The light or optical microscope is a common lab tool that can be used to visualize structures with sizes below that which can be seen by the human eye. Light microscopes are useful to size ranges down to roughly 1 micron (for comparison, the diameter of a human hair is approximately 100 microns). These microscopes are versatile in the types of materials and samples they can analyze (opaque or transparent, liquid or solid). A number of modular accessories have been developed which enhance the capability of the microscope, giving it, for example, improved contrast or the ability to image in three dimensions. When combined with a digital camera and image analysis software, light microscopes can also be used to collect quantitative information.
Light microscopy is ideally suited for imaging biological specimens, because the resolution of the microscope is within the size range of key cellular structures, and because using it requires only a minimal amount of perturbation of the specimen. For these reasons, several techniques have been developed specifically for biological applications. Examples include the diverse range of fluorescent probes that can be used to selectively highlight structures and processes within cells.
There is uncertainty surrounding the inventor of the first light microscope. However, magnifying microscopes consisting of collapsing tubes, similar to a telescope, date back to as early as the late 16th or early 17th century. Credit for inventing this type of microscope is sometimes given to Zacharias Jansen (1580–1638). Antony van Leeuwenhoek (1632–1723) invented the simple microscope in 1670, which had a magnification up to 200x, and doubled the resolution compared to the best compound microscopes of those days. He observed the individual cells, protozoans, bacteria, muscle cells, and sperm cells for the first time. Englishman Robert Hooke (1635–1703) added a stage to hold the specimen, an illuminator, and fine focus controls to improve the compound microscope further. The magnification of commonly available microscopes was limited to roughly 30-50x until the 1800s, and the images exhibited blurry edges and rainbow-like distortions. These issues were eliminated by Carl Zeiss (1816–1888) and Ernst Abbe (1840–1905), who added the sub-stage condenser and developed the superior lenses that provided improved resolution and higher magnification.
This article will describe the key parts of the light microscope, general steps for analyzing a specimen and caring for the microscope, a number of variations on it that are commonly used, and finally a comparison with other types of microscopes.
Parts Of A Common Light Microscope
All modern light microscopes have several parts in common. Following the path of the light, the parts are:
- Light source: there are a variety of different types of lamps which can be used as the light source in optical microscopy, including tungsten filament, various types of arc lamps, and LEDs. The light source is generally chosen based on a number of factors including illumination intensity, emitted spectrum, and temperature.
- Condenser, which collects light from the source and projects it on to or through the sample.
- Sample stage, which holds the specimen and usually has multiple axes of motion.
- Objective, which collects light from the sample. The objective is a critical component, because, it determines the magnification and quality of the image. Most microscopes will have a number of selectable objectives mounted on a rotatable nosepiece, where each objective is contained in a metallic cylinder. Common objective magnifications are between 4X to 100X. Due to optical limitations, higher magnification objectives may require the use of an immersion fluid placed between the specimen and the objective.
- Eyepiece, or ocular, which further magnifies and focuses the image. The total magnification of the microscope is calculated by multiplying the magnifying power of the objective by the eyepiece (for example, a 40X objective with 10X eyepiece will magnify a specimen by 400X).
All of these parts are mounted on a microscope frame, which will also include focusing and positioning knobs or digital controllers to position the components relative to one another.
Light microscopes can be used to visualize materials by transmitting light through them (known as diascopic illumination, as in the case of looking at cells in a fluid mounted on a microscope slide with coverslip), or by using reflected/scattered light (episcopic illumination, for example visualizing an insect or small electronic components).
Modifications Available On Light Microscopes
The section above described the basic components of the common microscope. There can be modifications made to these basic components to specialize the microscope for certain applications. There are many possible modifications available, many of which can be combined to increase the capabilities of a single microscope. Five of the most common are listed below.
Phase Contrast and Differential Interference Contrast (DIC)
In these techniques, imaging is based on phase shifts in the light passing through the specimen due to variation in the optical path length (distance multiplied by refractive index) in the specimen. The refractive index may vary across a specimen due to small material differences, for example, the difference in the liquid environments inside and outside a cell. Measuring the phase shift provides an additional improvement in contrast not available in simple transmitted light microscopy. The main difference between the two techniques is that phase contrast measures simple differences in the optical path length, whereas DIC measures the rate of change (or gradient) in the path length as the light passes through the specimen.
For imaging in biology, phase contrast causes the cells to appear dark against a light background, while DIC gives a pseudo–three-dimensional (3D) shaded appearance to the cells. Regular brightfield, transmitted light microscopy is sufficient to observe the general outlines of cells, but phase contrast or DIC is vital to get detailed, high-contrast images (Thorn, 2016).
