While scientists have a variety of optical instruments with which to perform routine laboratory procedures and sophisticated research, the compound brightfield micro-scope is the "workhorse" and is commonly found in all biological laboratories. Although you should be familiar with the basic principles of microscopy, you probably have not been exposed to this diverse array of complex and expensive equipment. Therefore, only the compound brightfield microscope will be discussed in depth and used to examine specimens.

What ismicroscope in science

Effective illumination is required for efficient magnification and resolving power. Since the intensity of daylight is an uncontrolled variable, artificial light from a tungsten lamp is the most commonly used light source in microscopy. The light is passed through the con-denser located beneath the stage. The condenser contains two lenses that are necessary to produce a maximum numerical aperture. The height of the condenser can be adjusted with the con-denser knob. Always keep the condenser close to the stage, especially when using the oil-immersion objective.

The minimum sampling rate (i.e., the number of sensors per inch) that produces a signal that still contains all of the original signal’s information is known as the Nyquist rate, which is twice the maximum frequency in the original signal. The top plot in the figure below shows a 1Hz sine wave sampled at the Nyquist rate, which for this sine wave is 2Hz. The resulting discrete signal, shown in red, contains the same information as the discrete signal plotted beneath it, which was sampled at a frequency of 10Hz. While a slight over simplification, it is essentially true that no information is lost when the original sample rate is known, and the highest frequency in the original signal is less than half the sample rate.

On completion of the laboratory exercise, return the microscope to its cabinet in its original condition. The following steps are recommended:

8. During microscopic examination of microbial organisms, it is always necessary to observe several areas of the preparation. This is accomplished by scanning the slide with-out the application of additional immersion oil. This will require continuous, very fine adjustments by the slow, back-and-forth rotation of the fine adjustment knob only.

The most commonly used microscopes are equipped with a revolving nosepiece containing four objective lenses possessing different degrees of magnification. When these are combined with the magnification of the ocular lens, the total or overall linear magnification of the specimen is obtained.

4. If this is the first specimen of the day, you should Kohler your microscope at this point (while it is in focus). Otherwise, if your microscope has already been Kohlered you won't need to do it again

The reason all of this matters for images is that through application of the Fourier Series, any signal of finite length can be represented as a sum of sinusoids. This means that even if a picture has no discernable wave pattern, it can still be represented as a sequence of sinusoids of different frequencies. The highest frequency that can be represented in the image is half the Nyquist rate (sampling frequency).

microscope: definition biology

In digital photography, a light pattern is focused by the lens onto the image sensor. The image sensor is made up of millions of tiny light-sensitive sensors whose measurements are combined to form a 2-dimential array of pixels. Each tiny sensor produces a single light intensity measurement. For simplicity, I will look at the 1-dimensional case. (Think of this as a slice that looks at only a single row of pixels).

Above the stage and attached to the arm of the microscope is the body tube. This structure houses the lens system that magnifies the specimen. The upper end of the tube contains the ocular or eyepiece lens. The lower portion consists of a movable nosepiece containing the objective lenses. Rotation of the nosepiece posi-tions objectives above the stage opening. The body tube may be raised or lowered with the aid of coarse-adjustment and fine-adjustment knobs that are located above or below the stage, depending on the type and make of the instrument.

Although magnification is important, you must be aware that unlimited enlargement is not possible by merely increasing the magnifying power of the lenses or by using additional lenses, because lenses are limited by a property called resolving power. By definition, resolving power is the ability of a lens to show two adjacent objects as discrete entities. When a lens cannot discriminate, that is, when the two objects appear as one, it has lost resolu-tion. Increased magnification will not rectify the loss, and will, in fact, blur the object. The resolv-ing power of a lens is dependent on the wave-length of light used and the numerical aperture, which is a characteristic of each lens and imprinted on each objective. The numerical aper-ture is defined as a function of the diameter of the objective lens in relation to its focal length. It is doubled by use of the substage condenser; which illuminates the object with rays of light that pass through the specimen obliquely as well as directly. Thus, resolving power is expressed mathematically, as follows:

The instruments are housed in special cabinets and must be moved by users to their laboratory benches. The correct and only acceptable way to do this is to grip the microscope arm firmly with the right hand and the base with the left hand, and lift the instrument from the cabinet shelf. Carry it close to the body and gently place it on the laboratory bench. This will prevent collision with furniture or co-workers and will protect the instrument against damage.

