Bright fieldmicroscope application

Hot spots are highly reflective portions of a field within a more diffuse reflecting field. In Figure 8, a polarizer is placed in front of the lens of a camera as well as over the light source illuminating the scene to reduce hot spots.

Edmund Optics® offers a wide variety of polarizers, waveplates, polarizing beamsplitters, and other polarization-manipulating optics.

Figure 11 shows a photo taken of Edmund Optics Headquarters and the variation in the color of the sky, grass, and foliage from using or not using a polarizer in front of a camera lens. Because electrons in air molecules scatter light in many directions, the appearance of the sky without a polarizer is a lighter shade of blue, as seen in the left image (without polarizer). Additionally, the surface of leaves of trees and on blades of grass are very slightly reflective. Using a polarizer filters out some of the light reflected from these surfaces, darkening the perceived color of these surfaces.

With a single ocular, there is nothing to do with the eyepiece except to keep it clean. With a binocular microscope (preferred) you need to adjust the eyepiece separation just like you do a pair of binoculars. Binocular vision is much more sensitive to light and detail than monocular vision, so if you have a binocular microscope, take advantage of it.

Light is an electromagnetic wave, and the electric field of this wave oscillates perpendicularly to the direction of propagation. Light is called unpolarized if the direction of this electric field fluctuates randomly in time. Many common light sources such as sunlight, halogen lighting, LED spotlights, and incandescent bulbs produce unpolarized light. If the direction of the electric field of light is well defined, it is called polarized light. The most common source of polarized light is a laser.

The angular difference between the axes of polarization of the two polarizers is directly related to the amount of overall light attenuation of the set of polarizers. By changing the angle offset, the optical density of the polarizer set can be varied, achieving a similar effect to using a neutral density filter. This ensures that the overall field is evenly illuminated.

Bright field microscopyprinciple

The bright field microscope is best known to students and is most likely to be found in a classroom. Better equipped classrooms and labs may have dark field and/or phase contrast optics. Differential interference contrast, Nomarski, Hoffman modulation contrast and variations produce considerable depth of resolution and a three dimensional effect. Fluorescence and confocal microscopes are specialized instruments, used for research, clinical, and industrial applications.

Reflective polarizers transmit the desired polarization while reflecting the rest. Wire grid polarizers are a common example of this, consisting of many thin wires arranged parallel to each other. The light that is polarized along these wires is reflected, while light that is polarized perpendicular to these wires is transmitted. Other reflective polarizers use Brewster’s angle. Brewster’s angle is a specific angle of incidence under which only s-polarized light is reflected. The reflected beam is s-polarized and the transmitted beam becomes partially p-polarized.

In Figure 5, a linear polarizer was placed in front of the lens in a machine vision system to remove obfuscating glare such that an electronic chip could be clearly seen. The left image (without polarizer) shows randomly polarized light scattering off of the many glass surfaces between the object and the camera sensor. Much of the chip is obscured by Fresnel reflection of the unpolarized light. The image on the right (with polarizer) shows the chip without glare obscuring any of the object details, allowing the chip to be viewed, analyzed, and measured without obstruction.

Darkfieldmicroscope

A disadvantage of having to rely solely on an aperture diaphragm for contrast is that beyond an optimum point the more contrast you produce the more you distort the image. With a small, unstained, unpigmented specimen, you are usually past optimum contrast when you begin to see the image.

Move up in magnification by steps. Each time you go to a higher power objective, re-focus and re-center the specimen. Higher magnification lenses must be physically closer to the specimen itself, which poses the risk of jamming the objective into the specimen. Be very cautious when focusing. By the way, good quality sets of lenses are parfocal, that is, when you switch magnifications the specimen remains in focus or close to focused.

A good quality microscope has a built-in illuminator, adjustable condenser with aperture diaphragm (contrast) control, mechanical stage, and binocular eyepiece tube. The condenser is used to focus light on the specimen through an opening in the stage. After passing through the specimen, the light is displayed to the eye with an apparent field that is much larger than the area illuminated. The magnification of the image is simply the objective lens magnification (usually stamped on the lens body) times the ocular magnification.

