Contrast is the difference in luminance or color that makes an object (or its representation in an image or display) visible against a background of different luminance or color.[1] The human visual system is more sensitive to contrast than to absolute luminance; thus, we can perceive the world similarly despite significant changes in illumination throughout the day or across different locations.[2]

Illumination: Compound microscopes often have built-in illumination systems, such as a substage light source, condenser, and diaphragm, to provide transmitted light through the specimen. Other microscopes, like dissecting or fluorescence microscopes, may utilize different lighting techniques or illumination configurations.

Crumey[23] showed that Hecht's formula fitted the data very poorly at low light levels, so was not really suitable for modelling stellar visibility. Crumey instead constructed a more accurate and general model applicable to both the Blackwell and Knoll et al data. Crumey's model covers all light levels, from zero background luminance to daylight levels, and instead of parameter-tuning is based on an underlying linearity related to Ricco's law. Crumey used it to model astronomical visibility for targets of arbitrary size, and to study the effects of light pollution.

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Diaphragm or Iris: Many microscopes have a rotating disk under the stage. This diaphragm has different sized holes and is used to vary the intensity and size of the cone of light that is projected upward into the slide. There is no set rule regarding which setting to use for a particular power. Rather, the setting is a function of the transparency of the specimen, the degree of contrast you desire and the particular objective lens in use.

There are many possible definitions of contrast. Some include color; others do not. Russian scientist N. P. Travnikova [d] laments, "Such a multiplicity of notions of contrast is extremely inconvenient. It complicates the solution of many applied problems and makes it difficult to compare the results published by different authors."[6][7]

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A separate study by Knoll et al[25] investigated thresholds for point sources by requiring subjects to vary the brightness of the source to find the level at which it was just visible. A mathematical formula for the resulting threshold curve was proposed by Hecht,[26] with separate branches for scotopic and photopic vision. Hecht's formula was used by Weaver[27] to model the naked-eye visibility of stars. The same formula was used later by Schaefer[28] to model stellar visibility through a telescope.

Eyepiece/Ocular: Compound microscopes commonly have a pair of eyepieces that provide binocular vision. Other microscopes may have a single eyepiece or sometimes no eyepieces at all.

How to Focus Your Microscope: The proper way to focus a microscope is to start with the lowest power objective lens first and while looking from the side, crank the lens down as close to the specimen as possible without touching it. Now, look through the eyepiece lens and focus upward only until the image is sharp. If you can't get it in focus, repeat the process again. Once the image is sharp with the low power lens, you should be able to simply click in the next power lens and do minor adjustments with the focus knob. If your microscope has a fine focus adjustment, turning it a bit should be all that's necessary. Continue with subsequent objective lenses and fine focus each time.

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with I {\displaystyle I} and I b {\displaystyle I_{\mathrm {b} }} representing the luminance of the features and the background, respectively. The measure is also referred to as Weber fraction, since it is the term that is constant in Weber's Law. Weber contrast is commonly used in cases where small features are present on a large uniform background, i.e., where the average luminance is approximately equal to the background luminance.

Contrastexamples

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Objective Lenses: Compound microscopes have multiple objective lenses mounted on a rotating nosepiece, typically with magnifications ranging from 4x to 100x or higher. Other microscopes, such as dissecting or stereo microscopes, usually have fixed magnification lenses.

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The high-frequency cut-off represents the optical limitations of the visual system's ability to resolve detail and is typically about 60 cpd. The high-frequency cut-off is also related to the packing density of the retinal photoreceptor cells: a finer matrix can resolve finer gratings.

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In general, m f {\displaystyle m_{f}} refers to the contrast of the periodic signal f {\displaystyle f} relative to its average value. If m f = 0 {\displaystyle m_{f}=0} , then f {\displaystyle f} has no contrast. If two periodic functions f {\displaystyle f} and g {\displaystyle g} have the same average value, then f {\displaystyle f} has more contrast than g {\displaystyle g} if m f > m g {\displaystyle m_{f}>m_{g}} .[10]

Decreased contrast sensitivity arises from multiple etiologies, including retinal disorders such as age-related macular degeneration (ARMD), amblyopia, lens abnormalities, such as cataract, and by higher-order neural dysfunction, including stroke and Alzheimer's disease.[21] In light of the multitude of etiologies leading to decreased contrast sensitivity, contrast sensitivity tests are useful in the characterization and monitoring of dysfunction, and less helpful in detection of disease.