3. Now, while looking through the ocular lens, turn the coarse focus knob carefully, and slowly move the stage away from the lens until the specimen comes into vague focus. Then, use the fine focus knob to bring the specimen into sharp focus.

The light source is positioned in the base of the instrument. Some microscopes are equipped with a built-in light source to pro-vide direct illumination. Others are provided with a mirror; one side flat and the other concave.

Light microscope

Observation of microorganisms in an unstained state is possible with this microscope. Its optics include special objectives and a condenser that make visible cellular components that differ only slightly in their refractive indexes. As light is transmitted through a specimen with a refractive index different from that of the surrounding medium, a portion of the light is refracted (bent) due to slight varia-tions in density and thickness of the cellular components. The special optics convert the difference between transmitted light and refracted rays, resulting in a significant vari-ation in the intensity of light and thereby producing a discernible image of the struc-ture under study. The image appears dark against a light background.

Based on this formula, the shorter the wave-length, the greater the resolving power of the lens. Thus, short wavelengths of the electromag-netic spectrum are better suited than longer wavelengths in terms of the numerical aperture.

Microscope parts and functions

7. Once you have brought the specimen into sharp focus with a low-powered lens, preparation may be made for visualizing the spec-imen under oil immersion. Place a drop of oil on the slide directly over the area to be viewed. Rotate the nosepiece until the oil-immersion objective locks into position. Care should be taken not to allow the high-power objective to touch the drop of oil.The slide is observed from the side as the objective is rotated slowly into position. This will ensure that the objective will be properly immersed in the oil. The fine-adjustment knob is readjusted to bring the image into sharp focus.

If the sample frequency were less than 2 times the maximum frequency of the signal, then the signal is said to be under sampled. In that case, it is not possible to reconstruct the original continuous signal from the discrete one. An illustration of why this is the case can be found in the figure below. There, two sine waves of different frequencies sampled at the same rate produce the same set of discrete points. These two sine waves are called aliases of each other.

Enlargement or magnification of a specimen is the function of a two-lens system; the ocular lens is found in the eyepiece, and the objective lens is situated in a revolving nose-piece. These lenses are separated by the body tube. The objective lens is nearer the specimen and magnifies it, producing the real image that is projected up into the focal plane and then magnified by the ocular lens to produce the final image.

Our row of tiny sensors, each of which is measuring a single point of light, is performing sampling of a continuous signal (the light coming through the lens) to produce a discrete signal (light intensity values at each evenly spaced pixel).

A fixed platform with an opening in the center allows for the passage of light from an illu-minating source below to the lens system above the stage. This platform provides a surface for the placement of a slide with its specimen over the central opening. In addition to the fixed stage, most microscopes have a mechanical stage that can be moved vertically or horizontally by means of adjustment controls. Less sophisticated micro-scopes have clips on the fixed stage, and the slide must be positioned manually over the central opening.

These two terms are two sides of the same coin. The first gives you a bound on sampling rate as a function of max frequency. The second gives you the max possible frequency as a function of sampling rate. See Wikipedia: Nyquist frequency for further reading.

An external light source, such as a lamp, is placed in front of the mirror to direct the light upward into the lens system. The flat side of the mirror is used for artificial light, and the concave side for sunlight.

The Nyquist Limit is frequently mentioned in the context of lens and sensor resolution. What is it and what is its significance to photographers?

You will be responsible for the proper care and use of microscopes. Since microscopes are expensive, you must observe the following regu-lations and procedures.

This component is found directly under the stage and contains two sets of lenses that collect and concentrate light passing upward from the light source into the lens sys-tems. The condenser is equipped with an iris diaphragm, a shutter controlled by a lever that is used to regulate the amount of light entering the lens system.

Microbiology is a science that studies living organisms that are too small to be seen with the naked eye. Needless to say, such a study must involve the use of a good compound microscope. Although there are many types and variations, they all fundamentally consist of a two-lens system, a variable but controllable light source, and mechanical adjustable parts for determining focal length between the lenses and specimen.

