3.1: Introduction to the Microscope - Biology

3.1: Introduction to the Microscope - Biology

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Learning Outcomes

  • Review the principles of light microscopy and identify the major parts of the microscope.
  • Learn how to use the microscope to view slides of several different cell types, including the use of the oil immersion lens to view bacterial cells.

Early Microscopy

The first microscope was developed in 1590 by Dutch lens grinders Hans and Zacharias Jansen. In 1667, Robert Hooke described the microscopic appearance of cork and used the term cell to describe the compartments he observed. Anton van Leeuwenhoek was the first person to observe living cells under the microscope in 1675—he described many types of cells, including bacteria. Since then more sophisticated and powerful scopes have been developed that allow for higher magnification and clearer images.

Microscopy is used by scientists and health care professionals for many purposes, including diagnosis of infectious diseases, identification of microorganisms (microscopic organisms) in environmental samples (including food and water), and determination of the effect of pathogenic (disease-causing) microbes on human cells. This exercise will familiarize you with the microscopes we will be using to look at various types of microorganisms throughout the semester.

The Light Microscope

What does it mean to be microscopic? Objects are said to be microscopic when they are too small to be seen with the unaided eye—they need to be magnified (enlarged) for the human eye to be able to see them. This includes human cells and many other types of cells that you will be studying in this class. The microscope you will be using uses visible light and two sets of lenses to produce a magnified image. The total magnification will depend on which objective lens you are using—the highest magnification possible on these microscopes is typically 1000X—meaning that objects appear 1000X larger than they actually are.

Resolution vs. Magnification

Magnification refers to the process of making an object appear larger than it is; whereas resolution is the ability to see objects clearly enough to tell two distinct objects apart. Although it is possible to magnify above 1000X, a higher magnification would result in a blurry image. (Think about magnifying a digital photograph beyond the point where you can see the image clearly). This is due to the limitations of visible light (details that are smaller than the wavelength of light used cannot be resolved).

The limit of resolution of the human eye is about 0.1 mm, or 100 microns (see Table 1 for metric review). Objects that are smaller than this cannot be seen clearly without magnification. Since most cells are much smaller than 100 microns, we need to use microscopes to see them.

The limit of resolution of a standard brightfield light microscope, also called the resolving power, is ~0.2 µm, or 200 nm. Biologists typically use microscopes to view all types of cells, including plant cells, animal cells, protozoa, algae, fungi, and bacteria. The nucleus and chloroplasts of eukaryotic cells can also be seen—however smaller organelles and viruses are beyond the limit of resolution of the light microscope (see Figure 1).

Resolution is the ability of the lenses to distinguish between two adjacent objects as distinct and separate.

A compound light microscope has a maximum resolution of 0.2 µm, this means it can distinguish between two points ≥ 0.2 µm, any objects closer than 0.2um will be seen as 1 object. Shorter wavelengths of light provide greater resolution. This is why we often have a blue filter over our light source in the microscope, it helps to increase resolution since its wavelength is the shortest in the visible light spectrum. Without resolution, no matter how much the image is magnified, the amount of observable detail is fixed, and regardless of how much you increase the size of the image, no more detail can be seen. At this point, you will have reached the limit of resolution or the resolving power of the lens. This property of the lens is fixed by the design and construction of the lens. To change the resolution, a different lens is often the only answer.

Table 1: Metric units commonly used in Microbiology
The basic unit of measurement of length in the metric system is the meter.
There are 1000 millimeters (mm) in one meter. 1 mm = 10-3 meter.
There are 1000 micrometers (microns, or µm) in one millimeter. 1 µm = 10-6 meter.
There are 1000 nanometers in one micrometer. 1 nm = 10-9 meter.

The microscope is one of the microbiologist's greatest tools. It allows for the visualization of small particles, including microbes, which individually are too small to be seen with the human eye. With the help of proper illumination, a microscope can magnify a specimen and optically resolve fine detail. This introduction to microscopy will include an explanation of features and adjustments of a compound brightfield light microscope, which magnifies images using a two lens system.

Before reading the following discussion of the theory of the microscope, please familiarize yourself with the names of the microscope parts shown in Figure 2 and their function.

