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6.13: Introduction to Microscopes - Biology

6.13: Introduction to Microscopes - Biology


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Understand why and how the light microscope and electron microscope are used in biology

A cell is the smallest unit of a living thing. Thus, cells are the basic building blocks of all organisms.

Several cells of one kind that interconnect with each other and perform a shared function form tissues, several tissues combine to form an organ (your stomach, heart, or brain), and several organs make up an organ system (such as the digestive system, circulatory system, or nervous system). Several systems that function together form an organism (like a human being). Here, we will examine the structure and function of cells.

There are many types of cells, all grouped into one of two broad categories: prokaryotic and eukaryotic. For example, both animal and plant cells are classified as eukaryotic cells, whereas bacterial cells are classified as prokaryotic. Regardless of the type of cell, be it a component in a multicelled eukaryotic animal’s nervous system or a single-celled prokaryotic lifeform, biologists will use microscopes to study them.

What You’ll Learn to Do

  • Identify the general applications of light microscopes in biology
  • Discuss the advantages and disadvantages of electron microscopes for biological studies

Learning Activities

The learning activities for this section include the following:

  • Microscopy
  • Self Check: Microscopes

Project Report on Microscope

A project report on microscope. This project report will help you to learn about: 1. Definition of Microscope 2. Types of Microscope 3. Components 4. Use 5. Working System 6. Measurement of Magnification.

  1. Project Report on the Definition of Microscope
  2. Project Report on the Types of Microscope
  3. Project Report on the Components of Microscope
  4. Project Report on How to Use Microscope
  5. Project Report on the Working System of Microscope
  6. Project Report on the Measurement of Magnification of Microscope

Project Report # 1. Microscope:

Microscope is an instrument to get an en­larged image of the object. The smallest thing visible to our naked eyes is about 1/5 of a cm or 2.2 millimetre, and that also not in details. To study smaller objects they are to be magni­fied. For ordinary use, the length ‘millimetre’ is quite sufficient, but this is too big for scientific studies. Instead we use the ‘μm’. ‘m’ is short for a metre, ‘μ’ is short for the word ‘micron’.

If metre is taken as the unit of length ‘μ.m’ is a millionth part of a metre (a thousand times a thousand makes a million). The microscope in which visible light is used for observation is called optical microscope. For general work optical microscopes are used.

Project Report # 2. Types of Microscope:

It is used in field studies. It has only one lens. The magnification is about fivefold.

ii. Binocular Microscope:

In shape it is almost similar to a compound microscope, without a nose piece and it is of less height. The objectives and eye pieces are changeable. The base is either a horse-shoe or rectangle in shape.

Both the object to be stu­died and the microscope are placed directly on the dissecting tray in case of a horse-shoe base. In others the object is put on a small dissecting tray, placed on a thick glass plate mounted on a circular metallic frame serving as the stage. These microscopes give about ten to twentyfold magnification.

iii. Compound Microscope:

Project Report # 3. Components of Microscope:

It is a supporting stand, rests on the table and bears the weight of the microscope.

It is a curved, solid piece, movably adjusted with the base at the inclination joint. The arm holds the body tube and the stage.

It holds the body of the microscope with the base and permits inclina­tion of the upper part to adjust to eye level.

It is a metal tube blackened inside. A revolving nose piece carrying the ob­jectives is attached to the lower end. At the top of the body tube is screwed a draw tube which houses the eye piece.

The revolving inclined mono or binocular.

It is a sort of squarish or rectangular platform with 10 to 1 2 cm sides and having a circular hole at the centre. It is made up of highly polished metal or bakelite and provided with two spring clips for holding the slide or a sliding bar. The stage is firmly secured with the arm.

It is attached to the arm. The adjustment has a large head. Clock­wise turn of the pinion head moves the body tube downward and anticlockwise turn moves the body tube upward.

It is also attached to the arm, parallel to the coarse adjustment. It oper­ates in the way as in the coarse adjustment, but the movement of the body tube is only slight with the turning of the pinion. Usually ten turns of the fine adjustment pinion are equal to one turn of that of the coarse adjustment.

