Microscopy is used to visualize objects that are too small to see with the naked eye. In biology, this technique enables us to examine things like bacteria and cells at a magnification of up to 10000 times their original size (BIO1000F practical manual, 2017). In light microscopy, visible light is used to detect such small objects – with bright field microscopy being the most common form of light microscopy. In bright field microscopy, the image you see is formed mainly by absorption of light by the specimen (for example a cell) (Lackie, 2010).
A simple microscope is really nothing more than a magnifying glass, where a convex lens is used to magnify an image. A compound microscope, on the other hand uses a minimum of two magnifying lenses or lens arrays, called the objective and the eyepiece (BIO1000F practical manual, 2017; Encyclopædia Britannica, 2019).
The coordinated system of lenses is arranged in such a way that a magnified image of a specimen can be viewed with increased resolution and contrast. Resolution is how well one can distinguish between two points on a specimen – the better the resolution, the sharper the image. Resolution is a function of the microscope, its lens design and source of radiation. Contrast, on the other hand, is the difference in intensity perceived between different parts of an image. Histological stains that bind selectively to different parts of the specimen can enhance the contrast of an image (BIO1000F practical manual, 2017; Lackie, 2010). Contrast is thus mainly dependent on intrinsic qualities of the specimen and how it was prepared for microscopy.
The typical upright compound microscope consists of the following parts (see figure 1, from the bottom up) (BIO1000F practical manual, 2017; New York Microscope Company, 2019):
Light intensity control: Knob used to adjust the amount of light that reaches the specimen or slide from the base illumination.
Light (or Illumination): The light used to illuminate the specimen from the base of the microscope. Low voltage halogen bulbs or LED are the most commonly used sources of illumination for compound microscopes.
Fine and coarse adjustment controls: Adjust the focus of the microscope by moving the stage vertically. The coarse adjustment knob is moved to its highest position stop (forward rotation). The fine adjustment knob is used to bring the image into sharp focus.
Condenser: Condenses the light from the base illumination and focuses it onto the stage. The condenser has an iris diaphragm (a circular opening where light can pass through), which can be adjusted to match the effective numerical aperture of each objective lens. To open and close the iris diaphragm, the condenser ring can be rotated so that the amount of light permitted through matches the requirements of the objective in use.
Stage adjustment: Adjusts the position of the mechanical stage horizontally in the X and Y plane, in order to position the slide so that a portion of the specimen is under the objective.
Stage (or platform): The platform upon which the specimen or slide is placed.
Stage clips (or slide holder): Clips on the stage that hold the slide in place on the mechanical stage.
Objective lenses: There are usually 3-5 optical lens objectives on a compound microscope, each with different magnification levels – most commonly 4x, 10x, 40x, and 100x. The total magnification of a compound microscope is calculated by multiplying the objective lens magnification by the eyepiece magnification level. So, a compound microscope with a 10x eyepiece magnification looking through the 40x objective lens has a total magnification of 400x (10 x 40).
Nosepiece: Holds the objective lenses and attaches them to the microscope head. The nosepiece rotates to change which objective lens is in working position.
Eyepiece (or ocular): The part that you looked through at the top of the compound microscope. Eyepieces typically have ocular lenses with magnification between 5x and 30x. Many microscopes are fitted with foldable rubber eye guards that can help minimize ambient light.
The design described here is for a standard upright compound light microscope. Another design commonly used, especially when observing living cells, is the inverted microscope. The difference is in where the objectives sit, where the light source is, and which parts of the microscope move to bring the image into focus. In the conventional upright microscope (as in Figure 1), the objectives are attached to a nosepiece on the microscope body above the stage, the sample is illuminated from below, and the focus controls move the stage up and down to bring the image to its proper location of focus relative to the eyepiece. In inverted designs, the stage itself is fixed and the objectives are below the stage, in an inverted position. The sample is illuminated from above and the focus controls move the objectives up and down to focus the image in the eyepiece. Having a fixed stage allows better access to the specimen in circumstances where the specimen needs to be manipulated while being observed (such as microinjection) (Murphy and Davidson, 2012).
The light path
In a standard bright field microscope, light travels from the source of illumination through the condenser, through the specimen, through the objective lens, and through the eyepiece to the eye of the observer. Light thus gets transmitted through the specimen and it appears against an illuminated background. The observer can see objects in the light path because natural pigmentation or stains absorb light differentially, or because they are just thick enough (but not too thick) to absorb a significant amount of light despite being colorless (Caprette, 2012; New York Microscope Company, 2019).
