The Structure and Function of a Light Microscope

We may never know who made the first microscope. It could have been a spectacle maker, or a telescope maker who discovered that a telescope reversed magnified objects. However, two of the greatest pioneers in microscopy were Robert Hooke who used both a compound microscope and a single lens microscope around 1665 in England. In a compound microscope the magnification is the result of the eyepiece magnification multiplied by the objective magnification. The other microscopist was Antonie van Leeuwenhoek, a Dutch fabric merchant who created single lens microscopes that were able to magnify approximately 400X. Antonie discovered bacteria and protozoa around 1674 and he is known as the Father of Microbiology. Robert Hooke is known for having discovered cells in cork and gave “cells” their name. To learn more about the interesting history of the microscope see Brian J. Ford (1973) “The Revealing Lens - Mankind and the Microscope”.

An optical microscope until recently could resolve objects separated by about 0.2 microns where a micron = 0.001 mm. Super resolution optical microscopes were developed in the early 1990s and can visualize molecules down to about ~250 nm (nm = 0.000001 mm). Bacteria (prokaryotes cells without a nucleus) are about 1 micron and larger, whereas eukaryotic cells (cells with a nucleus) are about 6 microns in size or larger. The microscope is significant because it allowed scientists to identify contagion (bacteria) that could infect and harm humankind. Light microscopes are routinely used to examine blood samples and the structure of plant and animal cells. Below is an image of a large bacterium found in ponds magnified 630X.

The purpose of this article is to describe the structure and function of a modern light microscope and its’ components. For those new to microscopes they might appear complicated. However, only a few hours of instruction will reveal how to use one. An entry level microscope costs about $150. A super resolution microscope can cost hundreds of thousands. There are three common types of light microscope used in biology that magnify objects: stereo microscopes, compound microscopes and inverted microscopes.

First you need to be able to identify the major parts of a microscope and then learn what their function is. The parts of a polarizing light microscope are shown in the labelled diagram Fig. 3.

A polarizing microscope includes the use of two polarizing filters and an accessory filter (wave plate or retardation filter). Polarizing filters can be added to any light microscope allowing some specimens to be viewed in color (crystals, hair, and plants) without staining them. A polarizing microscope can also be used to see subjects by white light called bright field microscopy. Learning how to use a light microscope is easier when someone shows you, even children can learn how to use one.

Cells and tissues viewed by a light microscope often appear translucent because of their high water content. One solution is to stain tissues with various dyes. Unfortunately this requires thin sections or isolated cells that are fixed and stained before being viewed. Processing involves treating the cells with a fixative (e.g. alcohol or formalin), embedding the tissues in paraffin and cutting thin sections which are mounted on microscope slides. Sometimes thin sections can be made simply with just a razor blade.

Scientists have developed optical techniques that can add color and contrast to cells. These techniques include dark-field illumination, phase contrast, fluorescence, polarized light, differential interference contrast and others. When crystal-like subjects made up of more than one refractive index are observed with polarized light they display different colors which can reveal information about the molecular properties of the substance. The crystals may also form beautiful abstract patterns.

Parts of an Optical Microscope

Body

The body of a microscope consists of a head, arm and base. While most light microscopes can be carried by using the arm, heavier ones may need to be disassembled first. The base supports the microscope and should be placed on a secure table or bench. To prevent vibration place the microscope on top of a rubber mat. I use 1⁄4 inch thick rubber floor tiling from the hardware store cut to size with a sharp knife. The table, desk or bench that the microscope is placed onto should be rigid and minimize microscope vibration. Ideally a microscope room should allow the room to be darkened for viewing, especially for fluorescence subjects. The low light makes viewing more comfortable and easier to see faint subjects like bacteria in a blood sample (e.g. Lyme disease bacteria Borrelia burgdorferi).