A planoconcave round mirror is fitted below the stage at some distance from it. In artificial light the plain surface is used. In skylight the concave surface is used, as more light is reflected to the object from this surface. If required the mirror can be taken out from the hole in the base of the microscope and a sub-stage lamp fitted.

Illuminating apparatus (condenser):

It is fitted below the stage and provided with an iris The opening of the diaphragm can be reduced or even closed completely. The arrangement is to regulate the amount of light passing through the object.

Project Report # 4. How to Use a Microscope?

Before using the microscope one must be sure that the mechanical parts are perfectly clean. For cleaning the mechanical parts use a soft, clean linen and for the optical parts a clean, soft silk cloth or tissue paper. Remember microscope is a costly, precision instrument and must be handled with caution.

1. Turn the nose piece to bring the low power objective lens in a line with the body tube.

2. Focus the mirror for maximum illumina­tion of the object.

3. Clean the prepared slide and place it on the stage. Look through the eye piece and bring.

the object in the field of the microscope. Move the slide to bring the portion of the object to be studied to the centre of the field. Fix the slide with spring clips. Look through the eye piece and turn the coarse adjustment for focusing. For sharp focusing use fine adjustment.

For detailed study under high magnification, turn the nose piece to bring the objective with higher magnification in the line. Use only fine adjust­ment for focusing. If the light is strong, reduce it by narrowing the opening of the Irish dia­phragm. If poor, arrange for a brighter source of light. Study the object.

Project Report # 5. Working System of Microscope:

The working system of a compound micro­scope is magnification with double lenses. The initial magnification of the objective lens is fur­ther magnified by the eyepiece, and we get the total magnification (Fig. 21.2).

If the body tube is of standard length, i.e., 160 mm, only then the total magnification is equal to that obtained by multiplying the magnification of the objec­tive with the magnification by the eye piece. The body tube is always not of standard length and the magnification should be measured with a micro-metre.

Project Report # 6. Measurement of Magnification of Microscope:

A. To measure magnification under a microscope the tools required are an oculometre and a micrometer.

It is a piece of thin, circu­lar glass disc with a diameter of about 15 mm. A scale, the divisions of which are not to scale, is etched across the middle of the disc.

It is a thick glass slide. A scale, divisions of which are to the scale, is etched at the middle of the slide along its long axis.

The oculometre is put inside the eye piece by unscrewing the top part of the lens. It is screwed down again before use. The micrometer is placed on the stage of the micro­scope and the scale is brought to the centre of the hole of the stage.

Rotating the eye piece and also moving the micrometer the two scales are made to overlap. The divisions of the two scales are not equal, as a result perfect overlapping of lines marking the divisions of the scales appear only at a few points.

Note the number of divisions of the micrometre (MM) which correspond to the number in the oculometre (OM). The divisions of the micrometre are always smaller than those in the oculometre. Calculate the actual measurement of a division of the oculometre.

Assuming 5 divisions of OM correspond to 10 divisions of MM

One division of OM = 10/5 or 2 μm

One div. of OM = 10/5 or 2 μm

The magnifications of both the objective and eye piece, and the particular microscope used for ascertaining the measurement of oculometre scale are to be noted for future use, as the magnification varies with lens and the microscope.

Procedure for measurement:

To measure an object put the oculometre in the eye piece. Place the slide on the stage of the microscope, focus the object, and slowly move the slide and also rotate the eye piece to bring any structure of the object with sharp outlines, against the scale in the oculometre.

Read the divisions and calculate the actual size of the structure. To know the magnification of a camera lucida drawing or a photomicrograph, measure the figure with a scale and calculate the magnification from the actual size of the object.

B. An approximate magnification of the object may be calculated using the following formula:


Microscope, Scientific Method, Introduction to Biology Crossword Puzzle

Introduction to Biology Crossword Puzzle. This is a 45 word crossword puzzle that I use at the first of the school year after I cover my lessons on "Introduction to Biology". These are the terms that students need to know at the start of their biology class in order to be successful for the rest of the year.

