Optical methods biomedical systems.
The optical microscope, often referred to as the "light microscope", is a type of microscope which uses visible light and a system of lenses to magnify images of small samples. Optical microscopes are the oldest design of microscope and were possibly designed in their present compound form in the 17th century. Basic optical microscopes can be very simple, although there are many complex designs which aim to improve resolution and sample contrast. Historically optical microscopes were easy to develop and are popular because they use visible light so that samples may be directly observed by eye.
The image from an optical microscope can be captured by normal light-sensitive cameras to generate a micrograph. Originally images were captured by photographic film but modern developments in CMOS and charge-coupled device (CCD) cameras allow the capture of digital images. Purely digital microscopes are now available which use a CCD camera to examine a sample, showing the resulting image directly on a computer screen without the need for eyepieces.
Basic optical transmission microscope elements (1990s)
All modern optical microscopes designed for viewing samples by transmitted light share the same basic components of the light path. In addition, the vast majority of microscopes have the same 'structural' components (numbered below according to the image on the right):
- Eyepiece (ocular lens) (1)
- Objective turret, revolver, or revolving nose piece (to hold multiple objective lenses) (2)
- Objective lenses (3)
- Focus knobs (to move the stage)
- Coarse adjustment (4)
- Fine adjustment (5)
- Stage (to hold the specimen) (6)
- Light source (a light or a mirror) (7)
- Diaphragm and condenser (8)
- Mechanical stage (9)
Two basic types of microscopes are used in biology: the compound light microscope and the electron microscope. Compound light microscopes use ordinary light to magnify, compared to electron microscopes which use a stream of electrons. Although different in appearance, both of these microscopes perform the same function: to magnify objects.
The development of the microscope can be traced back to the first century A.D. Roman author and philosopher Seneca, who noted that a jar of water positioned properly would make writing appear clearer and larger in size. The ancients at Nineva, the Romans, Chinese, and Greeks may have used lenses for reading. In 1558, Swiss naturalist Conrad Gesner wrote of the use of magnification in biological investigation.
Perhaps the best known of the early microscopists was Antony van Leeuwenhoek. A master lens grinder, Leeuwenhoek made his own microscopes and spent most of his life observing and describing the then virtually unknown microscopic world (microscopic being any substance or structure too small to be viewed with the naked eye). His contributions include sketching the three major groups of bacteria; capillary circulation in an eel’s tail; muscle fibers; insect anatomy; plant tissues; and one of the first people to view human sperm beneath a microscope. Leeuwenhoek also made observations of tartar taken from his teeth, in which he described living “beasties” in this material (we know today that this is bacteria). In one of his poorer choices for observation, Leeuwenhoek viewed a small gunpowder explosion beneath one microscope. It is reported that this experiment left him blinded for several days!
Care of the Microscope
The microscopes you will be using are compound light microscopes. They are sophisticated and expensive instruments and must be used with care. Mistreatment of microscopes may result in the need for costly repair work as well as your loss of user privileges.
To ensure the continued use of these microscopes, the following rules will be strictly adhered to.
1. Carry the microscope upright with two hands. Once removed from the storage cabinet, the microscope must always be held upright. Place your dominant hand on the arm of the microscope with your other hand supporting the base of the microscope. Care should be taken not to bump your microscope on objects such as chairs, tables, or walls. Gently place your microscope on your laboratory table and remove the protective plastic cover.
2. Inspect the microscope before use. All parts should move easily. If they do not, do not force them! Do not attempt any repairs! Report all damage, missing parts, or anything else to your laboratory instructor.
3. Only clean the lenses with lens paper. The optical parts of the microscope are precision lenses and scratch easily. The only acceptable method of cleaning them is through the use of lens paper. Lens paper can be found on the bench at the front of the room. If you cannot find any, ask your instructor. DO NOT use paper towels, shirtsleeves, handkerchiefs, or Kleenex to clean lenses. Never remove oculars or other parts from the body tube of the microscope.
4. Do not push the microscope across the table. When sharing a microscope with others, never push the microscope across the table top. Instead, trade places with them. Sliding results in vibrations to the microscope that may result in the loosening of screws, or misalignment of parts.
5. Unplug carefully. Use care when plugging in and unplugging the microscope from electrical outlets. When unplugging, always pull gently on the plug at the outlet. Do not attempt to unplug the microscope by pulling on the cord away from the plug.
6. Replace microscope properly. When finished with the microscope, turn off the light, remove the last slide from the stage, and wipe any water or other materials from the stage. Next, be certain that the lowest power objective is clicked into position, and racked up to its highest point. Neatly wrap your electrical cord (your instructor will demonstrate the proper way to do this), and place the plastic cover over the microscope. Return the microscope to its proper numbered slot in the cabinet.