Bright field microscopyadvantages and disadvantages

Bright field microscopy is best suited to viewing stained or naturally pigmented specimens such as stained prepared slides of tissue sections or living photosynthetic organisms. It is useless for living specimens of bacteria, and inferior for non-photosynthetic protists or metazoans, or unstained cell suspensions or tissue sections. Here is a not-so-complete list of specimens that might be observed using bright-field microscopy, and appropriate magnifications (preferred final magnifications are emphasized).

Advantages ofbright-fieldmicroscope

For linearly polarized light with intensity I0, the intensity transmitted through an ideal polarizer, I, can be described by Malus’ law,

The light microscope, so called because it employs visible light to detect small objects, is probably the most well-known and well-used research tool in biology. Yet, many students and teachers are unaware of the full range of features that are available in light microscopes. Since the cost of an instrument increases with its quality and versatility, the best instruments are, unfortunately, unavailable to most academic programs. However, even the most inexpensive "student" microscopes can provide spectacular views of nature and can enable students to perform some reasonably sophisticated experiments.

By cross-polarizing light with two linear polarizers that are oriented perpendicularly, hot spots can be reduced or eliminated altogether.

Start with the lowest magnification objective lens, to home in on the specimen and/or the part of the specimen you wish to examine. It is rather easy to find and focus on sections of tissues, especially if they are fixed and stained, as with most prepared slides. However it can be very difficult to locate living, minute specimens such as bacteria or unpigmented protists. A suspension of yeast cells makes a good practice specimen for finding difficult objects.

In amorphous solids such as glass and plastic, stress from temperature and pressure profiles in the material imparts localized variations and gradients in the material properties, making the material birefringent and nonhomogeneous. This can be quantified in transparent objects using the photoelastic effect, as stress and its related birefringence can be measured with polarized light methodologies.

It is a lot harder to find something when you have no expectations as to its apprearance. How big is it? Will it be moving? Is it pigmented or stained, and if so what is its color? Where do you expect to find it on a slide? For example, students typically have a lot of trouble finding stained bacteria because with the unaided eye and at low magnifications the stuff looks like dirt. It helps to know that as smears dry down they usually leave rings so that the edge of a smear usually has the densest concentration of cells.

The smallest objects that are considered to be living are the bacteria. The smallest bacteria can be observed and cell shape recognized at a mere 100x magnification. They are invisible in bright field microscopes, though. These pages will describe types of optics that are used to obtain contrast, suggestions for finding specimens and focusing on them, and advice on using measurement devices with a light microscope.

Bigger is not always better. All specimens have three dimensions, and unless a specimen is extremely thin you will be unable to focus with a high magnification objective. The higher the magnification, the harder it is to "chase" a moving specimen.

Other than the compound microscope, a simpler instrument for low magnification use may also be found in the laboratory. The stereo microscope, or dissecting microscope usually has a binocular eyepiece tube, a long working distance, and a range of magnifications typically from 5x to 35 or 40x. Some instruments supply lenses for higher magnifications, but there is no improvement in resolution. Such "false magnification" is rarely worth the expense.

Unpolarized light can be considered a rapidly varying random combination of p- and s-polarized light. An ideal linear polarizer will only transmit one of the two linear polarizations, reducing the initial unpolarized intensity I0 by half,

Bright fieldmicroscope image

Dichroic polarizers absorb a specific polarization of light, transmitting the rest; modern nanoparticle polarizers are dichroic polarizers.

Another characteristic way to see how polarizers reduce reflective glare is by viewing water surfaces. In Figure 7, the surface of the water appears reflective in the left image, obscuring what is below the surface. On the right, however, the rocky debris on the floor of the body of water is much more clearly visible.

The same phenomenon can be seen in the Figure 6. In the left image (without polarizer), unpolarized light from the sun is interacting with the windows of the Edmund Optics building and most of this light is reflecting off the windows. In the right image, a polarizing filter has been applied such that the reflected light, rich in one polarization type, is being blocked from the camera sensor and the photographer, using the other polarization type, can see into the building more easily.

