Figure 2.1.2 : (a) A Petri dish is made of transparent plastic or glass, which allows transmission of a high proportion of light.

This
transparency allows us to see through the sides of the dish to view the contents. (b) This slice of an iron meteorite is opaque (i.e., it
has opacity). Light is not transmitted through the material, making it impossible to see the part of the hand covered by the object.
(credit a: modification of work by Umberto Salvagnin; credit b: modification of work by “Waifer X”/Flickr)
Light waves can also interact with each other by interference, creating complex patterns of motion. Dropping two pebbles into a
puddle causes the waves on the puddle’s surface to interact, creating complex interference patterns. Light waves can interact in the
same way.
In addition to interfering with each other, light waves can also interact with small objects or openings by bending or scattering. This
is called diffraction. Diffraction is larger when the object is smaller relative to the wavelength of the light (the distance between two
consecutive peaks of a light wave). Often, when waves diffract in different directions around an obstacle or opening, they will
interfere with each other.

 Exercise 2.1.2
1. If a light wave has a long wavelength, is it likely to have a low or high frequency?
2. If an object is transparent, does it reflect, absorb, or transmit light?

Lenses and Refraction

In the context of microscopy, refraction is perhaps the most important behavior exhibited by light waves. Refraction occurs when
light waves change direction as they enter a new medium (Figure 2.1.3). Different transparent materials transmit light at different
speeds; thus, light can change speed when passing from one material to another. This change in speed usually also causes a change
in direction (refraction), with the degree of change dependent on the angle of the incoming light.

Figure 2.1.3 : (a) Refraction occurs when light passes from one medium, such as air, to another, such as glass, changing the
direction of the light rays. (b) As shown in this diagram, light rays passing from one medium to another may be either refracted or
reflected. (credit a: modification of work by “ajizai”/Wikimedia Commons).
The extent to which a material slows transmission speed relative to empty space is called the refractive index of that material.
Large differences between the refractive indices of two materials will result in a large amount of refraction when light passes from
one material to the other. For example, light moves much more slowly through water than through air, so light entering water from
air can change direction greatly. We say that the water has a higher refractive index than air (Figure 2.1.4).

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 Figure 2.1.4 : This straight pole appears to bend at an angle as it enters the water. This optical illusion is due to the large difference
between the refractive indices of air and water.
When light crosses a boundary into a material with a higher refractive index, its direction turns to be closer to perpendicular to the
boundary (i.e., more toward a normal to that boundary; Figure 2.1.5). This is the principle behind lenses. We can think of a lens as
an object with a curved boundary (or a collection of prisms) that collects all of the light that strikes it and refracts it so that it all
meets at a single point called the image point (focus). A convex lens can be used to magnify because it can focus at closer range
than the human eye, producing a larger image. Concave lenses and mirrors can also be used in microscopes to redirect the light
path. Figure 2.1.5 shows the focal point (the image point when light entering the lens is parallel) and the focal length (the distance
to the focal point) for convex and concave lenses.

Figure 2.1.5 : (a) A lens is like a collection of prisms, such as the one shown here. (b) When light passes through a convex lens, it is
refracted toward a focal point on the other side of the lens. The focal length is the distance to the focal point. (c) Light passing
through a concave lens is refracted away from a focal point in front of the lens.
The human eye contains a lens that enables us to see images. This lens focuses the light reflecting off of objects in front of the eye
onto the surface of the retina, which is like a screen in the back of the eye. Artificial lenses placed in front of the eye (contact
lenses, glasses, or microscopic lenses) focus light before it is focused (again) by the lens of the eye, manipulating the image that
ends up on the retina (e.g., by making it appear larger).
Images are commonly manipulated by controlling the distances between the object, the lens, and the screen, as well as the
curvature of the lens. For example, for a given amount of curvature, when an object is closer to the lens, the focal points are farther
from the lens. As a result, it is often necessary to manipulate these distances to create a focused image on a screen. Similarly, more
curvature creates image points closer to the lens and a larger image when the image is in focus. This property is often described in
terms of the focal distance, or distance to the focal point.

