The human eye is sensitive to the narrow band of electromagnetic radiation called the visible light spectrum, the source of color. The visible light of the sun appears to be colorless, or white. White light is not the light of a single color, or frequency. It is made up of many color frequencies.
Isaac Newton demonstrated how white light works. Newton passed sunlight through a glass prism to separate the colors into a rainbow spectrum. He then passed sunlight through a second glass prism and combined the two rainbows. The combination produced white light.
Red, green, and blue are the primary colors. An equal mix of all three creates white light, while a mix of varying degrees creates virtually any color. When mixed in equal proportions, red and blue produce magenta, red and green produce yellow, and green and blue produce cyan.
Cyan, yellow and magenta are called the complementary colors. They are also called the primary subtractive colors because each can be formed by subtracting one of the primary additives (red, green, and blue) from white light. For example, yellow light is produced when blue light is removed from white light, magenta is produced when green is removed, and cyan is produced when red is removed. The color observed by subtracting a primary color from white light results because the brain adds together the colors that are left to produce the respective complementary or subtractive color.
White light can be made by other combinations other than mixing all colors together, such as yellow with blue, magenta with green, cyan with red, and by mixing all of the colors together. Computer monitors are often called RGB monitors because they produce colors by mixing various combinations of red, green and blue. The printing industry relies on a 4-color separation process using cyan, magenta, yellow, and black dyes to reproduce artwork and photographs.
Colors are also created when some of the frequencies of light are absorbed. The absorbed colors are the ones not seen. The colors seen are the ones that are reflected back to the eye. Absorption is how paints and dies work. The paint or dye molecules absorb specific frequencies reflect other frequencies. The reflected frequency (or frequencies) is perceived as the color of the object. Another example is the color of leaves. The leaves of green plants contain a pigment called chlorophyll, which absorbs the blue and red colors of the spectrum and reflects the green.
Pigments and dyes are responsible for most of the color humans see. Eyes, skin, and hair contain natural protein pigments that reflect colors (including colors used in facial makeup and hair dyes). Books, magazines, signs, and billboards are printed with colored inks that create colors through the process of color subtraction.
Cars, airplanes and houses are coated with paints containing a variety of pigments. The concept of color subtraction is responsible for most of the color produced by the objects just described. For many years, artists and printers have searched for substances containing dyes and pigments that are particularly good at subtracting specific colors.
When a light wave hits an object, what happens to it depends on the energy of the light wave, the natural frequency at which electrons vibrate in the material and the strength with which the atoms in the material hold on to their electrons. The waves can be reflected, scattered, absorbed, refracted, or pass through an object. More than one of these possibilities can happen at once.
If the frequency or energy of the incoming light wave is much higher or much lower than the frequency needed to make the electrons in the material vibrate, then the electrons will not capture the energy of the light. The wave will pass through the material unchanged. As a result, the material will be transparent to that frequency of light. Most materials are transparent to some frequencies, but not to others. High frequency light, such as gamma rays and X-rays, will pass through ordinary glass, but lower frequency ultraviolet and infrared light will not.
In absorption, the frequency of the incoming light wave is at or near the vibration frequency of the electrons in the material. The electrons take in the energy of the light wave and start to vibrate. What happens next depends upon how tightly the atoms hold on to their electrons.
Absorption occurs when the electrons are held tightly, and they pass the vibrations along to the nuclei of the atoms. This makes the atoms speed up, collide with other atoms in the material, and then give up as heat the energy they acquired from the vibrations. The absorption of light makes an object dark or opaque to the frequency of the incoming wave. Wood is opaque to visible light. Some materials are opaque to some frequencies of light, but transparent to others. Glass is opaque to ultraviolet light, but transparent to visible light.
The atoms in some materials hold on to their electrons loosely. In other words, the materials contain many free electrons that can jump readily from one atom to another within the material. When the electrons in this type of material absorb energy from an incoming light wave, they do not pass that energy on to other atoms.
