eye open. When working with a binocular head, first adjust the interpupillary distance to match the distance between the observer's eyes so that the fields of view of both eyepieces merge into one.

Always carry the microscope with both hands: hold the arm with one hand and the base with the other. Protect the microscope from shocks and contact with aggressive chemicals (acids, alkalis, etc.).

Do not remove the eyepiece from the body tube to avoid getting dust inside the tube and objectives.

Lenses must be kept clean at all times. Never Touch optical surfaces with your fingers.

The microscope should be stored in its protective cover.

Working with the immersion system. When using an immersion objective (V = 90*; A = 1.25), set the mirror with its flat side facing up and raise the condenser.

Apply a drop of immersion liquid (cedar oil) directly onto the specimen slide without spreading it across the glass. Only immersion objectives (not dry ones!) should be immersed in the liquid.

While looking at the glass slide from the side, lower the objective until it touches the oil drop. Then, looking through the eyepiece, carefully lower the objective using the coarse adjustment knob while watching for the image to appear.

Once the object comes into view, switch to the fine adjustment knob. If the image is blurry, dim, or drifting, an error has been made: the front lens of the objective is dirty, air bubbles are trapped in the oil, the diaphragm is accidentally closed, or the lamp or mirror has shifted. The cause of poor image quality must be resolved.

When finished, raise the body tube, remove the slide, and gently wipe the front lens of the objective with a cotton cloth dampened in refined gasoline.

Immersion liquid (cedar oil) should be stored in specialized dual-chamber oilers. The outer chamber contains xylene or refined gasoline for cleaning oil off the objectives, while the inner chamber holds the cedar oil. The oil chamber must be tightly sealed with a stopper equipped with a glass rod used for applying a drop of oil to the specimen.

Setting up the illumination. It is more convenient to use an artificial light source—it is more stable than daylight and provides better illumination of the specimen, which is especially important when working with high-magnification objectives (90x).

Köhler illumination is based on the principle that the apertures of the collector, condenser, and objective must be evenly illuminated.

The Procedure for setting up Köhler illumination is as follows:

1) place the illuminator with its low-voltage bulb at a distance of 25–30 cm from the microscope using a connecting bar (cross bracket);

2) place the specimen on the stage, select the 8x objective, raise the condenser as high as it will go, fully open its iris diaphragm, almost completely close the field iris diaphragm of the illuminator leaving only a small aperture (1.0–1.2 cm in diameter), move the frosted glass aside, and position the flat mirror;

3) turn on the illuminator and adjust the light intensity so that the lamp filament does not overheat (which is harmful to the eyes). Place a blank sheet of white paper on the mirror and focus the image of the illuminator lamp filament onto it;

4) looking through the eyepiece, adjust the mirror to project the light beam into the microscope's field of view, then focus on the specimen by lowering the condenser until the image of the illuminator's field diaphragm appears as a bright circular patch. Use the mirror to center this light patch in the field of view. The larger the illuminator diaphragm opening, the larger the light patch. If it takes up most of the field of view, reduce it by closing down the diaphragm (do this while looking through the eyepiece);

5) observing through the microscope, focus on the specimen within the illuminated area while continuing to lower the condenser slightly. If done correctly, the light patch, visible simultaneously with the specimen, should be evenly illuminated. Otherwise, slightly rotate the illuminator housing;

6) continuing to look through the eyepiece, open the illuminator diaphragm until the light spot fills the entire field of view. Ideally, the illuminated circle should extend slightly beyond the edges of the field of view.

The positions of the mirror, condenser, and illuminator diaphragm should not be adjusted further. The condenser diaphragm is only used when switching objective lenses.

Köhler illumination is also recommended for dark-field and Phase-contrast microscopy.

Measuring objects. Microorganism Cells can be measured (in µm) in both fixed and live preparations using an ocular micrometer scale, or ocular ruler. For cocci, The Cell diameter is determined; for Bacteria of other shapes, the length and width are measured.