In this type of microscopy, a specimen is prepared, using a stain or dye for example, so that certain parts of it will absorb one wavelength of light, and emit another, different wavelength (the property of fluorescence). The modification required to the microscope for this type of analysis is a filter on the light source, so that the sample is illuminated only by a specific wavelength, and a second filter between the specimen and the observer, so that only the specific emitted wavelength of light reaches the observer. In this way, individual parts of very complex structures can be selectively illuminated to the observer. This is used, for example, in visualizing specific parts of human cells. This technique was recognized in the Nobel Prize in chemistry in 2014.
This type of microscope filters the light source, and the light passing between the specimen and the observer, by polarization state. An example of this is cross-polarized illumination. In this technique, polarization filters (fine gratings) offset by 90 degrees are placed on the light source, and between the specimen and observer. The only light that can pass through both filters is light which has had its polarization state affected by the specimen. Crystalline materials that alter the polarization state will show up bright in this technique.
In confocal microscopy, an additional set of optical elements (sometimes including a laser) are used so that only light from a specific point, within a narrow depth in the sample, reaches the observer. This point is then scanned, or rastered, over the sample to develop a full image. This provides a number of benefits. First, since only in-focus light is detected, the resolution of the image is significantly improved. This is particularly useful for fluorescence microscopy, where extraneous out of focus light is emitted when the entire sample is illuminated at the excitation wavelength. Second, since light is selectively collected from a narrow focal plane, the information collected consists of a series of two-dimensional sections which can be analyzed individually, or assembled to construct a three-dimensional image.
The disadvantages of using confocal microscopy include the requirement of more complex and expensive optical elements to scan and focus the illumination. Also, since the technique is inherently a point-by-point analysis, there can be a trade-off between scanning speed and image quality.
In this type of microscope, two slightly offset optical paths exist between the specimen and the eyepiece, so that the user has depth resolution of the sample. This technique is useful when work is being done on the specimen, for example dissection or assembly of small pieces.
Additional optics are available to improve the contrast in certain types of samples, which include phase contrast, differential interference (Nomarski) contrast, and dark field techniques. These techniques require specialized condenser and objective optics. Further modifications can be made to collect spectroscopic data on specimens in parallel to optical images, infrared or Raman data for example, which can be useful in determining material composition.
Steps For Analyzing A Specimen With A Light Microscope
As with any lab analysis, the first step to using light microscopy is to develop a plan and set of goals for the analysis. One way of doing this is to list the questions you hope to answer using this technique.
The next step is sample preparation. For normal optical microscopy, this is relatively straightforward. For liquid samples that will be analyzed using transmitted light, common steps are:
- Clean the microscope slide and cover slip
- Agitate the sample if any particles or solids have fallen to the bottom of the container
- Using a pipette, place a small droplet of the sample on the slide
- Gently place a cover slip over the droplet, and allow time for the sample to settle and for any air bubbles to be forced out
- Place the slide on the stage, and secure with stage clips
- Begin by observing the sample at low magnification, and adjusting the focus and illumination as needed
- Progressively increase the magnification by rotating between objectives on the nosepiece, adjusting focus and illumination at each magnification. This method is advisable since higher magnifying power objectives have a smaller working distance (the distance between the end of the objective and the sample), so there is a risk that the objective can contact the coverslip if the rough focus is not properly set. If a digital camera is installed, representative images should be captured and systematically named to provide a record of the analysis.
The analysis of opaque samples by reflected light microscopy is similar, but instead of preparing a slide, the specimen is mounted on a support that will prevent it from moving during analysis.
Care Of A Light Microscope
The most common form of damage to a light microscope is contamination or scratches on the sensitive precision lenses and other optical components. This can be avoided by:
- Placing a dust cover over the microscope when not in use.
- Avoiding touching any of the optical components with bare hands, and using non-powdered gloves whenever there is a potential for contact.
- Placing the objective canisters and other optical components in the cases provided by the manufacturer when not in use.
- Not removing condensers, objectives, and oculars from the microscope except when needed
- Periodically checking the distance between the objective and the slide to avoid contact while focusing
- Cleaning it only with lens cloths or paper meant for this purpose when any optical component becomes dirty. When cleaning, hold the lens paper flat and parallel to the lens surface.
Comparisons With Other Microscopes
- The main strength of the light microscope is its simplicity of use, and versatility to be able to analyze many different types of materials.
- It is affordable and easy to use as an imaging tool for tissue sections and microorganisms.
- It is lightweight and small, making it portable for the researcher.
- The availability of additional components to adapt the microscope to specific purposes further increases its versatility.
- The main limitation of the optical microscope is the minimum length scales that can be visualized. The limitation is due to diffraction, which becomes a dominant effect at length scales of roughly the wavelength of visible light, about 0.4 to 0.7 microns (1 micron = 10-6 m). If structures smaller than 1 micron need to be analyzed, different types of microscopes are required, for example, electron microscopes (scanning electron or transmission electron microscopes) or scanning probe microscopes (atomic force or scanning tunneling types). However, these microscopes tend to be much more specialized and expensive.
- The internal processes in living cells are difficult to observe without dyes under the light microscope.