 Exercise 2.1.3
1. Explain how a lens focuses light at the image point.
2. Name some factors that affect the focal length of a lens.

Electromagnetic Spectrum and Color

Visible light is just one form of electromagnetic radiation (EMR), a type of energy that is all around us. Other forms of EMR
include microwaves, X-rays, and radio waves, among others. The different types of EMR fall on the electromagnetic spectrum,
which is defined in terms of wavelength and frequency. The spectrum of visible light occupies a relatively small range of
frequencies between infrared and ultraviolet light (Figure 2.1.6).

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 Figure 2.1.6 : The electromagnetic spectrum ranges from high-frequency gamma rays to low-frequency radio waves. Visible light is
the relatively small range of electromagnetic frequencies that can be sensed by the human eye. On the electromagnetic spectrum,
visible light falls between ultraviolet and infrared light. (credit: modification of work by Johannes Ahlmann).

Whereas wavelength represents the distance between adjacent peaks of a light wave, frequency, in a simplified definition,
represents the rate of oscillation. Waves with higher frequencies have shorter wavelengths and, therefore, have more oscillations
per unit time than lower-frequency waves. Higher-frequency waves also contain more energy than lower-frequency waves. This
energy is delivered as elementary particles called photons. Higher-frequency waves deliver more energetic photons than lower-
frequency waves.
Photons with different energies interact differently with the retina. In the spectrum of visible light, each color corresponds to a
particular frequency and wavelength (Figure 2.1.6).The lowest frequency of visible light appears as the color red, whereas the
highest appears as the color violet. When the retina receives visible light of many different frequencies, we perceive this as white
light. However, white light can be separated into its component colors using refraction. If we pass white light through a prism,
different colors will be refracted in different directions, creating a rainbow-like spectrum on a screen behind the prism. This
separation of colors is called dispersion, and it occurs because, for a given material, the refractive index is different for different
frequencies of light.
Certain materials can refract nonvisible forms of EMR and, in effect, transform them into visible light. Certain fluorescent dyes, for
instance, absorb ultraviolet or blue light and then use the energy to emit photons of a different color, giving off light rather than
simply vibrating. This occurs because the energy absorption causes electrons to jump to higher energy states, after which they then
almost immediately fall back down to their ground states, emitting specific amounts of energy as photons. Not all of the energy is
emitted in a given photon, so the emitted photons will be of lower energy and, thus, of lower frequency than the absorbed ones.
Thus, a dye such as Texas red may be excited by blue light, but emit red light; or a dye such as fluorescein isothiocyanate (FITC)
may absorb (invisible) high-energy ultraviolet light and emit green light (Figure 2.1.7). In some materials, the photons may be
emitted following a delay after absorption; in this case, the process is called phosphorescence. Glow-in-the-dark plastic works by
using phosphorescent material.

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 Figure 2.1.7 : The fluorescent dyes absorbed by these bovine pulmonary artery endothelial cells emit brilliant colors when excited
by ultraviolet light under a fluorescence microscope. Various cell structures absorb different dyes. The nuclei are stained blue with
4’,6-diamidino-2-phenylindole (DAPI); microtubles are marked green by an antibody bound to FITC; and actin filaments are
labeled red with phalloidin bound to tetramethylrhodamine (TRITC).

 Exercise 2.1.4
1. Which has a higher frequency: red light or green light?
2. Explain why dispersion occurs when white light passes through a prism.
3. Why do fluorescent dyes emit a different color of light than they absorb?