An ocular ruler is a circular glass plate with a scale etched in the middle (50 or 100 divisions) with a total length of 5 mm. Insert the ocular ruler scale-side up onto the eyepiece diaphragm after unscrewing the eyepiece lens. Then, place the specimen and determine how many Divisions of the ruler correspond to the length and width of the cell. Measure at least 10–20 cells.

To calculate the true dimensions of the cells, determine the calibration value of the ocular ruler divisions using a stage micrometer, which is a metal plate shaped like a microscope slide with a central aperture containing a glass insert with a scale (100 divisions). The total length of the stage micrometer scale is 1 mm, and the value of a single division is 10 µm (0.01 mm).

To determine the calibration value of the ocular ruler, place the stage micrometer on the microscope stage instead of the specimen and focus the image of the scale at low magnification. Next, center the scale in the field of view and switch to the objective lens that will be used for measuring the cells. By moving the microscope stage and rotating the eyepiece, align the stage and ocular micrometers so their scales are parallel and overlap one another. The calibration of the ocular micrometer is performed using the vernier principle: align one of the lines on the ocular micrometer scale with a line on the stage micrometer scale, and find the next point of alignment. For example, if two divisions of the stage micrometer (20 µm) span five divisions of the ocular micrometer, then one division of the ocular micrometer at that magnification equals 4 µm (20:5). Knowing how many divisions of the ocular ruler correspond to the length and width of the studied cells, multiply these numbers by the ocular micrometer calibration value.

The obtained values for the ocular ruler divisions are valid only for that specific eyepiece-objective combination.

1.3. Types of Microscopy

The Main characteristics of a microscope are its total magnification and resolving power.

Total magnification does not reflect image quality, which can be either sharp or unsharp.

The clarity of the resulting image is determined by the resolving power of the microscope—that is, the minimum distance between objects or details that can be distinguished as separate entities using the instrument. Resolving power depends on the wavelength of light passing through the object, the refractive index of the optical medium (refractive index of air is 1.0; immersion oil, 1.516; glass, 1.520), and the numerical aperture of the objective. This relationship was derived by the German physicist Ernst Abbe In the second half of the 19th century:

d = λ/2 n sina,

where d is the minimum distance between two points seen as distinct; λ is the wavelength of light passing through the specimen; n sina is the numerical aperture, where n is the refractive index of the optical medium, and a is the aperture angle of the objective.

E. Abbe proved that endlessly increasing the magnification of a Light Microscope is pointless. When illuminating a specimen with light of a wavelength of 550 nm (to which the human eye is most sensitive) using a microscope with an aperture angle of 90° (the limiting angle where sina = 1), the minimum distance between two points is approximately 300 nm for a dry system and about 200 nm for an immersion system.

Thus, the resolving power of a microscope can be increased by:

- decreasing the wavelength of light passing through the specimen;

- using an immersion system;

- increasing the aperture angle to its maximum (up to 90°).

Dark-Field Microscopy

Used to examine very small, low-contrast living specimens. This method employs a specialized dark-field condenser with an opaque center. As a result, the central beam of light rays does not enter the objective lens, leaving the microscope field of view dark. The specimen is illuminated solely by oblique rays striking it from the side. Upon interacting with the specimen, some of these rays are scattered and enter the objective. The specimen appears as a glowing point against a dark Background. The dark-field method provides insight into the external Morphology of live, unstained specimens and their motility.

Dark-field microscopy increases the resolving power of the objective by approximately 10-fold, making it possible to observe objects whose dimensions lie beyond the limits of conventional light microscopy. The increase in resolving power is achieved by expanding the aperture angle.

Phase-Contrast Microscopy

Makes it possible to study living specimens without staining or fixation. The human eye responds to changes in light wave amplitude (intensity, contrast) and wavelength (color), but is insensitive to phase shifts. Biological preparations contain alternating areas that absorb light to varying degrees. As light waves pass through these areas, their amplitudes change. Such Regions of the specimen are called amplitude areas and appear darker under the microscope. Structural elements that are transparent in visible light transmit rays of equal wavelength and amplitude, but shift their phase. The magnitude of this shift depends on the thickness and refractive index of the structures, yet produces virtually no visible change on its own. Such specimens are considered low-contrast.