To use the microscope efficiently and with minimal frustration, you should understand the basic principles of microscopy: magnification, resolution, numerical aperture, illumination, and focusing.

10 uses of microscope

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Nyquist rate - The lowest possible sampling frequency that can be used while still guaranteeing the possibility of perfect reconstruction of the original continuous signal.

All discrete and digital signals have an infinite number of aliases, which correspond to all the sine waves that could produce the discrete signals. While the existence of these aliases may seem to present a problem when reconstructing the original signal, the solution is to ignore all signal content above the maximum frequency of the original signal. This is equivalent to assuming that the sampled points were taken from the lowest possible frequency sinusoid. Trouble arises when aliases overlap, which can happen when a signal is under sampled.

3.Clean all lens svstems; the smallest bit of dust, oil, lint, or eyelash will decrease the efficiency ot the microscope. The ocular; scan-ning, low-power, and high-power lenses may be cleaned by wiping several times with acceptable lens tissue. Never use paper tow-eling or cloth on a lens surface. If the oil-immersion lens is gummy or tacky, a piece of lens paper moistened with methanol is used to wipe it clean. If the lens is very dirty it may be cleaned with xylol however the xylol cleansing procedure should be performed only by the instructor, and only if necessary. Consistent use of xylol may loosen the lens.

This instrument contains two lens systems for magnifying specimens: the ocular lens in the eyepiece and the objective lens located in the nose-piece. The specimen is illuminated by a beam of tungsten light focused on it by a sub-stage lens called a condenser, and the result is that the specimen appears dark against a bright background. A major limitation of this system is the absence of contrast between the specimen and the surrounding medium, which makes it difficult to observe living cells. Therefore, most brightfield observations are performed on nonviable, stained preparations.

Between the light source and the condenser is the iris diaphragm, which can be opened and closed by means of a lever; thereby regulating the amount of light entering the condenser. Excessive illumination may actually obscure the specimen because of lack of contrast. The amount of light entering the microscope differs with each objec-tive lens used. A rule of thumb is that as the mag-nification of the lens increases, the distance between the objective lens and slide, called working distance, decreases, whereas the numerical aperture of the objective lens increases.

5. Routinely adjust the light source by means of the light source transformer setting, and/or the iris diaphragm, for optimum illumination for each new slide and for each change in magnification.

2. Rotate the scanning lens or the low power lens into position. While watching from the side to insure that the lens doesn't touch the specimen, turn the coarse focus knob to move the stage as close as it can get to the lens without touching the lens. (Always watch from the side whenever you move a specimen towards any objective lens to make sure the lens doesn't crash through the specimen and get damaged!)

Microbiology, the branch of science that has so vastly extended and expanded our knowledge of the living world, owes its existence to Antony van Leeuwenhoek. In 1673, with the aid of a crude microscope consisting of a biconcave lens enclosed in two metal plates, Leeuwenhoek introduced the world to the existence of microbial forms of life. Over the years, microscopes have evolved from the simple, single-lens instrument of Leeuwenhoek, with a magnification of 300, to the present-day electron microscopes capable of magnifications greater than 250,000. Microscopes are designated as either light microscopes or electron microscopes. The former use visible light or ultraviolet rays to illuminate specimens. They include brightfield, darkfield, phase-contrast, and fluorescent instruments. Fluorescent micro-scopes use ultraviolet radiations whose wavelengths are shorter than those of visible light and are not directly perceptible to the human eye. Electron microscopes use elec-tron beams instead of light rays, and magnets instead of lenses to observe submicro-scopic particles.

Compound microscope

You can see the effect of this limit in a few situations where there is a horisontal or vertical wave patterns in a photo, like for example taking a picture where there is a window at a distance with the blinds pulled. If the blades in the blind are closer than two pixels, you can't distinguish the separate blades. However, you are more likely to see a wave pattern that is not exactly horizontal of vertical; it is in that case you will instead see the effect of jagged edges or moiré patterns which occur before the Nyquist Limit.