Specimen preparation (wet mount) for viewing with an upright compound microscope
Because light needs to travel through it, the material you observe with the compound microscope must be very small, transparent, or cut in a thin section. Preparing specimens for viewing under the upright compound microscope involves placing them on a glass slide in a mounting medium, such as water or glycerine. A cover slip is then placed over the specimen to protect the lens from the mounting medium and to flatten the specimen slightly. Throughout the wet mount specimen preparation, care should be taken to minimize obstructions (like debris or bubbles) in the light path. The idea is to create as clear as possible, a path for the light to travel through. Here follows a protocol for preparing a wet mount:
- Use a clean glass slide.
- Place your specimen in the center of the slide.
- Place a drop of mounting medium on the specimen.
- Use a coverslip to overlay the specimen and medium. To prevent air bubbles from being trapped underneath, lower edge of the coverslip onto the slide and bring it into contact with the mounting medium. Support the other edge with a pipette tip/toothpick and lower the cover slip carefully until it covers the specimen.
- It may be necessary to put gentle pressure on the cover slip to separate cells.
- Absorb excess liquid around the cover slip with a piece of tissue.
Specimens are often stained to enhance contrast. When this applies, the specimen or tissue can either be stained before it is mounted on the slide, or the stain can be added to the mounting medium.
Specimen preparation for viewing with an inverted compound microscope
With the inverted microscope, living cells can be observed in their culture dishes with medium on the microscope stage. This is called live-cell imaging and it enables the observer to monitor a variety of dynamic intracellular events over time. Because the inverted microscope has a fixed stage onto which culture dishes or flasks can be viewed, no special specimen preparation is necessary. Live-cell imaging often makes use of fluorescent labeling to help the observer distinguish cellular structures and processes (Murphy and Davidson, 2012).
Light microscopy has become one of the most widely used methods in the life sciences since the invention of the microscope in the 1670’s by Antoni van Leeuwenhoek. Soon after the first microscope was built, a host of biological specimens (including protozoa, bacteria, spermatozoa, and red blood) were observed and described (Periasamy, 2014). Nowadays, no biological laboratory is complete without a microscope
Bright field microscopy allows one to observe the development, organization, and function of unicellular and higher organisms and to study structures and mechanisms at cellular and subcellular levels (Periasamy, 2014). A common application of the upright compound light microscope is in cytogenetics i.e. the study of chromosomes. As an example, the steps and materials used to prepare and observe metaphase chromosome spreads of a marine mollusc is described below (based on Van der Merwe and Roodt-Wilding, 2008):
- Mitotic inhibition: the tissue (marine mollusc larvae in this case) is treated with a mitotic inhibitor (for example 0.01% m/v colchicine) to arrest cell division at the metaphase stage where chromosomes can be easily observed
- Hypotonic treatment: the sample is next treated with a hypotonic solution (for example 0.075M KCl). This allows the cells to swell, which aids in spreading of chromosomes during slide preparation.
- Fixation: The sample is fixed in Carnoy’s solution (3:1; methanol:acetic acid) to terminate all biochemical reactions and increase the treated sample’s stability
- Dissociation: Fixed larvae are dissociated in 60% (v/v) acetic acid to obtain a cell suspension
- Slide preparation: the cell suspension is dropped onto pre-washed slides at 60 °C and allowed to dry overnight
- Staining: Slides are soaked in Giemsa (10%; m/v) for 25 min and allowed to dry for 2 hours. Giemsa is a dye that binds to phosphate groups of DNA, specifically in adenine-thymine rich areas.
- Mounting: Slides are mounted with coverslips.
- Observation: Chromosome metaphase spreads can be observed using 100× objective magnification.
Cytogenetics is just one of many applications of the microscope in the biology laboratory. Whether it is used for identifying cells and tiny organisms, or to study the organisation and processes within cells through live-cell imaging, there is no doubt that bright field microscopy has advanced the life sciences over the past decades. With increasing sophistication and technology, this technique promises to stay central to the study of life.
- BIO1000F practical manual. (2017). University of Cape Town.
- Caprette, D.R. (2012). Light Microscopy. Rice University. Retrieved from: https://www.ruf.rice.edu/~bioslabs/methods/microscopy/microscopy.html [5 December 2019]
- Encyclopædia Britannica, Inc. (2019). The compound microscope. Retrieved from: https://www.britannica.com/technology/microscope/Stereoscopic-microscopes [5 December 2019]
- Lackie, J. 2010. A Dictionary of Biomedicine. Oxford University Press.
- Murphy, D. B., & Davidson, M. W. (2012). Fundamentals of light microscopy and electronic imaging.
- New York Microscope Company. (2019). What is a Compound Microscope? Retrieved from: https://www.microscopeinternational.com/what-is-a-compound-microscope/ [5 December 2019]
- Periasamy, A. (2014). Advanced Light Microscopy. Methods (66): 121-123.
- Van der Merwe, M. and Roodt-Wilding, R., 2008. Chromosome number of the South African abalone Haliotis midae. African Journal of Marine Science, 30(1), pp.195-198.