3.1 Microscopy Cont.
Overview of Microscopic Technologies and Categorization
Laboratory visualization of cells and microbes has evolved significantly due to diverse microscope types and accompanying technological advancements.
There are three primary categories of microscopes utilized in laboratory settings:
Light Microscopes: The most widely used variety, employing visible or ultraviolet light.
Electron Microscopes: Significantly more expensive than light microscopes, requiring highly trained personnel and intensive sample preparation.
Probe Microscopes: The least commonly used category due to high cost and extremely difficult sample preparation.
Size and Resolution Progression:
Light Microscopes: Used to visualize whole cells and larger organelles.
Electron Microscopes: Capable of seeing smaller entities including prokaryotic cells, viruses, macromolecules, and large atoms.
Probe Microscopes: Allow for the visualization of three-dimensional (3D) surfaces of macromolecules and individual atoms.
Light Microscopy Varieties and Applications
Light microscopes are standard in basic clinical, applied microbiology, and teaching laboratories. Several specialized types exist, each with unique underlying technologies.
Bright Field Microscopy
Technology: These are the most common microscopes. They produce an image with a bright background and a clear or colored specimen.
Functionality:
Used to count microorganisms or cells.
Ideal for viewing stained specimens, which increases contrast.
Can be used to view live, unstained cells, though contrast is inherently low.
Limitations: Provides minimal detail without staining. For instance, in a large eukaryotic cell with a nucleus surrounded by bacteria, bright field may not distinguish if bacteria are inside or simply on the cell surface.
Enhancement: Staining (such as the Gram stain) improves contrast significantly. For example, a Gram stain can differentiate between purple and pink bacteria in the same sample.
Phase Contrast Microscopy
Technology: Uses specialized condensers and objective lenses to amplify differences in the refractive index between the sample and the background.
Functionality:
Highly distinguishes cells from their surroundings without the need for dyes or staining.
Because staining is unnecessary, specimens remain alive.
Ideal for observing cell behavior, motility, and movement.
Visual Output: Images typically show distinct light and dark areas of the microbe, offering more internal detail than standard bright field.
Dark Field Microscopy
Technology: Employs a specific filter that prevents light from passing directly through the organism. Instead, light is reflected by the organism into the objective.
Visual Output: Results in a bright specimen against a dark background.
Functionality: Best for viewing living, unstained specimens.
Drawback: It is not capable of visualizing intracellular structures.
Fluorescence Microscopy
Technology: Based on the use of fluorophores (fluorescent molecules) and UV light.
Functionality:
UV light excites fluorophores at specific wavelengths, which then emit light at different colors detectable by the microscope.
Some organisms possess natural fluorescence; others must be treated with fluorescent stains.
Clinical Application: In diagnosing Mycobacterium tuberculosis, clinical specimens (containing mucus and debris) are treated with a fluorescent dye called Auramine O. Under UV light, the bacteria fluoresce a distinctive green, making them easy to identify among debris.
Immunofluorescence
Technology: A specialized subset of fluorescence microscopy using antibodies conjugated (linked) to fluorescent dyes.
Mechanism: Antibodies are highly specific and bind only to a particular target antigen on a pathogen or cell structure.
Applications:
Localizing specific structures within cells.
Visualizing interactions between macromolecules (e.g., protein-protein interactions).
Measuring protein expression and location within a cell.
Assisting in disease diagnosis.
Confocal Microscopy
Technology: Also known as laser scanning microscopy because it uses a laser to focus on a single plane.
Functionality:
The laser focuses on a plane no thicker than approximately within the object.
It captures images as thin
Brightfield microscopes are the simplest form of light, or optical, microscopes. Light, most often emitted from a standard halogen bulb, enters the microscope from the base (bottom) and is reflected via mirrors towards the sample. As described in Lab 2, before the light reaches the sample, it first passes through a condenser converging the light beams into a focused area on the sample (see the hotspot image below). The iris diaphragm controls the amount of light that passes through the sample and into the objective lens. The objective lens is the lens closest to the sample and yields the greatest amount of magnification. (Note: The degree of magnification is directly proportional to the amount of light needed. Thus, to image samples clearly at higher magnifications, more light is required). Once light passes through the sample and the objective lens, it is directed through the ocular lens , or eyepiece, to your eye. The most common power of an ocular lens is 10x. For a microscope using two lenses (objective and ocular) the total magnification of a specimen is multiplicative. Thus, a 40x objective and a 10x ocular result in a total magnification of 400x.