Base

The microscope base usually contains an illuminator that can be tungsten, light emitting diode (LED) or even a mirror to use reflected light from a window or other light source. The base supports the body of the microscope and the stage. For photomicrography a bright light with a variable power source e.g. 100 Watt tungsten or a LED light emitting diode is recommended. To capture fast moving organisms like ciliates some investigators connect a flash to the microscope and camera. All light sources should allow you to vary the intensity so viewing is comfortable. Some light sources e.g. LED have a color temperature of about 5000 K and appear paper-white. A 3200 K light source makes the background appear yellow. Most tungsten bulbs give off a yellow light and can be color corrected by using a blue filter. A bright light source is important if you intend to use different types of illumination like polarizing, phase contrast or dark-field microscopy. The light source on the base usually has an iris diaphragm which helps you center the condenser, and provide optimum lighting for the microscope and photography. For optimum viewing and photography the microscope optics should be aligned according to Köhler illumination (A.K.A. Koehler, Berdan, 2020).

Stage

Above the base is a stage where you place your microscope slides with samples. Most stages are rectangular in shape with hole in them to allow light from the illuminator to shine through the specimen. Stages on polarizing microscopes are round so they may be rotated. The stage functions to support the microscope slide and sometimes other accessories. A microscope stage can have clips to hold microscope slides in place or a mechanical stage that grips the slide and allows it to be moved smoothly. A mechanical stage can be used to relocate objects, or precisely overlap parts of a specimen for making panoramic photos. A low cost mechanical stage costs about $25 while better ones cost several hundred dollars. Modern microscopes have a stage that is parallel with the table it sits on and this aids the viewing of fluids like pond water or cells in tissue culture. Microscopes that tilt are sometimes preferred for viewing prepared slides or using window light when there is no built in light source.

Microscope Slides

A standard microscope glass slide is 1 x 3 inches (25 x 76 mm) and usually 1-1.2 mm thick, though thickness varies between manufacturers. The correct thickness of a cover glass (A.K.A coverslip) is 0.17 mm (No. 1.5) as most objectives are optically corrected for this thickness. The number 0.17 is engraved on the objective barrel (Berdan, 2023). Sometimes investigators use a thinner No. 1 cover glass because it is the thickness of the cover glass and fluid above the specimen that is critical for getting the sharpest images. Some objectives have a correction ring on the barrel that can be rotated for use with different cover glass thicknesses, immersed directly into a solution, or are designed for optimum performance without a cover glass.

Condensers

Below a microscope stage is a condenser whose purpose is to focus light onto the specimen, and it can influence depth of field and contrast of the specimen image. The condenser may hold filters and it has an iris diaphragm to control the width of the illuminating cone of light. Condensers are located above the light source and under the sample in an upright microscope. On inverted microscopes the condenser is above the stage but below the light source (see Fig. 10).

The distance between the specimen and condenser is controlled by a rack and pinion focus knob. The distance of the condenser from the slide can be varied in order to alter contrast and depth of field. Most of the time the condenser is positioned close to the slide specimen for maximum resolution and some
condensers also require oil immersion fluid to be applied between the slide and condenser. Oil immersion objectives always require immersion oil between the top of the cover glass and on the objective lens. When an iris diaphragm is open too wide on a condenser, stray light can cause glare and lower contrast of the specimen.

There are several different types of condensers:

1. With a simple two lens Abbe chromatic condenser there is no attempt to correct the light source for changes in color introduced by the glass called spherical and chromatic aberration. These aberrations can introduce color fringing in the subject like purple and red. A chromatic condenser usually contains two to four lens elements and has a numerical aperture ranging from 0.95 to 1.4 to support objectives from 4X to 100X. Numerical aperture (NA) is related to the angle of the light cone which is formed between a point on the specimen and in front of the lens of an objective or condenser. Higher NA condensers and objectives are better and allow more light to enter. The common Abbe condenser is suitable for routine observations with modest numerical aperture objectives and magnifications and it works well with long working distance objectives.
   
   
2. An Aplanatic condenser is corrected for spherical aberration. When light travels through a lens not all the light rays come into the same focus in the central part of the lens. Rays passing through the lens close to its center are focused farther away than rays passing through a zone near its rim causing some rays to be out of focus resulting in spherical aberration. Aplanatic condensers are often used with a green filter for black and white photography so color fringing isn’t visible. In practice, color fringing around specimens can also be eliminated using software like Adobe Photoshop.
   