The energized electrons merely vibrate and then send the energy back out of the object as a light wave with the same frequency as the incoming wave. The overall effect is that the light wave does not penetrate deeply into the material. In most metals, electrons are held loosely, and are free to move around, so these metals reflect visible light and appear to be shiny. The electrons in glass have some freedom, though not as much as in metals. To a lesser degree, glass reflects light and appears to be shiny, as well.
A reflected wave always comes off the surface of a material at an angle equal to the angle at which the incoming wave hit the surface. In physics, this is called the Law of Reflectance. The Law of Reflectance states: “the angle of incidence equals the angle of reflection.” A mirror demonstrates this law. For instance, when a person looks at their image in a mirror, the colors in a mirror are the same as the colors on the person.
When light hits a rough surface, it scatters. Incoming light waves get reflected at all sorts of angles. The earth’s atmosphere acts as a rough surface. It contains molecules of many different sizes, including nitrogen, oxygen, water vapor, dust and a variety of pollutants. The mix of molecules scatters the higher energy light waves like blue light and, in part, explains why the sky is blue. Of course, the colors we see are also a function of the sensitivity of our eyes and how our brain processes color.
Refraction occurs when the energy of an incoming light wave matches the natural vibration frequency of the electrons in a material. The light wave penetrates deeply into the material, and causes small vibrations in the electrons. The electrons pass these vibrations on to the atoms in the material, and they send out light waves of the same frequency as the incoming wave.
The part of the wave inside the material slows down, while the part of the wave outside the object maintains its original frequency and speed. This has the effect of bending the portion of the wave inside the object toward what is called the normal line, an imaginary straight line that runs perpendicular to the surface of the object. The deviation from the normal line of the light inside the object will be less than the deviation of the light before it entered the object. The amount of bending, or angle of refraction, of the light wave depends on how much the material slows down the light.
Diamonds glitter because of how much they slow down incoming light. Light of different frequencies, or energies, will bend at slightly different angles. For example, in comparing violet light and red light when they enter a glass prism, violet light has more energy so it takes longer to interact with the prism. Because it is slowed down more than a wave of red light, it will bend more. Refraction explains the order of colors in a rainbow. It also explains why rainbows can be seen in diamonds. Soap bubbles and oil spills also produce rainbows.
When light waves pass through an object with two reflective surfaces, parts of the light waves are reflected from the top surface, while other parts of the light pass through the film and are reflected from the bottom surface. Because the parts of the waves that penetrate the film interact with the film longer, they get knocked out of sync with the parts of the waves reflected by the top surface. This is called being out of phase.
When the two sets of waves strike the photoreceptors in the eyes, they interfere with each other. Interference occurs when waves add together or subtract from each other and so form a new wave of a different frequency (color). When white light shines on a film with two reflective surfaces, the various reflected waves interfere with each other to form rainbow fringes. The fringes change colors when the angle of sight changes.
An object can directly emit light waves in the frequency of the observed color, or an object can absorb all other frequencies, reflecting back only the light wave, or combination of light waves, that appears as the observed color.
To see a yellow object, either the object is directly emitting light waves in the yellow frequency, or it is absorbing the blue part of the spectrum and reflecting red and green. When combined, red and green are perceived as yellow.
There are natural sources of light, like the sun, moon and stars, and artificial light born from such sources as room lights, flashlights, and car headlights. These sources of light utilize a wide wavelength spectrum. To narrow the wavelength range for specific applications that require a selected region of color or frequency, specialized filters are used that transmit some wavelengths and selectively absorb, reflect, refract, or diffract others
The concept of color temperature is of critical importance in photography and digital imaging, regardless of whether the image capture device is a camera, microscope, or telescope. A lack of proper color temperature balance between the microscope light source and the film emulsion or image sensor is the most common reason for unexpected color shifts in photomicrography and digital imaging.
If the color temperature of the light source is too low for the film, photomicrographs will have an overall yellowish or reddish cast and will appear warm. On the other hand, when the color temperature of the light source is too high for the film, photomicrographs will have a blue cast and will appear cool. The degree of mismatch will determine the extent of these color shifts, with large discrepancies leading to extremes in color variations.