Campbell and Robson (1968) showed that the human contrast sensitivity function shows a typical band-pass filter shape peaking at around 4 cycles per degree (cpd or cyc/deg), with sensitivity dropping off either side of the peak.[3] This can be observed by changing one's viewing distance from a "sweep grating" (shown below) showing many bars of a sinusoidal grating that go from high to low contrast along the bars, and go from narrow (high spatial frequency) to wide (low spatial frequency) bars across the width of the grating.

Sample Size and Depth of Field: Compound microscopes are designed to observe thin, transparent specimens placed on glass slides. They offer a narrow depth of field, allowing clear focus on one plane at a time. Other microscopes, like stereo or electron microscopes, can accommodate larger specimens or samples with more depth, providing a wider depth of field.

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Michelson contrast[8] (also known as the visibility) is commonly used for patterns where both bright and dark features are equivalent and take up similar fractions of the area (e.g. sine-wave gratings). The Michelson contrast is defined as[6]

Applications: Compound microscopes are commonly used in fields such as biology, medicine, and research, where detailed examination of small structures is required. Other microscopes, such as stereo microscopes, are utilized for examining larger objects or conducting dissections. Electron microscopes are used for high-resolution imaging of nanoscale structures.

For example, in the case of graphical computer displays, contrast depends on the properties of the picture source or file and the properties of the computer display, including its variable settings. For some screens the angle between the screen surface and the observer's line of sight is also important.

Visual acuity is a parameter that is frequently used to assess overall vision. However, diminished contrast sensitivity may cause decreased visual function in spite of normal visual acuity.[12] For example, some individuals with glaucoma may achieve 20/20 vision on acuity exams, yet struggle with activities of daily living, such as driving at night.

Contrast sensitivity is a measure of the ability to discern different luminances in a static image. It varies with age, increasing to a maximum around 20 years at spatial frequencies of about 2–5 cpd; aging then progressively attenuates contrast sensitivity beyond this peak. Factors such as cataracts and diabetic retinopathy also reduce contrast sensitivity.[11] In the sweep grating figure below, at an ordinary viewing distance, the bars in the middle appear to be the longest due to their optimal spatial frequency. However, at a far viewing distance, the longest visible bars shift to what were originally the wide bars, now matching the spatial frequency of the middle bars at reading distance.

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Compound microscopes and other types of microscopes differ in their design and functionality. Here are the key differences between compound microscope parts and those of other microscopes:

Illuminator: A steady light source (110 volts) used in place of a mirror. If your microscope has a mirror, it is used to reflect light from an external light source up through the bottom of the stage.

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Stage with Stage Clips: The flat platform where you place your slides. Stage clips hold the slides in place. If your microscope has a mechanical stage, you will be able to move the slide around by turning two knobs. One moves it left and right, the other moves it up and down.

The low frequency drop-off is due to lateral inhibition within the retinal ganglion cells.[4] A typical retinal ganglion cell's receptive field comprises a central region in which light either excites or inhibits the cell, and a surround region in which light has the opposite effects.

One experimental phenomenon is the inhibition of blue in the periphery if blue light is displayed against white, leading to a yellow surrounding. The yellow is derived from the inhibition of blue on the surroundings by the center. Since white minus blue is red and green, this mixes to become yellow.[5]

with I m a x {\displaystyle I_{\mathrm {max} }} and I m i n {\displaystyle I_{\mathrm {min} }} representing the highest and lowest luminance. The denominator represents twice the average of the maximum and minimum luminances.[9]

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It's important to note that the term "other microscope parts" is quite broad and can include various microscope types with different designs and features. The above differences are generalized and may not apply to every microscope outside the category of compound microscopes.

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The rationale behind this is that a small difference is negligible if the average luminance is high, while the same small difference matters if the average luminance is low (see Weber–Fechner law). Below, some common definitions are given.