   
3. A compound Aplanatic-achromatic condenser is corrected for both spherical and chromatic aberrations caused by lenses. Chromatic aberration is the result of different colors (wave lengths) not focusing at the same point by an uncorrected lens.  Aplanatic-achromatic condensers contain more lens elements in order to correct both kinds of spherical and chromatic aberration.
   
   
4. Special condensers are available for use with low magnification objectives like 1 and 2X and provide a broader light source. Alternatively some condensers have a flip-out top lens for use with low power objectives (e.g. Motic BA-310 polarizing microscope shown above in Fig. 3). Another solution is to use a diffusion filter over the light source when using low power objectives in order to spread the light beam. 

The maximum resolution that can be obtained is when the condenser is very close to the bottom of the microscope slide. Condensers with a NA above 0.95 perform best when oil immersion fluid is used between an oil immersion objective and cover glass and also between the bottom of the slide and condenser lens. The immersion fluid has a refractive index similar to glass. This ensures the maximum NA of the objective is supported. The condenser NA should be equal to or greater than the objective NA since the final resolution achieved depends on both. In routine analysis oil immersion fluid is not always used between the condenser and slide because of the time required to clean off the oil, but oil immersion fluid should always be used between an oil immersion objective and the cover glass.

According to Abramowitz and Davidson the thickness of the microscope slide is crucial for the certain condensers, like cover glass thickness is to the objective. Most commercial microscope slides are about 1 mm thick but vary between 0.95 to 1.2 mm. A slide thickness of 1.2 mm is too thick to be used with high NA condensers as they tend to have a short working distance. These authors recommend glass microscope slides should be 1.0 +\- 0.05 mm thick.

Objective turret

Above the microscope stage is a rotating turret which may hold several objectives. The turret allows microscopists to rotate different magnification objectives in place. The objectives have magnifications varying from 1 to 200X. If the objectives are parfocal on a microscope when you change magnification sequentially (e.g. 4x to 10x to 40x to 100x), it should only require a slight turn of the fine focus knob with each increase or decrease to get the image in focus. If the objectives are not parfocal (this happens if objectives are from different manufacturers or have different design finite vs infinity) rotating different objectives into place may damage the coverslip and/or objective lens if you are not careful. It’s always a good idea to look at the distance between the objective and slide to make sure they won’t collide, this is especially important with the highest power objectives. The turret that holds the objectives can accept objectives of a certain diameter. Standard objective thread size according to the Royal Microscopical Society is 20.32 mm but some microscope manufacturers use different thread sizes. Zeiss for instance uses a M27 x 0.75 thread size on its’ Axioscopes. Objective adapters are sold so that smaller threaded objectives will fit into microscope turrets. Most microscope turrets hold 3 to 6 objectives. 

Finite Mechanical tube length Microscopes vs Infinity Microscopes

Mechanical tube length of a microscope is the distance between the objective rear focal plane and the intermediate image at the fixed diaphragm of the eyepiece – roughly back of objective to the back of the eyepiece. The Royal Microscopical Society suggested the tube length be standardized at 160 mm and inscribed on the objective barrels. While most manufacturers designed their objectives for this finite tube length some decided to use different tube lengths (e.g. Leitz used 170 mm). When objectives and tube lengths mismatch image quality often suffers because of spherical aberrations. Similarly when the tube length is altered by the addition of accessories (Wollaston prisms, polarizers, epi-illumination) the tube length becomes greater and may introduce aberrations and additional expensive optical elements may be needed to correct the image.

In the 1930’s Carl Reichert experimented with the concept of infinity space microscopes but these designs only became popular in the 1980’s. Correction for optical aberrations in infinity microscope designs is accomplished either through the addition of a tube lens in the microscope light path, or in the objective design or both. Unfortunately objectives designed for infinity microscopes are usually not interchangeable and are not parfocal on different manufacturer’s microscopes. Also infinity objectives when used on a finite mechanical tube length microscope often have greater spherical aberration.