1. Eyepiece/Ocular lens: Lens in which the final magnification occurs. Often is at 10X magnification, but can be different.

2. Revolving nose piece: Holds multiple objective lenses in place. The base of the nose piece can rotate, allowing each of the lens to be rotated into alignment with the ocular lens.

3. Objective lenses: Initial magnification of your specimen occurs here. Most brightfield light microscopes have 3 objective lenses seated into the resolving nose piece base.

4. Coarse focusing knob: larger of the two knobs, the coarse adjustment knob moves the stage up or down to bring the specimen into focus. It is very sensitive, even small partial rotation of this knob can bring about a big change in the vertical movement of the stage. ONLY use coarse focusing at the beginning with the 4X, 10X low powered objectives in place. If you use it with the higher powered objectives, it can damage the objective if you crash the lens through your glass specimen slide.

5. Fine focusing knob: smaller of the two knobs, the fine adjustment knob brings the specimen into sharp focus under low power and is used for all focusing when using high power lenses such as the 100x oil immersion lens.

6/9. Stage & Mechanical stage: The horizontal surface where you place the slide specimen is called the stage. The slide is held in place by spring loaded clips and moved around the stage by turning the geared knobs on the mechanical stage. The mechanical stage has two perpendicular scales that can be used to record the position of an object on a slide, useful to quickly relocate an object.

7. Illuminator: contains the light source, a lamp made either of an incandescent tungsten-halogen bulb or an LED. There is normally a switch to turn on/off or a rheostat located on the side that you can use to adjust the brightness of the light.

8. Diaphragm and Condenser: the diaphragm controls the amount of light passing from the illuminator through the bottom of the slide, there is a small lever used to achieve the optimal lighting. The condenser is a lens system that focuses the light coming up from the illuminator onto objects on the slide.

Figure 2: Brightfield light microscope used in a Microbiology lab (Lumen)

The Optical System. The optical system of a compound microscope consists of two lens systems: one found in the objective(s) lens(es) (Fig. 2, part 3); the other in the ocular (eyepiece) (Fig. 2 part 1). The objective lens system is found attached to a rotating nosepiece (Fig. 2, part 2). A microscope usually has three or four objectives that differ in their magnification and resolving power. Magnification is the apparent increase in size of an object. Resolving power is the term used to indicate the ability to distinguish two objects as separate. The most familiar example of resolving power is that of car headlights at night: at a long distance away, the headlights appear as one light; as the car approaches, the light becomes oblong, then barbell-shaped, and finally it becomes resolved into two separate lights. Both resolution and magnification are necessary in microscopy in order to give an apparently larger, finely detailed object to view.

Look at the engravings on the objective lenses and note both the magnification (for example: 10X, 40X, 100X) and the resolution given as N.A. = numerical aperture, from which the limit of resolution can be calculated:

limit of resolution = wavelength

2 X numerical aperture

At a wavelength of 550 nm (0.55µm), the 100X objective lens with a N.A. of 1.25 has a resolving power of 0.22 µm. Visible light has of wavelength from about 400-750 nanometers (nm). Since the limit of resolution decreases at the shorter wavelengths, microscopes are usually fitted with a blue filter. The resolving power of the lens separates the details of the specimen, and the magnification increases the apparent size of these details so that they are visible to the human eye. Without both resolution and magnification, you would either see nothing (good resolution, no magnification) or a big blur (poor resolution, good magnification).

The objective lens system produces an image of the specimen, which is then further magnified by the ocular lens (eyepiece). The magnification of this lens is engraved on the ocular. The total magnification of the microscope is determined by the combination of the magnification of the objective lens and ocular lens that is in use, that is:

Total magnification = objective lens X ocular lens (eyepiece)

For example, with a 10X objective lens and a 10X ocular, the total magnification of the microscope is 100X. If the objective lens is changed to a 20X objective, then the total magnification is now 200X, whereas if a 10X objective is used with a 12.5X ocular lens, the total magnification is now 125X. The use of objective and ocular lenses with different magnifications allows greater flexibility when using the compound microscope. Due to the size of most bacteria (ranges widely from ~1um to over 100um), generally we require the use of the 100x oil immersion lens with a 10x ocular lense to view bacteria in a standard brightfield light microscope.

