1.3: Light Microscopy

Learning Goals

• Explain the basics of how a light microscope works, with a particular focus on how we can use that information to interpret micrographs.
• Compare and contrast the different types of light microscopy (i.e., brightfield versus fluorescence) and identify when each type would be useful.
• Identify the major advantages and disadvantages of light microscopy.

The very first microscope was made in the mid-1600s by a British man named Robert Hooke. That microscope had a mirror to direct sunlight into the glass lenses of the microscope so that the sample could be viewed. Microscopes stayed more or less the same for the next 300 years, until the Industrial Revolution of the early 1900s. It is at this point that the advances in technology allowed us to greatly improve how microscopes work. Now there are light microscopes that use various combinations of lasers, lenses, spinning discs, and computer algorithms to push beyond what should be possible given the physical limitations of visible light. We’ve also made significant advances in sample preparation, which have made additional contributions to what we can visualize. Even though we have incredibly advanced microscopy techniques with which to view cells, their essence remains the same. We will explore the two largest categories of light microscopes: those that collect transmitted light, originating from some kind of light source, to view the sample and those that collect light that is emitted by the sample itself.

Pros to Light Microscopy

Live cells can be viewed and recorded in real time. Specific structures can be labeled for viewing, thus reducing the “noise” in the sample and showing finer details that may otherwise be masked by other components of the sample.

Cons to Light Microscopy

The limit of conventional (brightfield and confocal microscopy) resolution is about 0.2 µm, which restricts the amount of detail we can see of internal cellular structures using light microscopy.

• There is a newer technique, known as superresolution microscopy, which is technically able to break this barrier. We will discuss this in more detail later.

Transmitted versus Emitted Light

Light microscopy can be further divided into several subtypes of light microscopy. The most notable of these subdivisions takes into account the types of light that you are viewing when you look at an image (Figure 01-04).

• Transmitted light microscopy (Figure 01-04A) is the traditional light microscopy that you most likely tried in high school biology classes. In this type of microscopy, the sample sits between the light source and the eyepiece, and the light you see is the light that was able to pass through the sample. The simplest version, which is the one we will discuss, is called brightfield light microscopy.
• In emitted light microscopy (Figure 01-04B), the sample is illuminated by a light source that is off to the side, and then the molecules in the sample get excited by this light and release their own photons. Thus, the light that you view in these kinds of microscopes comes from the samples themselves. This type of microscopy is also known as fluorescence light microscopy, which is what we will be calling it after this.

Figure 01-04: Examples of how light is directed through transmitted and emitted light microscopes. (A) A standard desktop brightfield light microscope. Light is transmitted from the light source (at the bottom), through various lenses to condense the light, and then passed through the sample to the eye via the eyepiece at the top. The light you see is the light that was transmitted by the light source but not absorbed by the sample. (B) A standard desktop fluorescence microscope. In this microscope, the light source is off to one side (labeled as the laser excitation source in the image). This light source is either light in a specific color range or, more commonly, a laser of a precise wavelength. In the middle is a special mirror that allows some wavelengths of light to pass but not others (known as a dichroic mirror). The light from the laser hits the mirror and is reflected toward the sample. The photons in the laser light excite photo-activated particles in the sample. The photo-activated particles in the sample will then emit photons of a second wavelength. This light leaves the sample and is able to pass through the dichroic mirror so that it can travel up the objective lens and into the eye of the observer. This image was created by Heather Ng-Cornish and is licensed under a CC BY-SA 4.0 license.

Brightfield and Other Forms of Transmitted Light Microscopy

One of the primary challenges of microscopy is that living cells are fairly transparent. Microscopists use a number of techniques to increase the contrast of their samples in order to see things more clearly. Sometimes we add chemical stains to the sample, such as toluidine blue or hematoxylin and eosin (H&E), which add color contrast (see Figure 01-05). We can also use optical techniques, in which certain light is included or excluded from viewing by the microscope, to increase the contrast of our samples. An example of an optical technique used in our daily lives is polarized sunglasses. The polarized glasses reduce glare by only allowing light to pass through if it is oriented in a specific way. We can do similar kinds of things with microscopes. Some of the subtypes of transmitted light microscopy are listed below:

• Brightfield microscopy: If you have ever used a light microscope in school, this is most likely the kind that you used. It was the first invented, way back in the 1600s, and uses a light source as simple as sunlight. Images from this kind of microscopy usually have a white background, and the sample is expected to be in color when we look through the eyepiece of the microscope. Figure 01-05 shows a few different samples that have been stained and observed using brightfield light microscopy. This is the type that we focus on in this textbook when discussing transmitted light microscopy.
• Phase-contrast microscopy: In this type of microscopy, shifts in the amplitude and phase of the light are converted into shifts in brightness so that edges and other structures become more visible. Differential interference contrast (DIC) is a variation on this.
• Darkfield microscopy: In darkfield microscopy, light is excluded by blocking the center of the beam but not the outer part. This kind of light microscopy has a dark background (unlike other kinds of light microscopy), which again helps the edges of cells and other structures in the sample to stand out.
• Polarized light microscopy: This type of microscopy enhances the contrast of specimen by shining light of a particular orientation that causes shadows on structures differentially depending on their composition. This kind of microscopy is especially useful for samples like bone, fibers, or mineral deposits.