Magnification, Resolution, and Contrast

Microscopes magnify images and use the properties of light to create useful images of small objects. Magnification is defined as
the ability of a lens to enlarge the image of an object when compared to the real object. For example, a magnification of 10⨯
means that the image appears 10 times the size of the object as viewed with the naked eye.
Greater magnification typically improves our ability to see details of small objects, but magnification alone is not sufficient to
make the most useful images. It is often useful to enhance the resolution of objects: the ability to tell that two separate points or
objects are separate. A low-resolution image appears fuzzy, whereas a high-resolution image appears sharp. Two factors affect
resolution. The first is wavelength. Shorter wavelengths are able to resolve smaller objects; thus, an electron microscope has a
much higher resolution than a light microscope, since it uses an electron beam with a very short wavelength, as opposed to the
long-wavelength visible light used by a light microscope. The second factor that affects resolution is numerical aperture, which is a
measure of a lens’s ability to gather light. The higher the numerical aperture, the better the resolution.
Even when a microscope has high resolution, it can be difficult to distinguish small structures in many specimens because
microorganisms are relatively transparent. It is often necessary to increase contrast to detect different structures in a specimen.
Various types of microscopes use different features of light or electrons to increase contrast—visible differences between the parts
of a specimen (see Instruments of Microscopy). Additionally, dyes that bind to some structures but not others can be used to
improve the contrast between images of relatively transparent objects (see Staining Microscopic Specimens).

 Exercise 2.1.5
1. Explain the difference between magnification and resolution.
2. Explain the difference between resolution and contrast.
3. Name two factors that affect resolution.

Key Concepts and Summary

Light waves interacting with materials may be reflected, absorbed, or transmitted, depending on the properties of the material.
Light waves can interact with each other (interference) or be distorted by interactions with small objects or openings
(diffraction).
Refraction occurs when light waves change speed and direction as they pass from one medium to another. Differences in the
refraction indices of two materials determine the magnitude of directional changes when light passes from one to the other.
A lens is a medium with a curved surface that refracts and focuses light to produce an image.
Visible light is part of the electromagnetic spectrum; light waves of different frequencies and wavelengths are distinguished as
colors by the human eye.

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 A prism can separate the colors of white light (dispersion) because different frequencies of light have different refractive indices
for a given material.
Fluorescent dyes and phosphorescent materials can effectively transform nonvisible electromagnetic radiation into visible light.
The power of a microscope can be described in terms of its magnification and resolution.
Resolution can be increased by shortening wavelength, increasing the numerical aperture of the lens, or using stains that
enhance contrast.

Glossary
absorbance
when a molecule captures energy from a photon and vibrates or stretches, using the energy

amplitude
the height of a wave

contrast
visible differences between parts of a microscopic specimen

diffraction
the changing of direction (bending or spreading) that occurs when a light wave interacts with an opening or barrier

dispersion
the separation of light of different frequencies due to different degrees of refraction

fluorescent
the ability of certain materials to absorb energy and then immediately release that energy in the form of light

focal length
the distance from the lens to the image point when the object is at a definite distance from the lens (this is also the distance to
the focal point)

focal point
a property of the lens; the image point when light entering the lens is parallel (i.e., the object is an infinite distance from the
lens)

frequency
the rate of vibration for a light wave or other electromagnetic wave

image point (focus)

a property of the lens and the distance of the object to the lens; the point at which an image is in focus (the image point is often
called the focus)
interference
distortion of a light wave due to interaction with another wave
magnification
the power of a microscope (or lens) to produce an image that appears larger than the actual specimen, expressed as a factor of
the actual size
numerical aperture
a measure of a lens’s ability to gather light
opacity
the property of absorbing or blocking light
phosphorescence
the ability of certain materials to absorb energy and then release that energy as light after a delay
reflection

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 when light bounces back from a surface
refraction
bending of light waves, which occurs when a light wave passes from one medium to another
refractive index
a measure of the magnitude of slowing of light waves by a particular medium
resolution
the ability to distinguish between two points in an image
transmittance
the amount of light that passes through a medium
transparency
the property of allowing light to pass through
wavelength
the distance between one peak of a wave and the next peak

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2.2: Peering into the Invisible World
 Learning Objectives
Describe historical developments and individual contributions that led to the invention and development of the microscope
Compare and contrast the features of simple and compound microscopes

Some of the fundamental characteristics and functions of microscopes can be understood in the context of the history of their use.
Italian scholar Girolamo Fracastoro is regarded as the first person to formally postulate that disease was spread by tiny
invisibleseminaria, or “seeds of the contagion.” In his book De Contagione (1546), he proposed that these seeds could attach
themselves to certain objects (which he called fomes [cloth]) that supported their transfer from person to person. However, since the
technology for seeing such tiny objects did not yet exist, the existence of the seminaria remained hypothetical for a little over a
century—an invisible world waiting to be revealed.