An infinity designed microscopes allows auxillary components to be added without introducing spherical aberration, ghost images or altering the magnification of the image. Infinity microscopes are mainly sold with research grade and industrial microscopes. Most manufacturers now design their microscopes to support infinity corrected objectives. Beware that infinity objectives may not be interchangeable between different manufacture’s and are not generally useable on finite tube length microscopes.

Infinity microscopes and objectives became popular in the 1980’s, but both finite and infinity objectives are sold today. Microscopes that have a finite focal length can be used with some older objectives whereas infinity objectives are designed for use on the newer infinity microscopes. Both types of objectives provide excellent images when used with the correct microscope designs. Infinity objectives have the ∞ symbol engraved on their barrels and many finite tube length microscopes have the tube length indicated on the objective barrels. In general mixing finite and infinity objectives is not a good idea and will often result in reduced image quality. Infinity objectives from different manufacturers may not work on different manufacturers infinity microscopes or be parfocal. Good results can be achieved with either finite or infinity objectives when used on the correct type of microscope. If you are using or considering adding additional components in the microscope light path, a microscope designed for infinity optics is recommended.

Objectives Achromats, Plan-Achromats and Apochromats

Objectives vary greatly in quality, price and type. Achromat objectives are the most economical, followed by Plan Achromats and finally Apochromat objectives. With Achromats only the center of the viewing field is sharp and in focus but may be satisfactory for viewing. Plan Achromats display a flat field of view with images sharp to the edge of the lens and are recommended for photomicrography. Plan Achromats are corrected for two colors but may display color fringing around specimens. In practice, the color fringes can be removed from images using software after photomicrography. The third type of objective is an Apochromat which eliminates most chromatic aberration, (corrected for three colors red, green, and blue), and are corrected spherically for two or three wavelengths. The Apochromats have higher numerical apertures. The NA number is engraved on the objective and those objectives with higher NA are significantly brighter and more expensive.  High NA objectives are particularly useful for examining fluorescent specimens.

Head

The microscope head contains prisms that split the light in binocular scopes and may contain a retractable prism in a trinocular head to direct light to a camera. Some microscope heads also include additional prisms and accessories that allow you to achieve additional magnification of 1.6 or 2X. In general a binocular head permits more comfortable viewing if the microscope is used for hours at a time.

Some trinocular prisms allow 100% of the light to be directed to the camera, others split the light 80/20 with 80% of the light going to the camera and 20% to the eyepieces during photography. The 80/20% prism has one advantage in that it permits a viewer to continue watching a moving specimen while taking pictures or movies. The alternative is to watch the specimen on a computer screen connected to the camera. Usually the monitor view only shows part of the field of microscope view and it may be difficult to follow moving specimens when taking movies at high magnification.

Eyepieces

Eyepieces are inserted into tubes attached to the microscope head. Different microscopes may use different diameter tubes so be sure to measure the eyepiece tube diameter on your microscope when purchasing new eyepieces. The total magnification is equal to the eyepiece magnification times the objective magnification. The eyepieces are usually 10X, but 5X, 15X and 20X eyepieces are readily available. For photomicrography usually lower magnification flat field eyepieces are used (1.5, 3.3 or 5X). These eyepieces are corrected for any curvature in the image. Some eyepieces are corrected and designed to match specific objectives. The lowest price eyepieces have a small diameter hole that you peer through. Wide field eyepieces are more comfortable, provide a wider-field of view and maybe used by persons with eye-glasses. Some eyepieces or microscope heads have rotating collars which allows the investigator to adjust the diopter settings on each eyepiece. The diopter correction will not however, correct the viewer’s astigmatism, for this the viewer should select high eye point eyepieces and wear glasses. Most binocular eyepiece holders allow you to change the interpupillary distance for greater comfort. Some people prefer viewing through monocular microscopes and may find the binocular heads difficult to use. The optical lenses: objectives, eyepieces and the condenser are the most important components of a light microscope.