Darkfield microscopy

With a conventional bright field microscope, light from an incandescent source is aimed toward a lens beneath the stage called the condenser, through the specimen, through an objective lens, and to the eye through a second magnifying lens, the ocular or eyepiece. We see objects in the light path because natural pigmentation or stains absorb light differentially, or because they are thick enough to absorb a significant amount of light despite being colorless. A Paramecium should show up fairly well in a bright field microscope, although it will not be easy to see cilia or most organelles. Living bacteria won't show up at all unless the viewer hits the focal plane by luck and distorts the image by using maximum contrast.

Birefringent polarizers rely on the dependence of the refractive index on the polarization of light. Different polarizations will refract at different angles and this can be used to select certain polarizations of light.

Unstressed clear objects between crossed polarizers should yield a completely dark field, however, when internal material stress is present, the localized changes in refractive index rotate the angle of polarization, resulting in transmission variations.

The apparent field of an eyepiece is constant regardless of magnification used. So it follows that when you raise magnification the area of illuminated specimen you see is smaller. Since you are looking at a smaller area, less light reaches the eye, and the image darkens. With a low power objective you may have to cut down on illumination intensity. With a high power you need all the light you can get, especially with less expensive microscopes.

While polarizers select certain polarizations of light, discarding the other polarizations, ideal waveplates modify existing polarizations without attenuating, deviating, or displacing the beam. They do this by retarding (or delaying) one component of polarization with respect to its orthogonal component. To help you determine which waveplate is best for your application, read Understanding Waveplates. Correctly chosen waveplates can convert any polarization state into a new polarization state and are most often used to rotate linear polarization, to convert linearly polarized light to circularly polarized light, or vice versa.

A light source should have a wide dynamic range, to provide high intensity illumination at high magnifications, and lower intensities so that the user can view comfortably at low magnifications. Better microscopes have a built-in illuminator, and the best microscopes have controls over light intensity and shape of the light beam. If your microscope requires an external light source, make sure that the light is aimed toward the middle of the condenser. Adjust illumination so that the field is bright without hurting the eyes.

Ring light guides are popular illumination sources due to their even, diffuse illumination. However, glare or reflection of the ring itself may occur. Polarizing the ring light output and the lens separately can reduce these effects, and bring out surface details as seen in Figure 9.

Understanding and manipulating the polarization of light is crucial for many optical applications. Optical design frequently focuses on the wavelength and intensity of light, while neglecting its polarization. Polarization, however, is an important property of light that affects even those optical systems that do not explicitly measure it. The polarization of light affects the focus of laser beams, influences the cut-off wavelengths of filters, and can be important to prevent unwanted back reflections. It is essential for many metrology applications such as stress analysis in glass or plastic, pharmaceutical ingredient analysis, and biological microscopy. Different polarizations of light can also be absorbed to different degrees by materials, an essential property for LCD screens, 3D movies, and glare-reducing sunglasses.

Polarization control is also very important in the chemical, pharmaceutical, and food and beverage industries. Many important organic chemical compounds, such as active pharmaceutical ingredients or sugars, have multiple orientations. The study of molecules with multiple orientations is called stereochemistry.

Bright field microscopyand darkfield microscopy

One or both of the eyepieces may be a telescoping eyepiece, that is, you can focus it. Since very few people have eyes that are perfectly matched, most of us need to focus one eyepiece to match the other image. Look with the appropriate eye into the fixed eyepiece and focus with the microscope focus knob. Next, look into the adjustable eyepiece (with the other eye of course), and adjust the eyepiece, not the microscope.

The two orthogonal linear polarization states that are most important for reflection and transmission are referred to as p- and s-polarization. P-polarized (from the German parallel) light has an electric field polarized parallel to the plane of incidence, while s-polarized (from the German senkrecht) light is perpendicular to this plane.

A beginner tends to think that the challenge of viewing small objects lies in getting enough magnification. In fact, when it comes to looking at living things the biggest challenges are, in order,

The analyzer only transmits light that has experienced a specimen-induced phase shift and continues to block all the unaffected light from the source which was originally polarized by the polarizer. If the birefringence of the specimen is known, it can then be used to determine the specimen thickness. If the specimen thickness is known, it can be used to deduce the birefringence of the specimen. A convenient chart used for this purpose is known as the Michel-Levy interference color chart in Figure 14.