In sound recording, everything is frequencies, so the Nyquist Limit is always relevant. In photography you don't often have wave patterns that are affected, so it's mostly used as a theoretical limit of the resolution of the sensor.

Who invented the microscope

Nyquist frequency - The highest frequency continuous signal that can be represented by a discreet signal (for a given sampling frequency).

The relationship between wavelength and numerical aperture is valid only for increased resolving power when light rays are parallel. Therefore, the resolving power is dependent on another factor, the refractive index. This is the bending power of light passing through air from the glass slide to the objective lens. The refractive index of air is lower than that of glass, and as light rays pass from the glass slide into the air, they are bent or refracted so that they do not pass into the objective lens. This would cause a loss of light, which would reduce the numerical aperture and diminish the resolving power of the objective lens. Loss of refracted light can be compensated for by interposing mineral oil, which has the same refractive index as glass, between the slide and the objective lens. In this way, decreased light refraction occurs and more light rays enter directly into the objective lens, producing a vivid image with high resolution.

(This passage was adapted from Microbiology: A Laboratory Manual, 5th edition, Cappuccino, J.S. and Sherman, N., Benjamin/Cummings Science Publishing.)

This instrument provides a revolutionary method of microscopy, with magnifications up to one million. This permits visualization of submicroscopic cel-lular particles as well as viral agents. In the electron microscope, the specimen is illu-minated by a beam of electrons rather than light, and the focusing is carried out by elec-tromagnets instead of a set of optics. These components are sealed in a tube in which a complete vacuum is established. Transmission electron microscopes require speci-mens that are thinly prepared, fixed, and dehydrated for the electron beam to pass freely through them. As the electrons pass through the specimen, images are formed by direct-ing the electrons onto photographic film, thus making internal cellular structures visi-ble. Scanning electron microscopes are used for visualizing surface characteristics rather than intracellular structures A narrow beam of electrons scans back and forth, producing a three-dimensional image as the electrons are reflected off the specimen's surface.

Just to add to the previous answers... if you have a pattern beyond the Nyquist limit, you may experience aliasing — i.e. it may show as a lower frequency pattern in the image. This used to be very apparent on things like checked jackets on TV. Therefore, you do need a low pass anti-aliasing filter before sampling so that this artifact is not a problem.

This is similar to the ordinary light microscope; however, the condenser system is modified so that the specimen is not illuminated directly. The con-denser directs the light obliquely so that the light is deflected or scattered from the spec-imen, which then appears bright against a dark background. Living specimens may be observed more readily with darkfield than with brightfield microscopy.

This microscope is used most frequently to visualize speci-mens that are chemically tagged with a fluorescent dye. The source of illumination is an ultraviolet (UV) light obtained from a high-pressure mercury lamp or hydrogen quartz lamp. The ocular lens is fitted with a filter that permits the longer ultraviolet wavelengths to pass, while the shorter wavelengths are blocked or eliminated. Ultraviolet radiations are absorbed by the fluorescent label and the energy is re-emitted in the form of a different wavelength in the visible light range. The fluorescent dyes absorb at wavelengths between 230 and 350 nanometers (nm) and emit orange, yellow, or greenish light. This microscope is used primarily for the detection of antigen-antibody reactions. Antibodies are conjugated with a fluorescent dye that becomes excited in the presence of ultraviolet light, and the fluorescent portion of the dye becomes visible against a black background.

Types of microscope

However; as with magnification, resolving power also has limits. You might rationalize that merely decreasing the wavelength will automati-cally increase the resolving power of a lens. Such is not the case, because the visible portion of the electromagnetic spectrum is very narrow and borders on the very short wavelengths found in the ultraviolet portion of the spectrum.

6. Our microscopes are parfocal, which means that when one lens is in focus, other lenses will also have the same focal length and can be rotated into position without further major adjustment. In practice, however; usually a half-turn of the fine-adjustment knob in either direction is necessary for sharp focus.

In digital sound recording, the highest frequency sound that you can possibly record is half of the sampling frequency. A sound recording av 44100 kHz can not record any sound frequencies above 22050 Hz.

In photography it means that you can't possibly capture a wave pattern where the waves are closer together than two pixels.