Coarse and Fine Focus Controls

Most good microscopes (except stereomicroscopes) have both a coarse and a fine focus knob. The course focus knob allows you to quickly focus on a specimen and the fine focus knob is used mainly at higher magnification. Depth of field becomes very small at higher magnification and fine focus is frequently used. The fine focus knob often has graduations marked on them to indicate the depth when you change focus. The depth of the fine focus knob is usually calibrated to 1 or 2 micron increments so it can be used to calculate the height and depth of a specimen by focusing on the top and then the bottom. The coarse focus knob may have a lock so that you can prevent the objective from crashing into the slide accidently. On some microscopes there may be a way to tighten or adjust the tension of the coarse focus knob (e.g. focus torque adjustment Fig. 3). On other microscopes turning the focus knobs counterclockwise may loosen them but check with your microscope manual first. On many microscopes the focus knobs move the stage up or down and on others it will move the objectives up or down. Finally, note that most microscope manuals are on the Internet as a PDF, including those of older microscopes.

Bertrand Lens

A Bertrand lens, also called as a conoscopic lens, is an important component of polarizing microscopes. A Bertrand lens is inserted just above the objective lens and focuses on the back focal plane of the objective. The Bertrand lens is used to observe interference patterns created when crystals are illuminated with polarized light. These patterns provide information about the crystallographic features of the sample including its birefringence, optic sign, and composition. To learn how to interpret interference patterns see books on optical minerology.

Polarizing microscopes also use objectives designed for polarized light observation and they are distinguished from ordinary objectives with the inscription P, PO, or Pol on the barrel. They use strain free glass in their lenses. Other objective types may work, but the background will only be completely black with crossed polarizers using objectives designed for polarized light microscopy.

Stereo Microscopes

Stereo microscopes complement light microscopes and most biologists own both types. They are often referred to as a dissecting microscope. They are easier to use than a light microscope and offer plenty of working space above the specimen unlike compound microscopes. This allows investigators to interact with the specimen. Their main advantages are that they can be used to view opaque specimens, and the specimens require little or no preparation. Inside many stereoscopes head there are two side by side lenses mounted at an angle to each other which permits viewing the subject in three dimensions.

Some stereoscopes come with a trinocular head in order to attach a camera or monitor. To test a stereo scope for clarity use a specimen (e.g. penny), also tap on the stand to see how much vibration there might be. The other factor to consider is the weight if you intend to transport the stereoscope. Some stereoscopes come with lights in the base and/or built into the head. Many investigators prefer using fiber optic lighting so they can change the direction of the light source or add the ability to use UV light. Simple stereoscopes use fixed magnification lenses to produce 4, 10, 20 and 40X etc. while others offer the ability to zoom from 1 to 50X or more. Stereoscopes generally only offer coarse focusing knobs. For fine focus I use a micrometer table for vertical adjustments of the specimen in small increments on the stage (see Fig 11). To learn more about stereomicroscopes see (Berdan, 2021).

Inverted microscopes

Inverted microscopes are often used to examine cells grown in culture or for viewing plankton samples. These microscopes can do anything a compound light microscope can. They are often chosen because they are easier to attach accessories to the stage such as micromanipulators and offer more working space on the stage. Super resolution microscopes are often modified inverted light microscopes. These microscopes are heavy and are often placed on special anti-vibration air tables. Inverted microscopes are used in fertility clinics or for electrophysiological recording from cells. Smaller inverted microscopes are used routinely to assess the growth of cells grown in culture and come with phase contrast microscopy. These microscopes are available as both finite and infinity models.

Summary

Optical microscopes are widely used in medicine, research, pharmaceutical industries, forensic labs, manufacturing, engineering, metallurgy, geology, surgery, chemistry, botany, zoology, veterinary science, museums, fisheries, jewelers, environmental science, and breweries. There are two main types of compound light microscopes one uses a finite mechanical tube length and the other uses infinity optics with tube lens that allows additional accessories to be inserted into the light path without degrading the image. Stereo microscopes and inverted microscopes complement the use of compound microscopes.  Knowing how to use a light microscope is an important skill. A microscope can also play an important role in education and encourages experiential learning.

References

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