In a simple polarization microscope system, a linear polarizer is placed in front of a microscope light source, below the specimen stage, to polarize the light entering the system. Another linear polarizer placed above the specimen stage is referred to as an “analyzer,” as this polarizer is rotated to achieve the desired effect when analyzing the sample and while the first polarizer is kept stationary. The analyzer is then rotated such that the polarization planes of the analyzer and polarizer are 90° apart. When this has been achieved, the microscope has a minimum transmission (crossed polarizers); the amount of light transmission will be proportional to the extinction ratio of the polarizer and analyzer.

To adjust and align the microscope, start by reading the manual. If no manual is available, try using these guidelines. If the condenser is focusable, position it with the lens as close to the opening in the stage as you can get it. If the condenser has selectable options, set it to bright field. Start with the aperture diaphragm stopped down (high contrast). You should see the light that comes up through the specimen change brightness as you move the aperture diaphragm lever.

Molecular compounds that have the same type and number of atoms, but different molecular arrangements are called stereoisomers. These stereoisomers are “optically active” and will rotate polarized light in different directions. The amount of rotation is determined by the nature and the concentration of the compound, allowing polarimetry to detect and quantify the concentration of these compounds. This is the premise for identifying which stereoisomer may be present in a sample, which is important because stereoisomers can have vastly different chemical effects. For example, the stereoisomer limonene is the chemical that gives oranges and lemons their characteristic scents.

The lowest power lens is usually 3.5 or 4x, and is used primarily for initially finding specimens. We sometimes call it the scanning lens for that reason. The most frequently used objective lens is the 10x lens, which gives a final magnification of 100x with a 10x ocular lens. For very small protists and for details in prepared slides such as cell organelles or mitotic figures, you will need a higher magnification. Typical high magnification lenses are 40x and 97x or 100x. The latter two magnifications are used exclusively with oil in order to improve resolution.

The cover slip must be up if there is one. High magnification objective lenses can't focus through a thick glass slide; they must be brought close to the specimen, which is why coverslips are so thin. The stage may be equipped with simple clips (less expensive microscopes), or with some type of slide holder. The slide may require manual positioning, or there may be a mechanical stage (preferred) that allows precise positioning without touching the slide.

Many different types of microscopy techniques such as differential interference contrast (DIC) microscopy utilize polarizers to achieve a variety of effects.

Once the analyzer has been aligned perpendicularly to the polarizer, an anisotropic, or birefringent, the specimen is placed on the specimen stage. The specimen rotates the polarized light a designated amount, proportional to the specimen thickness (and thus the optical path distance) and the specimen birefringence, before its light reaches the analyzer.

Where θ is the angle between the incident linear polarization and the polarization axis. We see that for parallel axes, 100% transmission is achieved, while for 90° axes, also known as crossed polarizers, there is 0% transmission. In real-world applications the transmission never reaches exactly 0%, therefore, polarizers are characterized by an extinction ratio, which can be used to determine the actual transmission through two crossed polarizers.

First, think about what you want to do with the microscope. What is the maximum magnification you will need? Are you looking at a stained specimen? How much contrast/resolution do you require? Next, start setting up for viewing.

Students are usually aware of the use of the coarse and fine focus knobs, used to sharpen the image of the specimen. They are frequently unaware of adjustments to the condenser that can affect resolution and contrast. Some condensers are fixed in position, others are focusable, so that the quality of light can be adjusted. Usually the best position for a focusable condenser is as close to the stage as possible. The bright field condenser usually contains an aperture diaphragm, a device that controls the diameter of the light beam coming up through the condenser, so that when the diaphragm is stopped down (nearly closed) the light comes straight up through the center of the condenser lens and contrast is high. When the diaphragm is wide open the image is brighter and contrast is low.

Implementing polarization control can be useful in a variety of imaging applications. Polarizers are placed over a light source, lens, or both, to eliminate glare from light scattering, increase contrast, and eliminate hot spots from reflective objects. This either brings out more intense color or contrast or helps to better identify surface defects or other otherwise hidden structures.

In order to select a specific polarization of light, polarizers are used. Polarizers can be broadly divided into reflective, dichroic, and birefringent polarizers. More detailed information on which type of polarizer is right for your application can be found in our Polarizer Selection Guide.