Light Microscopy

Microscopy

Microscopes allow for magnification and visualization of cells and cellular components that cannot be seen with the naked eye.

Learning Objectives

Compare and contrast light and electron microscopy.

Key Takeaways

Key Points

  • Light microscopes allow for magnification of an object approximately up to 400-1000 times depending on whether the high power or oil immersion objective is used.
  • Light microscopes use visible light which passes and bends through the lens system.
  • Electron microscopes use a beam of electrons, opposed to visible light, for magnification.
  • Electron microscopes allow for higher magnification in comparison to a light microscope thus, allowing for visualization of cell internal structures.

Key Terms

  • resolution: The degree of fineness with which an image can be recorded or produced, often expressed as the number of pixels per unit of length (typically an inch).
  • electron: The subatomic particle having a negative charge and orbiting the nucleus; the flow of electrons in a conductor constitutes electricity.

Microscopy

Cells vary in size. With few exceptions, individual cells cannot be seen with the naked eye, so scientists use microscopes (micro- = “small”; -scope = “to look at”) to study them. A microscope is an instrument that magnifies an object. Most photographs of cells are taken with a microscope; these images can also be called micrographs.

The optics of a microscope’s lenses change the orientation of the image that the user sees. A specimen that is right-side up and facing right on the microscope slide will appear upside-down and facing left when viewed through a microscope, and vice versa. Similarly, if the slide is moved left while looking through the microscope, it will appear to move right, and if moved down, it will seem to move up. This occurs because microscopes use two sets of lenses to magnify the image. Because of the manner by which light travels through the lenses, this system of two lenses produces an inverted image (binocular, or dissecting microscopes, work in a similar manner, but they include an additional magnification system that makes the final image appear to be upright).

Light Microscopes

To give you a sense of cell size, a typical human red blood cell is about eight millionths of a meter or eight micrometers (abbreviated as eight μm) in diameter; the head of a pin of is about two thousandths of a meter (two mm) in diameter. That means about 250 red blood cells could fit on the head of a pin.

Most student microscopes are classified as light microscopes. Visible light passes and is bent through the lens system to enable the user to see the specimen. Light microscopes are advantageous for viewing living organisms, but since individual cells are generally transparent, their components are not distinguishable unless they are colored with special stains. Staining, however, usually kills the cells.

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Light and Electron Microscopes: (a) Most light microscopes used in a college biology lab can magnify cells up to approximately 400 times and have a resolution of about 200 nanometers. (b) Electron microscopes provide a much higher magnification, 100,000x, and a have a resolution of 50 picometers.

Light microscopes, commonly used in undergraduate college laboratories, magnify up to approximately 400 times. Two parameters that are important in microscopy are magnification and resolving power. Magnification is the process of enlarging an object in appearance. Resolving power is the ability of a microscope to distinguish two adjacent structures as separate: the higher the resolution, the better the clarity and detail of the image. When oil immersion lenses are used for the study of small objects, magnification is usually increased to 1,000 times. In order to gain a better understanding of cellular structure and function, scientists typically use electron microscopes.

Electron Microscopes

In contrast to light microscopes, electron microscopes use a beam of electrons instead of a beam of light. Not only does this allow for higher magnification and, thus, more detail, it also provides higher resolving power. The method used to prepare the specimen for viewing with an electron microscope kills the specimen. Electrons have short wavelengths (shorter than photons) that move best in a vacuum, so living cells cannot be viewed with an electron microscope.

In a scanning electron microscope, a beam of electrons moves back and forth across a cell’s surface, creating details of cell surface characteristics. In a transmission electron microscope, the electron beam penetrates the cell and provides details of a cell’s internal structures. As you might imagine, electron microscopes are significantly more bulky and expensive than light microscopes.

General Staining Methods

To properly visualize a microbe under a microscope, microbiologists use an array of stains to highlight cells and structures.

Learning Objectives

Compare and contrast in vitro and in vivo staining

Key Takeaways

Key Points

  • In-vivo staining, which visualizes cells that are alive, and in-vitro staining, which visualizes fixed cells, both have important uses.
  • There is a vast array of stains that can be used on microbes that can highlight almost any characteristic of a cell, even organelles within a cell.
  • Staining protocols can be complex, but they share some basic steps: preparation, fixation, staining, and mounting.

Key Terms

  • surfactant: a surface active agent, or wetting agent, capable of reducing the surface tension of a liquid; typically organic compounds having a hydrophilic “head” and a hydrophobic “tail”
  • organelle: a specialized structure found inside cells that carries out a specific life process (e.g., ribosomes, vacuoles)

Staining is a technique used in microscopy to enhance contrast in a microscopic image. Stains and dyes are frequently used to highlight structures in microbes for viewing, often with the aid of different microscopes. Stains may be used to define and examine different types of microbes, various stages of cellular life (e.g., the mitotic cycle), and even organelles within individual cells (e.g., mitochondria or chloroplasts).

In-vivo staining is the process of dyeing living tissue — in vivo means “in life” (as contrasted to in-vitro staining). When a certain cell or structure takes on contrasting color(s), its form (morphology) or position within a cell or tissue can be readily seen and studied. The usual purpose is to reveal cytological details that might otherwise not be apparent; however, staining can also reveal where certain chemicals or specific chemical reactions are taking place within cells. In-vitro staining involves coloring cells or structures that have been removed from their biological context. Certain stains are often combined to reveal more details and features than a single stain could reveal alone, and a counterstain is a stain that increases visibility of cells or structures when the principal stain is not sufficient. Scientists and physicians can combine staining with specific protocols for fixation and sample preparation and can use these standard techniques as consistent, repeatable diagnostic tools.

There are an incredible number of stains that can be used in a variety of different methods. What follows here are some common aspects of the process of preparing for in-vitro staining.

  • Fixation: This can itself consist of several steps. Fixation aims to preserve the shape of the cells (in this case, microbes) as much as possible. Sometimes heat fixation is used to kill, adhere, and alter the cells so they will accept stains. Most chemical fixatives generate chemical bonds between proteins and other substances within the sample, increasing their rigidity. Common fixatives include formaldehyde, ethanol, methanol, and picric acid.
  • Permeabilization: This involves treatment of the cells with (usually) a mild surfactant. This treatment dissolves cell membranes, allowing larger dye molecules to enter the cell’s interior.
  • Mounting: This step usually involves attaching the samples to a glass microscope slide for observation and analysis. In some cases, cells may be grown directly on a slide. For samples of loose cells the sample can be directly applied to a slide.

At its simplest, the actual staining process may involve immersing the sample (before or after fixation and mounting) in dye solution, followed by rinsing and observation. Many dyes, however, require the use of a mordant — a chemical compound that reacts with the stain to form an insoluble colored precipitate. When the excess dye solution is washed away, the mordanted stain remains. There is an incredible array of stains that can be used at this step, from those that stain specific microbial types (see the figure below) to those that highlight sub-compartments or organelles of a cell, such as the nucleus or endoplasmic reticulum. Alternatively, negative staining can be employed. This is a simple staining method for bacteria, performed by smearing the cells onto the slide and then applying nigrosin (a black synthetic dye) or Indian ink (an aqueous suspension of carbon particles). After drying, the microorganisms may be viewed in bright field microscopy as lighter inclusions contrast well against the dark environment surrounding them

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Chlamydia Stain: Cells of the bacterial pathogen chlamydia (indicated by arrows) are highlighted by a stain called “geimsa. “

Live, in-vivo staining microscopy shares many of these steps, with the exception of fixation, which invariably kills the microbe to be examined.