Early Microscopes
Antonie van Leeuwenhoek, sometimes hailed as “the Father of Microbiology,” is typically credited as the first person to have
created microscopes powerful enough to view microbes (Figure 2.2.1). Born in the city of Delft in the Dutch Republic, van
Leeuwenhoek began his career selling fabrics. However, he later became interested in lens making (perhaps to look at threads) and
his innovative techniques produced microscopes that allowed him to observe microorganisms as no one had before. In 1674, he
described his observations of single-celled organisms, whose existence was previously unknown, in a series of letters to the Royal
Society of London. His report was initially met with skepticism, but his claims were soon verified and he became something of a
celebrity in the scientific community.

Figure 2.2.1 : (a) Antonie van Leeuwenhoek (1632–1723) is credited as being the first person to observe microbes, including
bacteria, which he called “animalcules” and “wee little beasties.” (b) Even though van Leeuwenhoek’s microscopes were simple
microscopes (as seen in this replica), they were more powerful and provided better resolution than the compound microscopes of
his day. (credit b: modification of work by “Wellcome Images”/Wikimedia Commons) (c) Though more famous for developing the
telescope, Galileo Galilei (1564–1642) was also one of the pioneers of microscopy.
While van Leeuwenhoek is credited with the discovery of microorganisms, others before him had contributed to the development
of the microscope. These included eyeglass makers in the Netherlands in the late 1500s, as well as the Italian astronomer Galileo
Galilei, who used a compound microscope to examine insect parts (Figure 2.2.1). Whereas van Leeuwenhoek used a simple
microscope, in which light is passed through just one lens, Galileo’s compound microscope was more sophisticated, passing light
through two sets of lenses.
Van Leeuwenhoek’s contemporary, the Englishman Robert Hooke (1635–1703), also made important contributions to microscopy,
publishing in his book Micrographia (1665) many observations using compound microscopes. Viewing a thin sample of cork
through his microscope, he was the first to observe the structures that we now know as cells (Figure 2.2.2). Hooke described these
structures as resembling “Honey-comb,” and as “small Boxes or Bladders of Air,” noting that each “Cavern, Bubble, or Cell” is
distinct from the others (in Latin, “cell” literally means “small room”). They likely appeared to Hooke to be filled with air because
the cork cells were dead, with only the rigid cell walls providing the structure.

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 Figure 2.2.2 : Robert Hooke used his (a) compound microscope to view (b) cork cells. Both of these engravings are from his
seminal work Micrographia, published in 1665.

 Exercise 2.2.1
1. Explain the difference between simple and compound microscopes.
2. Compare and contrast the contributions of van Leeuwenhoek, Hooke, and Galileo to early microscopy.

 Who Invented the Microscope?

While Antonie van Leeuwenhoek and Robert Hooke generally receive much of the credit for early advances in microscopy,
neither can claim to be the inventor of the microscope. Some argue that this designation should belong to Hans and Zaccharias
Janssen, Dutch spectacle-makers who may have invented the telescope, the simple microscope, and the compound microscope
during the late 1500s or early 1600s (Figure 2.2.3). Unfortunately, little is known for sure about the Janssens, not even the
exact dates of their births and deaths. The Janssens were secretive about their work and never published. It is also possible that
the Janssens did not invent anything at all; their neighbor, Hans Lippershey, also developed microscopes and telescopes during
the same time frame, and he is often credited with inventing the telescope. The historical records from the time are as fuzzy
and imprecise as the images viewed through those early lenses, and any archived records have been lost over the centuries.