The topics covered are:

  • What is Science?
  • Themes of Biology
  • Scientific Method
  • The Microscope
  • Characteristics of Life

This document is perfect for a homework assignment, a quiz, classwork, or test review.

The words included in the puzzle are:

Control, data, quantitative, qualitative, hypothesis, biology, cell, unicellular, colonial, tissue, organ, homeostasis, gene, DNA, sexual, asexual, evolution, natural selection, ecology, autotroph, heterotroph, scientific method, variables, theory, biogenesis, spontaneous generation, Redi, Pasteur, fine adjustment, coarse adjustment, stage clips, objectives, ocular, rotating nosepiece, body tube, stage, diaphragm, resolution, magnification, system, photosynthesis, cellular respiration, independent variable, dependent variable, and micrometer.


Fluorescence microscopes

Fluorescence microscopes produce widefield images. This is the basis of what happens in a fluorescence microscope:

  • The fluorescence source (from the arc lamp) produces bright white light which is passed through a coloured filter to produce the specific excitation light, in this case blue for GFP
  • This is reflected by the dichroic mirror, goes through the objective and to the sample
  • The sample (hopefully!) fluoresces as described above and some of the emission light goes back through the objective
  • This is the important bit: the dichroic mirror transmits the longer wavelength light so the very bright excitation and the relatively weak fluorescence are separated.
  • The emission is normally passed through another filter and then can be either seen with the naked eye or captured with a camera

This is what an upright fluorescence scope typically looks like. Notice the fluorescence light path is only above the stage (this is called epi-illumination and the objective is both the condenser and the imaging lens). The transmitted light path features a condenser on one side of the sample and the objective on the other.


Definition of Bright Field Microscopy

It is defined as the optical microscopy, which is the simplest of all the illumination techniques, wherein a smear (the stained or the dense part appears darker with a white or brighter background). Bright-field microscope is a compound light microscope, which illuminates the background against a stained specimen. It is commonly used in the practical labs to study organisms’ behaviour and characteristics like size, shape and arrangement.

It is a type of light microscopy, where a path of light is very simple, which requires a light source (like a halogen lamp), condenser lens, objective lens and ocular lens. Bright-field microscopy can use either critical or Koehler magnification or illumination system to add contrast to the image.

  • Critical illumination: It was the old method, in which a condenser forms an unevenly illuminated image.
  • Koehler illumination: It is an advance method, which uses additional optic systems like condenser diaphragm, a condenser lens, collector diaphragm and collector lens to produce an evenly illuminated image.

Resolution

It is the optical lens’s power, which distinguishes between the two particular bodies that held very close to each other. The highly magnified image will not give better results as the picture becomes gauzy. The resolving power increases, as the lines per unit area, appears as distinctive lines.

When there is a small distance between the two distinct objects, the resolving power can be best known. The resolution power can give 1000-1500 times magnified image. Resolving power of the microscope decides the quality of the picture by the objective lens.

Higher is the magnification power higher will be the resolution of the microscope. The bright field microscope’s resolution is represented as ‘r’ which is equal to the half of the light wavelength by the numerical aperture.

Thus, the resolution of the bright field microscope depends upon the two factors:

  • Numerical aperture: It is the object side aperture, which is equal to the product of refractive index (n) and the magnitude of the angular aperture (represented as sinƟ).

  • The wavelength of light: Shorter is the wavelength of light, higher will be the resolution compared to the longer wavelength.

Steps in Bright Field Microscopy

Let us summarize the steps in bright-field microscopy to visualize the specimen.

Preparation of smear

Firstly, the smear is prepared on a glass slide by mixing of inoculum with a drop of distilled water. The thin-film of smear is then heat-fixed and stained. To identify the bacteria, one can perform gram staining. Conversely, for the identification of the fungus, lactophenol cotton blue stain is extensively used. Thus, the “mounting of specimen” refers to the placement of the specimen over the glass slide.

Optimization of light

After air-drying of the glass slide, add oil immersion for the better resolution. Then, place a slide on the stage of a light microscope and adjust it by moving the stage clips. Through the eyepiece, observe the specimen to test whether it is visible or not.