Parts of the Microscope
In order to operate a microscope properly and effectively, it is necessary to have an understanding of some of the various parts of the microscope and their functions. The microscope you will be using is shown in either Figure 1 or Figure 2. With the help of your instructor, identify and learn the following parts listed in Table 1.
Ocular or Eyepiece
Revolving Nose Piece or Turret
Iris Diaphragm Lever
Coarse Adjustment Knob
Fine Adjustment Knob
Supports the body tube and lenses. Use the arm to carry your microscope.
Supports the entire microscope. Broad and heavy, the base gives the instrument stability.
The lens in the upper part of the microscope. Monocular microscopes have one ocular, while binocular microscopes have two oculars.
Holds the ocular at one end and the nosepiece at the other. A prism housed in the body tube helps to reflect light towards the eye.
Located at the lower end of the body tube. A revolving device that holds the objectives.
Located on the revolving nosepiece. There are four lenses: 4x or scanning power; 10x or low power; 40x or high power; and oil lens. Only one objective may be used at a time. The selected lens is rotated into position by turning the nosepiece.
The horizontal platform upon which the slide rests.
Lens found beneath the stage that concentrates light before it passes through the specimen to be viewed.
Small lever beneath the condenser. Allows the observer to regulate the amount of light passing through the specimen.
Provides illumination of the specimen. Located beneath the condenser and iris diaphragm.
Small circular knobs adjacent to or below the stage. Allow the observer to move the slide across the stage either forward or backwards or laterally.
Located on either side of the arm. Moves the stage (or body tube) up or down to the approximate correct distance. This knob should only be used when using the low powered objective.
Located within the coarse adjustment knob. Moves the stage (or body tube) up or down small distances. Allows fine focus of the specimen.
Figure 1: The binocular, compound, light microscope.
Figure 2: The monocular, compound, light microscope.
Focusing the Microscope
Before starting Exercise 1, there are a few important ideas and details to keep in mind when focusing the microscope.
A. Always begin viewing every slide using the scanning power (4x) objective. Never begin an observation with the low or high-powered objectives. Attempting to focus on specific objects without first finding the specimen under scanning power is usually a waste of time and effort. Once the object is found under scanning magnification, you may increase magnification by rotating the low powered objective into place.
B. As you view a specimen under scanning, low, and high magnifications, note that the image remains nearly in focus from one magnification to the next. Most light microscopes are parfocal, meaning that the image remains nearly in focus as you change lenses. Note also that the image remains centered after the high-powered objective is in place-the image is parcentered.
C. When focusing with the high-powered objective, never use the coarse adjustment knob. It is very easy to break slides or damage objectives by doing this. Only the fine focus knob can be used at this time. When using this lens, always focus away from the microscope slide. To do this correctly, rotate the coarse adjustment knob so that it nearly touches the slide while viewing this whole process from the side-not while you are peering into the ocular. Once you have done this, focus up and away from the slide.
D. Don’t forget the iris diaphragm! Adjusting light levels with the diaphragm will usually improve contrast, providing the observer a clearer view of the specimen.
E. When using a binocular microscope the distance between the two oculars (interocular width) can be adjusted by moving the ocular tubes towards or away from each other. Using the scanning lens, look through and adjust the oculars so that a single field is seen. Focus on the specimen. Close one eye and then the other. The specimen should be in focus through each ocular. If they are not equally in focus, it will be necessary to focus each ocular individually. If your instructor has not already demonstrated how to do this, ask for assistance.
Field (Field of View) - The circular area that can be seen when looking through the ocular.
Depth of Field - The thickness of an object which is all in sharp focus at the same time.
Working Distance - The space between the bottom of the objective and the top of the slide.
Resolution - The minimum distance where you can see the separation between two points. In practice, it refers to how clearly you can see details in the microscope.
Binocular light microscope: A microscope with two oculars—one for each eye. Using both eyes to see in your microscope will not only be easier on your eyes but will also give you more depth perception. However, your eyes have to both be in focus on the same item at the same time, or you will get a bit of a headache. To be sure you are in the best focus with both eyes, first only focus with your right eye using the fine focus knob. Then adjust the focus of your left eye with the adjustment that is on the left ocular itself and not with the focus knobs. Take your time to do this every time and you will get much more out of your microscope experience.
Compound light microscope: Modern microscopes that use two or more lenses to magnify an object are called compound light microscopes. Because the microscopes Leeuwenhoek used had only one magnifying lens, his microscopes were considered to be simple light microscopes. The appearance of a true compound microscope dates from the late 16th century when Zaccharias Janssens of Holland discovered that by combining lenses and manipulating distances between them, objects could be enlarged.