The maximum contrast of an image is termed the contrast ratio or dynamic range. In images where the contrast ratio approaches the maximum possible for the medium, there is a conservation of contrast. In such cases, increasing contrast in certain parts of the image will necessarily result in a decrease in contrast elsewhere. Brightening an image increases contrast in darker areas but decreases it in brighter areas; conversely, darkening the image will have the opposite effect. Bleach bypass reduces contrast in the darkest and brightest parts of an image while enhancing luminance contrast in areas of intermediate brightness.

Condenser Lens: The purpose of the condenser lens is to focus the light onto the specimen. Condenser lenses are most useful at the highest powers (400x and above). Microscopes with in-stage condenser lenses render a sharper image than those with no lens (at 400x). If your microscope has a maximum power of 400x, you will get the maximum benefit by using a condenser lenses rated at 0.65 NA or greater. 0.65 NA condenser lenses may be mounted in the stage and work quite well. A big advantage to a stage mounted lens is that there is one less focusing item to deal with. If you go to 1000x then you should have a condenser lens with an N.A. of 1.25 or greater. All of our 1000x microscopes use 1.25 Abbe condenser lens systems. The Abbe condenser lens can be moved up and down. It is set very close to the slide at 1000x and moved further away at the lower powers.

Historians credit the invention of the compound microscope to the Dutch spectacle maker, Zacharias Janssen, around the year 1590 (more history here). The compound microscope uses lenses and light to enlarge the image and is also called an optical or light microscope (versus an electron microscope). The simplest optical microscope is the magnifying glass and is good to about ten times (10x) magnification.

Rack Stop: This is an adjustment that determines how close the objective lens can get to the slide. It is set at the factory and keeps students from cranking the high power objective lens down into the slide and breaking things. You would only need to adjust this if you were using very thin slides and you weren't able to focus on the specimen at high power. (Tip: If you are using thin slides and can't focus, rather than adjust the rack stop, place a clear glass slide under the original slide to raise it a bit higher).

Studies have demonstrated that contrast sensitivity is maximum for spatial frequencies of 2-5 cpd, falling off for lower spatial frequencies and rapidly falling off for higher spatial frequencies. The upper limit for the human vision system is about 60 cpd. The correct identification of small letters requires the letter size be about 18-30 cpd.[14] Contrast threshold can be defined as the minimum contrast that can be resolved by the patient. Contrast sensitivity is typically expressed as the reciprocal of the threshold contrast for detection of a given pattern (i.e., 1 ÷ contrast threshold).[15]

Various definitions of contrast are used in different situations. Here, luminance contrast is used as an example, but the formulas can also be applied to other physical quantities. In many cases, the definitions of contrast represent a ratio of the type

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Root mean square (RMS) contrast does not depend on the spatial frequency content or the spatial distribution of contrast in the image. RMS contrast is defined as the standard deviation of the pixel intensities:[6]

To assess a patient's contrast sensitivity, one of several diagnostic exams may be used. Most charts in an ophthalmologist's or optometrist's office will show images of varying contrast and spatial frequency. Parallel bars of varying width and contrast, known as sine-wave gratings, are sequentially viewed by the patient. The width of the bars and their distance apart represent spatial frequency, measured in cycles per degree.

Objective Lenses: Usually you will find 3 or 4 objective lenses on a microscope. They almost always consist of 4x, 10x, 40x and 100x powers. When coupled with a 10x (most common) eyepiece lens, total magnification is 40x (4x times 10x), 100x , 400x and 1000x. To have good resolution at 1000x, you will need a relatively sophisticated microscope with an Abbe condenser. An Abbe condenser is composed of two lenses that control the light that passes through the specimen before entering the objective lens on the microscope. The shortest lens is the lowest power, the longest one is the lens with the greatest power. Lenses are color coded and if built to DIN standards are interchangeable between microscopes. "DIN" is an abbreviation of "Deutsche Industrial Normen". This is a German standard that has been adopted internationally as an optical standard used in most quality microscopes. A typical DIN standard microscope objective lens has a 0.7965" (20.1mm) diameter threads, 36 TPI (threads per inch), and a 55º Whitworth. Many high power objective lenses are retractable (i.e. 40XR). This means that if they hit a slide, the end of the lens will push in (spring loaded) thereby protecting the lens and the slide. All good quality microscopes have achromatic, parcentered, parfocal lenses.