The Illumination System. The objective and ocular lens systems can only perform well under optimal illumination conditions. To achieve these conditions, the light from the light source (bulb) must be centered on the specimen. (In most inexpensive microscopes, the manufacturer adjusts this centering. In more versatile microscopes, the centering becomes more critical and is a function performed by the operator.) The parallel light rays from the light source are focused on the specimen by the condenser lens system (see Fig. 2) The condenser can move up and down to affect this focus. Finally, the amount of light entering the condenser lens system is adjusted using the condenser diaphragm. It is critical that the amount of light be appropriate for the size of the objective lens receiving the light. This is important to give sufficient light, while minimizing glare from stray light, which could otherwise reduce image detail. The higher the magnification and resolving power of the lens, the more light is needed to view the specimen.

Objective lenses used for observing very small objects such as bacteria are almost always oil immersion lenses. With an oil immersion lens, a drop of oil is placed between the specimen and the objective lens so that the image light passes through the oil. Without the oil, light passing through the glass microscope slide and specimen would be refracted (bent) when it entered the air between the slide and the objective lens. This refracted light might still be able to contribute to the image of the specimen if the objective lens is large. However, at the higher magnification, the objective lens is small, so is unable to capture this light. The loss of this light leads to loss of image detail. Therefore, at higher magnifications, the area between the slide and the lens is modified to have the same (or nearly the same) refracting qualities (refractive index) as the glass and specimen by the addition of immersion oil. Watch this NC BioNetwork video ( on oil immersion. For more information, read this article (

To use an oil immersion lens, place a drop of oil on top of the dried specimen on the slide and carefully focus the microscope so that the objective lens is immersed in the oil. Any lens, which requires oil, is marked "oil" or "oil immersion." Conversely, any lens not marked "oil" should NOT be used with oil and is generally not sealed against oil seeping into and ruining the objective.

Watch this Video on how to use a Microscope, filmed at NC State Microbiology labs:

Key Terms

microorganism, magnification, resolution, working distance, parfocal, parcentric, prokaryotic, eukaryotic, bacillus, coccus, spirillum, spirochete, morphology, bacterial arrangements, depth of field, field of view, taxonomic classification


  • Contributed by Joan Petersen & Susan McLaughli: Associate Professors (Biological Sciences and Geology) at Queensborough Community College
  • Lumen Learning: Figure 3: Brightfield light microscope

3.1: Introduction to the Microscope - Biology

Microscopes are instruments designed to produce magnified visual or photographic images of objects too small to be seen with the naked eye. The microscope must accomplish three tasks: produce a magnified image of the specimen, separate the details in the image, and render the details visible to the human eye or camera. This group of instruments includes not only multiple-lens (compound microscopes) designs with objectives and condensers, but also very simple single lens instruments that are often hand-held, such as a loupe or magnifying glass.

The microscope illustrated in Figure 1 is a simple compound microscope invented by British microscopist Robert Hooke sometime in the 1660s. This beautifully crafted microscope has an objective lens near the specimen and is focused by turning the body of the microscope to move the objective closer to or farther from the specimen. An eyepiece lens is inserted at the top of the microscope and, in many cases, there is an internal "field lens" within the barrel to increase the size of the viewfield. The microscope in Figure 1 is illuminated through the oil lamp and water-filled spherical reservoir, also illustrated in Figure 1. Light from the lamp is diffused when it passes through the reservoir and is then focused onto the specimen with a lens attached to the reservoir. This early microscope suffered from chromatic (and spherical) aberration, and all images viewed in white light contained "halos" that were either blue or red in color.

Since so many microscope users rely upon direct observation, it is important to understand the relationship between the microscope and the eye. Our eyes are capable of distinguishing color in the visible portion of the spectrum: from violet to blue to green to yellow to orange to red the eye cannot perceive ultraviolet or infrared rays. The eye also is able to sense differences in brightness or intensity ranging from black to white and all the gray shades in between. Thus, for an image to be seen by the eye, the image must be presented to the eye in colors of the visible spectrum and/or varying degrees of light intensity. The eye receptors of the retina used for sensing color are the cone cells the cells for distinguishing levels of intensity, not in color, are the rod cells. These cells are located on the retina at the back of the inside of the eye. The front of the eye (see Figure 2), including the iris, the curved cornea, and the lens are respectively the mechanisms for admitting light and focusing it on the retina.