Interocular width: This is the distance between the two oculars of a binocular microscope. When you are using a binocular microscope, you need to adjust the distance between them to match the distance between your own two pupils. Every person has a particular spacing between their eyes, so if you want to see using both eyes, you need to be sure that your oculars are adjusted properly. Once you find the appropriate interocular width, you can write down the number for it so you can quickly adjust it in the future.
Monocular light microscope: A microscope with only one ocular. When using this type of microscope, try not to squint the eye you are not using. Instead, cover that eye with your hand, keeping your eye open as if to view the skin of your palm up close. This will help prevent you from getting a headache or from having temporary blurry vision in that unused eye.
Stereoscopic Dissecting Microscope
Due to the small working distance of the compound microscope, large or thick specimens are difficult to view. A dissecting (stereoscopic) microscope provides a much greater working distance and is able to accommodate large specimens such as insects, fungi, algae, or plants. With its larger working distance, there is room to move specimens around on the stage.
Due to the thickness of specimens observed with the dissecting microscope, light will not transmit through the specimen. Therefore, dissecting microscopes project light onto the specimen from above. You don't shine a light through it, you shine a light on it.
The dissecting microscope is a binocular scope. Each of the two oculars views the specimen at a different angle, providing a three-dimensional image with a large depth of field. Compound microscopes, in comparison, provide only a two-dimensional image. Advantages of a dissecting microscope are often offset by lower magnification and resolution than a compound microscope.
Examine the dissecting microscopes on display in the laboratory and review the various parts of the dissecting microscope in Figure 3.
Figure 3: The dissecting light microscope.
Eyepiece (ocular lens)
The eyepiece, or ocular lens, is a cylinder containing two or more lenses; its function is to bring the image into focus for the eye. The eyepiece is inserted into the top end of the body tube. Eyepieces are interchangeable and many different eyepieces can be inserted with different degrees of magnification. Typical magnification values for eyepieces include 2×, 50× and 10×. In some high performance microscopes, the optical configuration of the objective lens and eyepiece are matched to give the best possible optical performance. This occurs most commonly with apochromatic objectives.
Optical path in a typical microscope
The optical components of a modern microscope are very complex and for a microscope to work well, the whole optical path has to be very accurately set up and controlled. Despite this, the basic operating principles of a microscope are quite simple.
The objective lens is, at its simplest, a very high powered magnifying glass i.e. a lens with a very short focal length. This is brought very close to the specimen being examined so that the light from the specimen comes to a focus about 160 mm inside the microscope tube. This creates an enlarged image of the subject. This image is inverted and can be seen by removing the eyepiece and placing a piece of tracing paper over the end of the tube. By carefully focusing a brightly lit specimen, a highly enlarged image can be seen. It is this real image that is viewed by the eyepiece lens that provides further enlargement.
In most microscopes, the eyepiece is a compound lens, with one component lens near the front and one near the back of the eyepiece tube. This forms an air-separated couplet. In many designs, the virtual image comes to a focus between the two lenses of the eyepiece, the first lens bringing the real image to a focus and the second lens enabling the eye to focus on the virtual image.
In all microscopes the image is intended to be viewed with the eyes focused at infinity (mind that the position of the eye in the above figure is determined by the eye's focus). Headaches and tired eyes after using a microscope are usually signs that the eye is being forced to focus at a close distance rather than at infinity.
Types of Microscopes
Light Microscope - the models found in most schools, use compound lenses to magnify objects. The lenses bend or refract light to make the object beneath them appear closer. Common magnifications: 40x, 100x, 400x
Stereoscope - this microscope allows for binocular (two eyes) viewing of larger specimens.
Scanning Electron Microscope - allow scientists to view a universe too small to be seen with a light microscope. SEMs do not use light waves; they use electrons (negatively charged electrical particles) to magnify objects up to two million times.
Transmission Electron Microscope - also uses electrons, but instead of scanning the surface (as with SEM's) electrons are passed through very thin specimens.
Parts of the Microscope
Your microscope has 3 magnifications: Scanning, Low and High. Each objective will have written the magnification. In addition to this, the ocular lens (eyepiece) has a magnification. The total magnification is the ocular x objective
1. Make sure all backpacks and junk are out of the aisles.
2. Plug your microscope in to the extension cords. Each row of desks uses the same cord.
3. Store with cord wrapped around microscope and the scanning objective clicked into place.
4. Carry by the base and arm with both hands.
1. Always start with the scanning objective. Odds are, you will be able to see something on this setting. Use the Coarse Knob to focus, image may be small at this magnification, but you won't be able to find it on the higher powers without this first step. Do not use stage clips, try moving the slide around until you find something.