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Magnification: Compound microscopes are designed for higher magnifications, typically used for observing microscopic details. Other microscopes may have lower magnification capabilities, suitable for larger specimens or samples.

1. Ocular eyepiece lens to look through. 2. Objective lens, closest to the object. Before purchasing or using a compound microscope, it is important to know the functions of each part. This information is presented below. Links will take you to additional information and images.

Revolving Nosepiece or Turret: This is the part of the microscope that holds two or more objective lenses and can be rotated to easily change power.

where intensities I i j {\displaystyle I_{ij}} are the i {\displaystyle i} -th j {\displaystyle j} -th element of the two-dimensional image of size M {\displaystyle M} by N {\displaystyle N} . I ¯ {\displaystyle {\bar {I}}} is the average intensity of all pixel values in the image. The image I {\displaystyle I} is assumed to have its pixel intensities normalized in the range [ 0 , 1 ] {\displaystyle [0,1]} .

Using the results of a contrast sensitivity exam, a contrast sensitivity curve can be plotted, with spatial frequency on the horizontal, and contrast threshold on the vertical axis. Also known as contrast sensitivity function (CSF), the plot demonstrates the normal range of contrast sensitivity, and will indicate diminished contrast sensitivity in patients who fall below the normal curve. Some graphs contain "contrast sensitivity acuity equivalents", with lower acuity values falling in the area under the curve. In patients with normal visual acuity and concomitant reduced contrast sensitivity, the area under the curve serves as a graphical representation of the visual deficit. It can be because of this impairment in contrast sensitivity that patients have difficulty driving at night, climbing stairs and other activities of daily living in which contrast is reduced.[16]

As mentioned above, contrast sensitivity describes the ability of the visual system to distinguish bright and dim components of a static image. Visual acuity can be defined as the angle with which one can resolve two points as being separate since the image is shown with 100% contrast and is projected onto the fovea of the retina.[13] Thus, when an optometrist or ophthalmologist assesses a patient's visual acuity using a Snellen chart or some other acuity chart, the target image is displayed at high contrast, e.g., black letters of decreasing size on a white background. A subsequent contrast sensitivity exam may demonstrate difficulty with decreased contrast (using, e.g., the Pelli–Robson chart, which consists of uniform-sized but increasingly pale grey letters on a white background).

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Recent studies have demonstrated that intermediate-frequency sinusoidal patterns are optimally-detected by the retina due to the center-surround arrangement of neuronal receptive fields.[17] In an intermediate spatial frequency, the peak (brighter bars) of the pattern is detected by the center of the receptive field, while the troughs (darker bars) are detected by the inhibitory periphery of the receptive field. For this reason, low- and high-spatial frequencies elicit excitatory and inhibitory impulses by overlapping frequency peaks and troughs in the center and periphery of the neuronal receptive field.[18] Other environmental,[19] physiological, and anatomical factors influence the neuronal transmission of sinusoidal patterns, including adaptation.[20]

A large-scale study of luminance contrast thresholds was done in the 1940s by Blackwell,[22] using a forced-choice procedure. Discs of various sizes and luminances were presented in different positions against backgrounds at a wide range of adaptation luminances, and subjects had to indicate where they thought the disc was being shown. After statistical pooling of results (90,000 observations by seven observers), the threshold for a given target size and luminance was defined as the Weber contrast level at which there was a 50% detection level. The experiment employed a discrete set of contrast levels, resulting in discrete values of threshold contrast. Smooth curves were drawn through these, and values tabulated. The resulting data have been used extensively in areas such as lighting engineering and road safety.[24]

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This form of contrast is an effective way to quantify contrast for periodic functions f ( x ) {\displaystyle f(x)} and is also known as the modulation m f {\displaystyle m_{f}} of a periodic signal f {\displaystyle f} . Modulation quantifies the relative amount by which the amplitude (or difference) ( f max − f min ) / 2 {\displaystyle (f_{\text{max}}-f_{\text{min}})/2} of f {\displaystyle f} stands out from the average value (or background) ( f max + f min ) / 2 {\displaystyle (f_{\text{max}}+f_{\text{min}})/2} .