In order to visualize cells, samples are most often stained with specific combinations of dyes that are taken up by the cell. Staining is often required due to the limitation of resolution on unstained cells because the flat and transparent regions of a cell may appear invisible under bright field conditions (Figure 3.2B). By staining the cell with various dyes, these regions can become labeled and thus visualized. However, staining typically requires fixing the cells by heat or chemical methods before adding the dye. The cell fixation process produces its own challenges as it kills and may even distort the sample. The challenges and benefits of various staining procedures will be covered in the next section.
Phase contrast microscopes have a distinct advantage over bright field microscopy in that they are often able to visualize certain structures that would otherwise be invisible (Figure 3.3). Thus, a phase contrast microscope can provide detailed images of live cells without staining. By using specialized condensers and objectives, a phase contrast microscope amplifies the slight differences between cells and the surrounding medium (background) to make the cells highly distinguishable. For these reasons, phase contrast microscopy can be used to visualize cell movements (motility), such as swimming or gliding, without altering the cell morphology commonly brought about from treating the cells with a fixing agent.
Dark field microscopes can be used to greatly increase the contrast between a specimen and background, resulting in a dark background with bright objects in it (Figure 3.4). Unlike brightfield or phase contrast microscopy where light passes directly through the sample, dark field microscopy reflects light off of the specimen at an angle. This reflective approach does not permit the visualization of intracellular structures.
Fluorescence microscopes take advantage of fluorescent molecules called fluorophores to visualize cells on a dark background. Unlike brightfield, the energy of the incoming light is in the form of the ultraviolet (UV) spectrum. UV light excites different fluorophores at varying wavelengths, enabling scientists to use a wide array of colors during imaging. For instance, the green, yellow, and red fluorescent proteins (GFP, YFP, and RFP, respectively) have become important tools in microscopy (Figure 3.5). These fluorescent proteins alone can be expressed in a cell:
nonspecifically illuminating the cell as a whole
linked (coupled) to a normal cellular protein of interest whereby the fluorescent color is indicative of protein movement and localization
or used as tags on molecules or antibodies used to designate the presence (fluorescence detected) or absence (no fluorescence) of a specific protein target.
Confocal (laser scanning) microscopes combine the usefulness of fluorescence microscopy with the ability to visualize cells in 3-D. Unlike light or fluorescence microscopy where light is focused a single plane (2-D), confocal microscopy can capture images in either 2-D or 3-D. Rather than using halogen (brightfield) or UV (fluorescence) light, confocal microscopes use lasers to focus on a single plane within an object and with a higher degree of accuracy (Figure 3.6). Rendering a three-dimensional image is a sequential process whereby once an image is taken, the laser then moves to an adjacent plane, captures an image, then repeats this process until the desired depth of the sample has been covered. Computer programs process the stack of 2-D images acquired, digitally combines them, and renders a 3-D reconstruction of the sample. (Practically, you can think of each 2-D plane as a sheet of paper. Each subsequent image would be like stacking another sheet upon the original and then adding another, then another, etc. Thus, as you increase the number of sheets of paper you begin to form a three-dimensional object).
Electron microscope (TEM and SEM) is used to visualize incredibly small specimens. As light microscopy (brightfield, phase contrast, fluorescence, and confocal) is limited to a resolution of about 0.2 µM, it cannot efficiently visualize viruses or even some subcellular compartments. To circumvent this restriction, electron microscopes use beams of electrons (rather than light), which have significantly shorter wavelengths than light, to increases its resolution capacity to less than 1nm—that’s 200x better! However, EM is labor intensive, requires samples to be fixed (killed), and the process may alter the cell structure.
Transmission electron microscopes (TEM) use thin slices of a sample, heavily treated and coated in preservatives, and placed between the electron beam source and the detector. An image is formed from the interactions of the electrons as they pass through the thin sectioning of the sample. This process can be used to visualize subcellular organelles, substructures, and viral particles as shown in Figure 3.8A.
Scanning electron microscopes (SEM) also use a beam of electrons, but the image is obtained as the electrons reflect off (not through) the surface of the specimen. Samples are coated with either gold or palladium to enhance electron reflection. Thus, SEM can only be used to generate a detailed three-dimensional shell model of the surface of a specimen, as shown in Figure 3.8B. As with TEM, live samples cannot be viewed using SEM.
Scanning transmission electron holography microscopes (STEHM) also use an electron beam but coupled with a holography technique to study surfaces of proteins and subcellular structures. It has the capacity to magnify subatomic structures up to 20 million times larger than what can be viewed with the naked eye. While EM can resolve 1 nm (10-9), STEHM has the capacity to resolve 35 pm (a picometer is one-trillionth of a meter, or 10-12) and possibly even smaller.