For an image to be seen clearly, it must spread on the retina at a sufficient visual angle. Unless the light falls on non-adjacent rows of retinal cells (a function of magnification and the spreading of the image), we are unable to distinguish closely-lying details as being separate (resolved). Further, there must be sufficient contrast between adjacent details and/or the background to render the magnified, resolved image visible.

Interactive Java Tutorial
Human Eye Accommodation Accommodation of the eye refers to the physiological act of adjusting crystalline lens elements to alter the refractive power and bring objects that are closer to the eye into sharp focus. This tutorial explores changes in the lens structure as objects are relocated with respect to the eye.

Because of the limited ability of the eye's lens to change its shape, objects brought very close to the eye cannot have their images brought to focus on the retina. The accepted conventional viewing distance is 10 inches or 25 centimeters.

More than five hundred years ago, simple glass magnifiers were developed. These were convex lenses (thicker in the center than the periphery). The specimen or object could then be focused by use of the magnifier placed between the object and the eye. These "simple microscopes" could spread the image on the retina by magnification through increasing the visual angle on the retina.

The "simple microscope" or magnifying glass reached its highest state of perfection, in the 1600's, in the work of Anton von Leeuwenhoek who was able to see single-celled animals (which he called "animalcules") and even some larger bacteria with a simple microscope similar to the one illustrated in Figure 3. The image produced by such a magnifier, held close to the observer's eye, appears as if it were on the same side of the lens as the object itself. Such an image, seen as if it were ten inches from the eye, is known as a virtual image and cannot be captured on film.

Around the beginning of the 1600's, through work attributed to the Janssen brothers (see the microscope in Figure 4) in the Netherlands and Galileo in Italy, the compound microscope was developed. In its simplest form, it consisted of two convex lenses aligned in series: an object glass (objective) closer to the object or specimen and an eyepiece (ocular) closer to the observer's eye (with means of adjusting the position of the specimen and the microscope lenses). The compound microscope achieves a two-stage magnification. The objective projects a magnified image into the body tube of the microscope and the eyepiece further magnifies the image projected by the objective.

Compound microscopes developed during the seventeenth and eighteenth centuries were hampered by optical aberration (both chromatic and spherical), a flaw that is worsened by the use of multiple lenses. These microscopes were actually inferior to single lens microscopes of the period because of these artifacts. The images they produced were often blurred and had the colorful halos associated with chromatic aberrations that not only degrade image quality, but also hamper resolution. In the mid 1700's lens makers discovered that by combining two lenses made of glass with different color dispersions, much of the chromatic aberration could be reduced or eliminated. This discovery was first utilized in telescopes, which have much larger lenses than microscopes. It wasn't until the start of the 1800's that chromatically corrected lenses became commonplace in compound microscopes.

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Transmitted Microscopy Light Pathways Explore the basic pathways of light through a transmitted light microscope.

The eighteenth and nineteenth centuries witnessed a great improvement in the mechanical and optical quality of compound microscopes. Advances in machine tools allowed more sophisticated parts to be fabricated and, by the mid 1800's, brass was the alloy of choice for the production of high-quality microscopes. A number of British and German microscope manufacturers flourished during this time period. Their microscopes varied widely in design and production quality, but the overall principles defining their optical properties remained relatively constant. The microscope illustrated in Figure 5 was manufactured by Hugh Powell and Peter Lealand about 1850. The tripod base provided a sturdy support for the microscope, which many people consider the most advanced of its period.

By the end of the nineteenth century, there was a high degree of competition among microscope manufacturers and the development and production costs of microscopes became an important factor. Brass, the material of choice for microscope manufacturers, is very expensive and it was a lengthy task to machine, polish, and lacquer microscope bodies and other parts machined from this metal. To cut expenses, microscope manufacturers first started to paint the exterior portion of the microscope body and stand, as well as the stage and other non-moving parts.