Adjustment of the Condenser lens

To visualize the magnified image of the specimen, adjust the condenser lens of the light microscope. A condenser lens plays a significant role in transferring the incident light from the illuminator to the specimen. The condenser lens must be near to the specimen.

Focus the specimen

To get the specimen’s clear picture, focus the object or organism through the iris diaphragm (controls the diameter of the light coming from the condenser to the specimen).

When the diaphragm is nearly close to the condenser lens, it adds contrast to the specimen. When the diaphragm is farther apart from the condenser lens, it adds brightness to the organism.

The positioning of the specimen

After focusing, locate the specimen relative to the eyepiece by using stage control. The stage control consists of coarse and fine adjustment knobs, which help in the movement of the stage to up, down, left and right directions. The coarse and fine knobs also sharpen the image.

Selection of objective lens

Then, adjust the separation between an eyepiece and objective lens. The distance between the eyepiece and the objective lens is the separation distance, which is adjusted to view the image.

Select one type of objective lens that can give a best-magnified image through the objective revolver. The objective revolver holds three or four objective lenses with different magnification powers like 10x, 40x, 100x etc.

Adjust the Illuminator

The intensity of the light or illumination coming from an illuminator is adjusted by moving the mirror of the microscope for the specimen’s brightness.

Working

Bright-field microscopy is a technique used in the light microscope, which magnifies the dark specimen with the colourless background. To accomplish the bright field microscopy, transfer the prepared glass slide onto the microscope stage. The luminous light will come through the source of light or say through an illuminator device.

The light coming from the illuminator is aimed at the condenser lens (present beneath the specimen). The aperture diaphragm helps to focus and control the light source from the illuminator on the specimen. Some of the light will reflect the specimen, which is then collected by the objective lens. The objective lens first magnifies the light and then transmits it to the ocular lens.

Some of the light will get deflected, while some will get absorbed by the stain and the dense areas in the sample, during this whole method. The intense illumination can increase the magnification of the image. Therefore, the tested organism that has absorbed the part of the light will appear darker, and the remaining, i.e. the background will appear more radiant, in bright field microscopy.

Advantages

  • Bright-field microscopy is a simple method to perform.
  • It can quickly produce a magnified image of the fixed specimens and live cells.

Disadvantages

  • The bright-field microscopy produces low contrast to the image.
  • It can give magnification only up to 1300 X.
  • Bright-field microscopy has a low apparent optical resolution.

For Better Results

We can adjust the iris diaphragm for the better results, which can reduce or increase the amount of light source getting to the specimen. The use of oil immersion can improve the image’s resolution under an objective lens of power 100X. The use of staining methods adds contrast to the picture. One can also use a coloured or polarizing filter on the light source to highlight the features of the mineral samples.


Discoveries thanks to the microscope

Pollen seen through a microscope.

The importance of microscopes in the life sciences can never be overestimated. After the discovery of blood cells among other microorganisms, other discoveries were made through the use of advanced instruments. Some of the other discoveries made are:

  • The Cell Division of Walther Fleming (1879).
  • The Krebs Cycle of Hans Krebs (1937).
  • Neurotransmission: discoveries made between the end of the nineteenth century and the twentieth century.
  • The photosynthesis and cellular respiration of Jan Ingenhousz in the 1770s.

Many discoveries have been made since the 1670s and have contributed significantly in a variety of studies that have seen great advances in the treatment of diseases and the development of cures. It is now possible to study diseases and how they progress within the human body in order to better understand how to treat them.

Because of the many applications, the data used in cell biology have been significantly transformed from representative non-quantitative observations in fixed cells to high-throughput quantitative data in living cells.

Through ingenious inventions, the limit of what scientists could reveal from the occult, expanded continuously during the seventeenth and eighteenth centuries. Finally, at the end of the 19th century, the physical limits in the wavelength form of light stopped the search to see further in the microcosm.

With the theories of quantum physics, new possibilities arose: the electron with its extremely short wavelength could be used as a"light source"in microscopes with unprecedented resolution.

