Optical Microscopy and Cell Biology: A Practical Approach
Practice 1. First Part: Use of the Optical Microscope for the Observation of Biological Samples
The microscope’s function is threefold: 1) to produce a magnified image, 2) to separate the details, and 3) to make the details visible. The microscope includes a group of instruments that includes those made up of multiple lenses (compound microscopes) and elementary devices such as the magnifying glass (simple microscope). A typical simple microscope consists of a single biconvex lens that produces a magnification between 1.5 and 30 times. It has the advantage that it allows stereoscopic (three-dimensional) vision and manipulation of the sample during observation.
1.1.1 The Mechanical System
- Base: This is the part on which the entire appliance rests. It supports all the elements of the microscope and allows it to be kept in a vertical position.
- Arm: Supports the rest of the components.
- Stage: It is a square or round metal plate on which we place and fix the histological preparation, using the stage clip. The stage can be displaced anteroposteriorly and laterally using two screws (stage controller) that move it.
- Focusing knobs: These are the coarse and fine adjustment (micrometer) knobs. We use the second one to achieve a precise focus.
1.1.2 The Lighting System
- Light or light source, which is turned on by a switch.
- Intensity regulator, controlling the amount of light that will reach the sample.
- Diaphragm, which controls the diameter of the light beam.
- Filter holder, in some microscopes. Allows the use of color filters to highlight structures stained with a specific color.
1.1.3 The Optical System
- Condenser. It is a piece with a set of lenses, located under the stage, whose function is to focus the light on the preparation.
- Objectives. They are in a rotating piece called the revolver. They are the pieces that have the lenses that produce the first magnification of the sample’s image. Each objective has engraved the value of the magnification coefficient, that is, the ability to magnify the object’s image a determined number of times (expressed by a number and by a colored ring). Usually, the revolver has the following objectives: 4 (with a red ring); 10 (yellow), 40 (light blue), 63 (dark blue), 100 (white).
- Oculars or Eyepieces. They are the lenses through which the preparation is observed. They magnify the image a second time a certain number of times (5x, 10x, and 15x).
1.3 The Resolving Power
The characteristic that determines the optical microscope’s quality is the resolving power, the ability to distinguish two points that are very close to each other as distinct and separate. (resolving power = 1 / resolution limit)
Prokaryotic and Eukaryotic Cells
Prokaryotic Cells: DNA is not associated with histones, and they possess a mesosome. Polymers not found in the eukaryotic cell include peptidoglycans and teichoic acids. They may have flagella. Domains: Bacteria and Archaea. The RNA polymerase and ribosomal proteins of Archaea more closely resemble those of the eukaryotic cell than those of the prokaryotic cell. The shape of these cells is quite simple: spherical, cylindrical, or spiral (cocci, bacilli, and spirochetes). It is common for cells to remain grouped after cell division in a characteristic way according to the species, for which we will observe them forming chains, as is the case of the genus Streptococcus.
Eukaryotic Cells: Possess membrane-bound organelles with specific functions: the Golgi apparatus, the endoplasmic reticulum, lysosomes, mitochondria, plastids, and peroxisomes. They have a highly structured and dynamic cytoskeleton made up of several protein filaments that give them their shape and the ability to move.
Cell Wall: Present in plant and fungi cells. In prokaryotes and plants, the extracellular matrix is composed of cellulose, hemicellulose, and pectins, while in fungi, it is made up of chitin. In Whitaker’s classification, the best-known organisms are grouped into four kingdoms: Animalia, Plantae, Fungi, and Protista.
The Protista kingdom is made up of unicellular organisms or those with colonial organization. It includes protozoa, free-living eukaryotes, that are microscopic, with sexual or asexual reproduction depending on the groups, some of which have developed locomotor capacity through pseudopodia, flagella, or cilia. It also includes unicellular eukaryotic algae, photosynthetic autotrophic organisms of aquatic life, some of which present flagella.