Using a phase-contrast device, phase shifts in light waves passing through transparent specimens are converted into amplitude changes, making the details of the examined objects visible and high-contrast.

The phase-contrast device makes it possible to study cellular structures, such as bacterial flagella and cell walls, as well as Yeast and fungal nuclei and Mitochondria.

Thus, although the resolution of phase-contrast microscopy remains unchanged compared to bright-field microscopy, image quality is significantly enhanced through increased contrast.

Fluorescence Microscopy

Fluorescence microscopy allows for the Study of Living cells, the visualization of membrane structures, and the acquisition of high-contrast color images of microorganisms.

The core principle of luminescence is that certain molecules within cellular structural elements (such as pigments, Vitamins, and Alkaloids) can absorb a portion of the energy from incident light of a specific wavelength, transition into an electronically excited state, and emit light of a different wavelength. The excitation source can be ultraviolet rays (300-400 nm) or short-wavelength visible light (400-460 nm).

Microorganism cells exhibit weak intrinsic (primary) fluorescence. This can be enhanced by pre-staining the preparations with non-toxic Dyes known as fluorochromes (such as acridine orange, neutral red, auramine, and fluorescein), resulting in secondary fluorescence. To excite this fluorescence, it is sufficient to use the blue-violet region of the spectrum, producing a high-contrast color image of the specimen under observation.

Thus, when using fluorescence microscopy, the resolving power of the microscope increases compared to bright-field microscopy due to the shorter wavelength of the light passing through the specimen.

Electron microscopy

The maximum resolving power of optical microscopes is approximately 0.2 µm and depends on the wavelength of the light rays used. Resolution can be increased 100-fold or more by replacing light or ultraviolet rays with a stream of moving electrons that exhibit wave properties (with a wavelength of about 0.04 nm).

The electron beam travels in a vacuum from an electron source (a heated tungsten filament) toward a fluorescent screen, causing it to glow uniformly. When a specimen is placed in the path of the electrons, they are scattered or absorbed to a greater or lesser degree depending on the specimen's density, resulting in lighter or darker areas on the screen. This straightforward operating principle of the modern Electron microscope is complemented by the deflection of electron beams in a magnetic field—much like light rays are bent by magnifying glass lenses—using electromagnetic lenses for this purpose.

The high resolving power of modern electron microscopes makes it possible to observe and study objects invisible under optical microscopes, including Viruses, phages, Mycoplasmas, the ultrastructure of prokaryotic and Eukaryotic cells, and their macro- and microstructural elements. Preparations for electron microscopy are prepared as extremely thin sections using specialized ultramicrotomes or placed on ultra-thin collodion support films. Consequently, microorganisms are examined in electron microscopes not in a living state, but as fixed preparations.

Presentation and Analysis of Research Results

In their reports, students should provide a brief Summary of the theoretical material.

Upon completing this topic, students will gain an understanding of the microscope, the rules for operating it, and the various types of microscopy.

Review Questions

1. What is the design and Structure OF THE microscope?

2. List the main CHARACTERISTICS OF THE microscope.

3. Which parts and mechanisms comprise the mechanical system of the microscope?

4. What is meant by the resolving power of a microscope? How is it determined?

5. What components make up the Optical System of the microscope?

6. What is the difference between dry objectives and immersion objectives?

7. How is the total magnification of the microscope calculated?

8. What elements comprise the illumination system of the microscope?

9. How should the illumination system be adjusted when working with an immersion objective?

10. List the fundamental rules for working with the microscope.

11. What are the design features and operating principles of dark-field, phase-contrast, fluorescence, and electron microscopes?

12. What determines the resolution of the resulting image?