Perhaps the best example is daylight film used in a microscope equipped with a tungsten-halogen illumination source without the benefit of color balancing filters. In this case, the photomicrographs will have a quite large color shift towards warmer reddish and yellowish hues. As problematic as these color shifts may seem, they are always easily corrected by the proper use of conversion and light balancing filters.
The color temperature model is based on the relationship between the temperature of a theoretical standardized material, known as a black body radiator, and the energy distribution of its emitted light as the radiator is brought from absolute zero to increasingly higher temperatures.
As the name implies, black body radiators completely absorb all radiation, without any transmission or reflection, and then re-emit all incident energy in the form of a continuous spectrum of light representing all frequencies in the electromagnetic spectrum. Although the black body radiator does not actually exist, many metals behave in a manner very similar to a theoretical radiator.
The overall color of a digital image captured with an optical microscope is dependent not only upon the spectrum of visible light wavelengths transmitted through or reflected by the specimen, but also on the spectral content of the illuminator. In color digital camera systems that employ either charge-coupled device (CCD) or complementary metal oxide semiconductor (CMOS) image sensors, white and/or black balance (baseline) adjustment is often necessary in order to produce acceptable color quality in digital images.
A majority of the common natural and artificial light sources emit a broad range of wavelengths that cover the entire visible light spectrum, with some extending into the ultraviolet and infrared regions as well. For simple lighting applications, such as interior room lights, flashlights, spot and automobile headlights, and a host of other consumer, business, and technical applications, the wide wavelength spectrum is acceptable and quite useful.
However, in many cases it is desirable to narrow the wavelength range of light for specific applications that require a selected region of color or frequency. This task can be easily accomplished through the use of specialized filters that transmit some wavelengths and selectively absorb, reflect, refract, or diffract unwanted wavelengths.
Filters are constructed in a wide variety of shapes and physical dimensions, and can be employed to remove or pass wavelength bands ranging in size from hundreds of nanometers down to a single wavelength. In other words, the amount of light excluded or limited by filters can be as narrow as a small band of wavelengths or as wide as the entire visible spectrum.
Many filters work by absorbing light, while others reflect unwanted light, but pass a selected region of wavelengths. The color temperature of light can be fine-tuned with filters to produce a spectrum of light having the characteristics of bright daylight, the evening sky, indoor tungsten illumination, or some variation in between.
Filters are useful for adjusting the contrast of colored regions as they are represented in black and white photography or to add special effects in color photography. Specialized dichroic filters can be used to polarize light, while heat-absorbing filters can limit infrared wavelengths (and heat), allowing only visible light to pass through.
Harmful ultraviolet rays can be exclusively removed from visible light by filters, or the intensity of all wavelengths (ultraviolet, visible, and infrared) can be reduced to specific ranges by neutral density filters. The most sophisticated filters operate by the principles of interference and can be adjusted to pass narrow bands (or even a single wavelength) of light while reflecting all others in a specific direction.
Photography through the microscope is complicated by a wide spectrum of unexpected color shifts and changes that affect how the image is rendered on the film emulsion or electronic image capturing device. These unexpected imaging results are caused by a number of factors ranging from incorrect color balance between the light source and the film emulsion to optical artifacts such as aberration and lamp voltage fluctuations.
A wide spectrum of filters is available to assist the microscopist in achieving the highest quality images in terms of color balance and saturation. These include color compensating and conversion filters, neutral density filters, didymium filters, filters to block ultraviolet light, and heat-absorbing filters.
In black and white photography through the microscope, filters are used primarily to control contrast in the final image captured either on film or with a CCD digital camera system. Specimens that are highly differentiated with respect to colored elements from biological stains are translated into shades of gray on black & white film and will often appear to have equal brightness. When this occurs, important specimen details may be lost through a lack of contrast. Filtration techniques for black and white film are significantly different from those employed in color photomicrography.
A wide variety of synthetic and naturally occurring biological dyes are available to the microscopist for selective staining of intracellular organelles in cells and tissues. Biological stains dramatically improve specimen contrast in brightfield illumination, and have been utilized for many years in histological preparations targeted at studies in anatomy, pathology, physiology, and similar disciplines.