The first prototype of the electron microscope was built around 1930. In the following decades, more and smaller things could be studied. Viruses were identified and with increases of up to one million, even the atoms eventually became visible.

The microscope has facilitated the studies of scientists, bringing as a result discoveries of causes and ways of curing diseases, studies of agents that can be used in the manufacturing process of inputs for agriculture, livestock and industry in general.

People who manage the microscope should have training in use and care for being in high cost equipment. It is a fundamental tool to make technical decisions that can help the profitability of a product and in health helps the development of human activities.


Microscope Introduction – “e” Lab

This lab is similar to the “e” lab used with freshman biology, but designed for students in the vocational track. It has less reading and more detailed steps for using the microscope as well as a larger font and bigger spacing. This helps students who are not good readers or are English language learners. Small compact text which has too many steps can discourage beginners from attempting to complete the activity.

This version also has a photo of a microscope for a quick reference rather than asking students to use their notes or other sources to answer the questions. Like the original version, students are also asked to look at “common things” to practice focusing the microscope. This section of the activity is popular, students do enjoy looking at things under the microscope, but may need extra direction to stay on track to complete the lab.

2 Comments

Carrie Ren

where can I find the answer for true false kind of problem in this worksheet?

Admin

The answers are in the section of the lab which includes the instructions. You just need to read carefully. Cheers!


Introduction to Microscopes Lab

This Introduction to Microscopes lab is a classic activity for introducing microscopy to high school students, or a review for students who have used scopes before. It's perfect at the beginning of the school year to get all of your students up to speed!

Using easily acquired materials, this lab will allow students to make their own wet mount slides of paper letter e's and view them under low and high power. The questions in the lab help students to understand what they're doing as they focus on their slides and answer basic questions about how the microscope works. They'll draw their "e" under low and high power . very helpful for introducing students to proper technique for lab drawings.

Students are always amazed at what a simple thing like paper looks like under the microscope!

The lab can be completed in a class period. An answer key for the lab and suggestions for the teacher are included.

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Why Phase Contrast Microscope?

The Phase Contrast Microscope is used to visualize unstained living cells. Most of the stains or staining procedures will kill the cells. Phase contrast microscopy enables the visualization of living cells and life events.

History of Phase Contrast Microscope

The Phase Contrast Microscope was developed by Zernike in early 1930s. The invention of this microscope enables us to visualize live cells and cellular processes. Due to the remarkable contribution of phase contrast microscopy in biological sciences, the inventor was awarded Nobel Prize in Physics in 1953.


Wet mounts are used for living samples, transparent liquids, and aquatic samples. A wet mount is like a sandwich. The bottom layer is the slide. Next is the liquid sample. A small square of clear glass or plastic (a coverslip) is placed on top of the liquid to minimize evaporation and protect the microscope lens from exposure to the sample.

To prepare a wet mount using a flat slide or a depression slide:

  1. Place a drop of fluid in the middle of the slide (e.g., water, glycerin, immersion oil, or a liquid sample).
  2. If viewing a sample not already in the liquid, use tweezers to position the specimen within the drop.
  3. Place one side of a coverslip at an angle so that its edge touches the slide and the outer edge of the drop.
  4. Slowly lower the coverslip, avoiding air bubbles. Most problems with air bubbles come from not applying the coverslip at an angle, not touching the liquid drop, or from using a viscous (thick) liquid. If the liquid drop is too large, the coverslip will float on the slide, making it hard to focus on the subject using a microscope.

Some living organisms move too quickly to be observed in a wet mount. One solution is to add a drop of a commercial preparation called "Proto Slow." A drop of the solution is added to the liquid drop before applying the coverslip.

Some organisms (like Paramecium) need more space than what forms between a coverslip and a flat slide. Adding a couple of strands of cotton from tissue or swab or else adding tiny bits of broken coverslip will add space and "corral" the organisms.

As the liquid evaporates from the edges of the slide, living samples may die. One way to retard evaporation is to use a toothpick to coat the edges of the coverslip with a thin rim of petroleum jelly before dropping the coverslip over the sample. Press gently on the coverslip to remove air bubbles and seal the slide.