Figure 2.2.3 : Zaccharias Janssen, along with his father Hans, may have invented the telescope, the simple microscope, and the
compound microscope during the late 1500s or early 1600s. The historical evidence is inconclusive.
By contrast, van Leeuwenhoek and Hooke can thank ample documentation of their work for their respective legacies. Like
Janssen, van Leeuwenhoek began his work in obscurity, leaving behind few records. However, his friend, the prominent
physician Reinier de Graaf, wrote a letter to the editor of the Philosophical Transactions of the Royal Society of London calling
attention to van Leeuwenhoek’s powerful microscopes. From 1673 onward, van Leeuwenhoek began regularly submitting

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 letters to the Royal Society detailing his observations. In 1674, his report describing single-celled organisms produced
controversy in the scientific community, but his observations were soon confirmed when the society sent a delegation to
investigate his findings. He subsequently enjoyed considerable celebrity, at one point even entertaining a visit by the czar of
Russia.
Similarly, Robert Hooke had his observations using microscopes published by the Royal Society in a book called
Micrographia in 1665. The book became a bestseller and greatly increased interest in microscopy throughout much of Europe.

Summary
Antonie van Leeuwenhoek is credited with the first observation of microbes, including protists and bacteria, with simple
microscopes that he made.
Robert Hooke was the first to describe what we now call cells.
Simple microscopes have a single lens, while compound microscopes have multiple lenses.

Glossary
compound microscope
a microscope that uses multiple lenses to focus light from the specimen

simple microscope
a type of microscope with only one lens to focus light from the specimen

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2.3: Instruments of Microscopy
 Learning Objectives
Identify and describe the parts of a brightfield microscope
Calculate total magnification for a compound microscope
Describe the distinguishing features and typical uses for various types of light microscopes, electron microscopes, and
scanning probe microscopes

The early pioneers of microscopy opened a window into the invisible world of microorganisms. But microscopy continued to
advance in the centuries that followed. In 1830, Joseph Jackson Lister created an essentially modern light microscope. The 20th
century saw the development of microscopes that leveraged nonvisible light, such as fluorescence microscopy, which uses an
ultraviolet light source, and electron microscopy, which uses short-wavelength electron beams. These advances led to major
improvements in magnification, resolution, and contrast. By comparison, the relatively rudimentary microscopes of van
Leeuwenhoek and his contemporaries were far less powerful than even the most basic microscopes in use today. In this section, we
will survey the broad range of modern microscopic technology and common applications for each type of microscope.

Light Microscopy
Many types of microscopes fall under the category of light microscopes, which use light to visualize images. Examples of light
microscopes include brightfield microscopes, darkfield microscopes, phase-contrast microscopes, differential interference contrast
microscopes, fluorescence microscopes, confocal scanning laser microscopes, and two-photon microscopes. These various types of
light microscopes can be used to complement each other in diagnostics and research.

Brightfield Microscopes
The brightfield microscope, perhaps the most commonly used type of microscope, is a compound microscope with two or more
lenses that produce a dark image on a bright background. Some brightfield microscopes are monocular (having a single eyepiece),
though most newer brightfield microscopes are binocular (having two eyepieces), like the one shown in Figure 2.3.1; in either case,
each eyepiece contains a lens called an ocular lens. The ocular lenses typically magnify images 10 times (10⨯). At the other end of
the body tube are a set of objective lenses on a rotating nosepiece. The magnification of these objective lenses typically ranges
from 4⨯ to 100⨯, with the magnification for each lens designated on the metal casing of the lens. The ocular and objective lenses
work together to create a magnified image. The total magnification is the product of the ocular magnification times the objective
magnification:

ocular magnification × objective magnification

For example, if a 40× objective lens is selected and the ocular lens is 10×, the total magnification would be

(40×)(10×) = 400×

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