Figure 01-05: Brightfield microscopy in plants and animals. These images are at roughly the same magnification. (A) Developing seed coat of the model plant Arabidopsis thaliana stained with toluidine blue. Microscopy was done by Dr. Robin Young and is licensed under a CC BY-SA 4.0 license. (B) Epithelial cells of a human gall bladder, stained with hematoxylin and eosin (H&E) Attribution: William Karkow (2011), CIL:34859, doi.org/10.7295/W9CIL34859. This image is in the public domain.

Fluorescence Light Microscopy

Fluorescence microscopy was first developed in the 1980s and has continued to revolutionize cell biology to this day. Not only does it allow us to view live samples, as other forms of light microscopy do, but it also allows us to label (or tag) specific macromolecules / cell structure so that we can track them within the cell (Figure 01-04B). We tag the structures using fluorescent molecules (called fluorochromes or fluorophores). Then, using our fluorescent microscope, we illuminate the sample with light of a particular wavelength, and the fluorophore responds by emitting light at a second known wavelength that we can then detect. The great advantage of this technique is that only the molecule or structure of interest shows up in the image, and the rest of the sample, which is not emitting light, is dark.

Much like transmitted light microscopy, there are several subtypes of fluorescent light microscopy. Most of these have been developed to increase the resolution of the images that are being viewed. The simplest type of fluorescence microscopy is known as epifluorescence, which uses powerful halogen lightbulbs and colored filters to produce light of specific wavelengths. Confocal laser scanning microscopy (or confocal for short) is very similar to epifluorescence, except a laser is used as the source of light. Using a laser allows us to focus the light very specifically and then use computer algorithms to remove out-of-focus light. There are also a number of extremely advanced techniques that allow us to seemingly “break” the laws of physics. Using a combination of computer algorithms and specific image collection protocols, microscopists have been able to resolve structures that should be much too small to see in light microscopy. These techniques are collectively known as superresolution microscopy.

Unlike the different kinds of transmitted light microscopy, the images that you create from the different subtypes of fluorescence light microscopy are very similar. It’s not always easy to tell what kind of fluorescence microscope was used to create the image just by looking at it. For this reason, we will not differentiate between the different types in this textbook. Instead, we will treat the images that are created using fluorescence microscopy as a single group. Examples of different fluorescence microscopy images are shown in Figure 01-06.

Figure 01-06: Three different fluorescence micrographs. Live imaging of (A) Lepidozia reptans, a small liverwort collected in the Pacific Northwest. This sample is unstained and is imaged using a special fluorescence technique known as two-photon imaging, which amplifies intrinsic fluorescence within the sample. Image collected by Dr. Robin Young. (B) Baker’s yeast (Saccharomyces cerevisiae) showing a vacuolar hydrolase (magenta) overlapping with Rab5 (green) signal at endosomes. The yeast vacuole was stained in blue. Image collected by Shawn Shortill, a PhD student at the University of British Columbia in Vancouver, British Columbia, Canada. (C) Human astrocytes showing a nucleus stained with blue and the actin cytoskeleton labeled in red. Image collected by Kyle Nguyen, a graduate student at Oregon State University in Corvallis, Oregon, USA. This image is shared under a CC BY-SA 4.0 license.

Sample Preparation for Fluorescence Microscopy

There are several approaches to sample preparation in fluorescence microscopy. It depends on both the scientific question you’re trying to answer and the fluorescent stains you have available. There are a number of chemical stains that can be added to live cells that latch onto specific components of the cell and then fluoresce. This is known as direct staining. An example of this is DAPI, which fluoresces when bound to double-stranded DNA. This is great when you’re interested in exploring a sample for which a fluorescent live-cell stain exists. Sadly, that is not always the case.

Another option is immunolabeling. In this case, antibodies bound to a fluorophore that have been specifically created to bind to your protein or structure of interest are used to label the cell. If your structure/molecule of interest is on the cell surface, then this works just fine. However, if your structure is in the interior of the cell, then the antibody will have difficulty accessing it. In this case, the cell is usually treated with fixatives, which kill it but also hold everything in place. Then the membrane can be disrupted just enough to allow the antibodies to access the interior of the cell.

One of the most important advances in fluorescence microscopy was the development of genetic engineering protocols so that one could add a fluorescent tag to any protein in the cell. There are a number of naturally occurring proteins that fluoresce and are used by organisms to produce bioluminescence. Some of the fluorescent proteins have been isolated and attached to other, nonfluorescent proteins. The most commonly used tag is known as green fluorescent protein (GFP), which was isolated from bioluminescent jellyfish. As its name states, GFP is a protein that fluoresces in the green range of visible light (~550 nm) when it is illuminated with blue light (~450 nm). The genetically engineered protein usually functions like the normal protein, except it now fluoresces its location under a particular wavelength of light. Thus, we can now track structures that include the fluorescent protein.

GFP was first isolated in the 1980s, but since then, we have genetically modified it to create versions in virtually every wavelength of the visible spectrum. We’ve even been able to produce forms that are split in half and only fluoresce when the two halves come together so as to identify when two proteins of interest interact with each other.