During the first quarter of the twentieth century, many microscope manufacturers had begun substituting cast iron for brass in microscope frames and stages. Iron was much cheaper and could be not be distinguished from brass when painted black. They also started to electroplate many of the critical brass components such as knobs, objective barrels, nosepieces, eyepieces, and mechanical stage assemblies (illustrated in Figure 6). These early twentieth century microscopes still subscribed to a common design motif. They were monocular with a substage mirror that was used with an external lamp to illuminate the specimen. A typical microscope of the period is the Zeiss Laboratory microscope pictured in Figure 6. This type of microscope is very functional and many are still in use today.

Modern microscopes far exceed the design specifications of those made prior to the mid 1900's. Glass formulations are vastly improved allowing greater correction for optical aberration than ever before, and synthetic anti-glare lens coatings are now very advanced. Integrated circuit technology has allowed manufacturers to produce "smart" microscopes that incorporate microprocessors into the microscope stand. Photomicrography in the late twentieth century is easier than ever before with auxiliary attachments that monitor light intensity, calculate exposure based on film speed, and automatically perform complicated tasks such as bracketing, multiple exposure, and time-lapse photography.

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Microscope Assembly Discover how various parts are assembled into a state-of-the-art microscope with this tutorial.

The microscope illustrated in Figure 7 is an Olympus Provis AX70 research microscope. This microscope represents the latest state-of-the-art design that incorporates multiple illuminators (episcopic and diascopic), analyzers and polarizers, DIC prisms, fluorescence attachments, and phase contrast capabilities. The photomicrography system is the ultimate in sophistication and performance featuring spot measurement, automatic exposure control, and zoom magnification for flexible, easy framing. The Y-shaped frame is designed to be user-friendly by offering the maximum in operator comfort and ease of use.

The previous discussion addressed the basic concept of what a microscope is and touched upon an abbreviated history beginning in the seventeenth century and progressing through modern times. There are a number of additional topics that are of paramount importance towards gaining a complete understanding of microscopes and microscopy. These topics will be discussed in subsequent sections of the primer.

Practically everyone has, at one time or another, viewed the world through an optical microscope. For most people, this experience occurs during biology training in high school or college, although some scientific entrepreneurs have purchased their own microscopes either individually or as part of a science kit. Photography through the microscope, or more commonly, photomicrography, has long been a useful tool to scientists. For many years, the biological and medical sciences have relied heavily on microscopy to solve problems relating to the overall morphological features of specimens as well as a quantitative tool for recording specific optical features and data. In this respect, the optical microscope has proven useful in countless investigations into the mysteries of life.

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More recently, microscopy has enjoyed an explosive growth as a tool in the physical and materials sciences as well as the semiconductor industry, due to the need to observe surface features of new high-tech materials and integrated circuits. Microscopy is also becoming an important tool for forensic scientists who are constantly examining hairs, fibers, clothing, blood stains, bullets, and other items associated with crimes. Modern advances in fluorochrome stains and monoclonal antibody techniques have heralded an explosive growth in the use of fluorescence microscopy in both biomedical analysis and cell biology.

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The basic differences between biomedical and materials microscopy involves how the microscope projects light onto the sample. In classical biological microscopy, very thin specimens are prepared and the light is passed or transmitted through the sample, focused with the objective and then passed into the eyepieces of the microscope. For observing the surface of integrated circuits (that comprise the internal workings of modern computers) light passed through the objective and is then reflected from the surface of the sample and into the microscope objective. In scientific nomenclature, transmitted and reflected light microscopy are known as diascopic and episcopic illuminated microscopy, respectively. The photomicrographs in our photo galleries are derived from both transmitted and reflected optical microscopic scientific investigations.

One of the most serious problems in microscopy is the poor contrast produced when light is passed through very thin specimens or reflected from surfaces with a high degree of reflectivity. To circumvent this lack of contrast, various optical "tricks" have been perfected by scientists to increase contrast and to provide color variations in specimens. The assortment of techniques in the microscopists bag include: polarized light, phase contrast imaging, differential interference contrast, fluorescence illumination, darkfield illumination, Rheinberg illumination, Hoffman modulation contrast, and the use of various gelatin optical filters. A thorough discussion of these techniques is provided in the Specialized Microscopy Techniques section of this primer. References are provided in both classical bibliographic form and as website links in the front-end page of the microscopy primer. These should serve to provide more details of microscopy and photomicrography to interested readers as well as links to additional material on the World Wide Web.

Mortimer Abramowitz - Olympus America, Inc., Two Corporate Center Drive., Melville, New York, 11747.

Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

Introduction to compound light microscope - Biology bibliographies - in Harvard style

Your Bibliography: Cochran, P., 2011. Veterinary anatomy & physiology. Clifton Park, NY: Delmar, Cengage Learning.

David B, F.

Immersion Oil Microscopy

In-text: (David B, 2015)

Your Bibliography: David B, F., 2015. Immersion Oil Microscopy. [online] Available at: <> [Accessed 16 November 2015].

Kent, M.

Advanced biology

2000 - Oxford University Press - Oxford

In-text: (Kent, 2000)

Your Bibliography: Kent, M., 2000. Advanced biology. Oxford: Oxford University Press.

Parts of a Compound Microscope with Diagram and Functions

In-text: (Parts of a Compound Microscope with Diagram and Functions, 2015)

Your Bibliography: 2015. Parts of a Compound Microscope with Diagram and Functions. [online] Available at: <> [Accessed 13 November 2015].

Refractive Index, Total Internal Reflection, Optical Fibres - Pass My Exams: Easy exam revision notes for GSCE Physics

In-text: (Refractive Index, Total Internal Reflection, Optical Fibres - Pass My Exams: Easy exam revision notes for GSCE Physics, 2015)

Your Bibliography: 2015. Refractive Index, Total Internal Reflection, Optical Fibres - Pass My Exams: Easy exam revision notes for GSCE Physics. [online] Available at: <> [Accessed 19 November 2015].

Roberts, M., Reiss, M. J. and Monger, G.

Advanced biology

2000 - Nelson - Walton-on-Thames

In-text: (Roberts, Reiss and Monger, 2000)

Your Bibliography: Roberts, M., Reiss, M. and Monger, G., 2000. Advanced biology. Walton-on-Thames: Nelson.

Light microscopy

In-text: (Light microscopy, 2015)

Your Bibliography: 2015. Light microscopy. [online] Available at: <

bioslabs/methods/microscopy/microscopy.html> [Accessed 13 November 2015].

Smith, P.

Looking Down and Through: Microscope Optics 3: Oil Immersion Objectives | Agar Scientific

In-text: (smith, 2015)

Your Bibliography: smith, P., 2015. Looking Down and Through: Microscope Optics 3: Oil Immersion Objectives | Agar Scientific. [online] Available at: <> [Accessed 19 November 2015].

Taylor, D. J., Green, N. P. O., Stout, W. and Soper, R. C.

Biological science

1998 - Cambridge University Press - Cambridge

In-text: (Taylor, Green, Stout and Soper, 1998)

Your Bibliography: Taylor, D., Green, N., Stout, W. and Soper, R., 1998. Biological science. Cambridge: Cambridge University Press.

Image Analysis

There is an enormous amount of image analysis that can be done and we have various programs for doing so.

  • Measuring the intensity of various regions within your images
  • Measuring the area of objects
  • Counting the number of objects
  • Tracking the position of an object over time

Frequently Asked Questions

Which microscope has the best resolution?

Resolution is a term that is often misused. Resolution means the smallest distance between two objects that can be seen (resolved) as two objects not one. Confocals have slightly better resolution than widefield systems but resolution is not usually the most important factor. Choosing the right microscope for your particular sample and aim is important and different modalities and systems have different optimal capabilities.

How much does a microscope cost?

A lot. A good fluorescence scope system might be about $100k, a confocal more like $400k. It's worth remembering a confocal costs more than one of these, so please try not to break them. Objectives range from $1000 to $14,000, (cf one of these), so try not to break those either.

Watch the video: Chapter: Introduction to Cells and Microscopy (May 2022).