2. Once you've focused on Scanning, switch to Low Power. Use the Coarse Knob to refocus. Again, if you haven't focused on this level, you will not be able to move to the next level.
3. Now switch to High Power. (If you have a thick slide, or a slide without a cover, do NOT use the high power objective). At this point, ONLY use the Fine Adjustment Knob to focus specimens.
4. If the specimen is too light or too dark, try adjusting the diaphragm.
5. If you see a line in your viewing field, try twisting the eyepiece, the line should move. That's because its a pointer, and is useful for pointing out things to your lab partner or teacher.
1. Use pencil - you can erase and shade areas
2. All drawings should include clear and proper labels (and be large enough to view details). Drawings should be labeled with the specimen name and magnification.
3. Labels should be written on the outside of the circle. The circle indicates the viewing field as seen through the eyepiece, specimens should be drawn to scale - ie..if your specimen takes up the whole viewing field, make sure your drawing reflects that.
Making a Wet Mount
1. Gather a thin slice/peice of whatever your specimen is. If your specimen is too thick, then the coverslip will wobble on top of the sample like a see-saw, and you will not be able to view it under High Power.
2. Place ONE drop of water directly over the specimen. If you put too much water, then the coverslip will float on top of the water, making it hard to draw the specimen, because they might actually float away. (Plus too much water is messy)
3. Place the coverslip at a 45 degree angle (approximately) with one edge touching the water drop and then gently let go. Performed correctly the coverslip will perfectly fall over the specimen.
Light as an Electromagnetic Wave
Light is an electromagnetic wave; a transverse wave that travels through empty space at the speed of or 186.000 miles/s. The relation between the wavelength , frequency , and speed c of the light is given by the fundamental equation of wave propagation as
The wavelength of visible light varies from about for violet light to the longer wavelength of red light at . Since the wavelength of light is so small, we commarly use the nanometer (nm), where .
So visible light varies from about 380.0 nm to about 720.0 nm. A nonstandard unit is the angstran abbreviated .
So visible light is from 3800 to 7200 .
A great deal of research went into optics, the study of light, before its electromagnetic character was known. A light wave is represented in figure
The electric and magnetic vectors are not shown and the magnetic portion of the wave is completely missing.
If a monochromatic point source of light (one of a single wavelength) is turned on at a particular instant, then a spherical wave emanates from the source. A two dimensional view of the wave is shown in figure.
Far away from the source of light the circular fronts look more like plane fronts. The waves then called plane waves. These plane waves can then be shown in figure.
A line drawn perpendicular to the wave front is called a ray of light and represent the direction of propagation of the light wave. Note that the ray of light travels in a straight line. The wavelength of light is so small that in a relatively short distance away from the point source of light, the waves appear plane. Even if the source of light is not a point, then we are sufficiently far away from the source, the waves are effectively plane. In all the subsequent discussions we will assume that all the light waves are plane waves.
This analysis of light into waves and rays allows us two different descriptions of light.
When only the light rays are dealt with in the analysis of an optical system, the description is called geometrical optics. When the analysis of an optical system is done in terms of waves, the description is called wave optics or physical optics. Still another description of light is possible by treading light as little bundles of electromagnetic energy, called photons. Such a description is called quantum optics.
Huygens’ principle states that each point on a wave front may be considered as a source of secondary spherical wavelets. These secondary wavelets propagate in the forward direction at the same speed as the initial wave. The new position of the wave front at a later time is found by drawing the tangent to all of these secondary wavelets at the later time.
The Law of Reflection
Consider a plane wave advancing toward a smooth surface such as a glass or a mirror, as in figure.
The First Law of reflection says that the angle of incidence is equal to the angle o reflection r.
The Second Law of reflection says that the incident ray, the normal, and the reflected ray all lie in the same plane.
Law refraction ;
The wavelength of the light in the second medium ;
The speed of the light in the second medium .
Air at STP
Water at 20 C
Sugar solution (80%)
Typical crown glass
Spectacle crown, C-1
Heavy flint glass
Extra dense flint, EDF-3
Rare earth flint
Arsenic trisulfide glass
The Plane Mirror
An object O is placed a distance p (called the object distance) in front of a plane mirror RT, as shown in Figure
As you know, if you are the object O, you will see your image in the mirror. How is this image formed and how far behind the mirror is the image located?
From figure you can see that q=p. This is, image is as far behind the mirror as the object is in front of it.
In general, to describe an optical image three words are necessary: its nature (real or virtual), its orientation (erect, inverted, perverted), and its size (enlarged, true, or reduced). Recal that when you look into a mirror your image is reversed. That is, if you hold up your right hand in front of the mirror, your image appears as though the left hand was held up. This inversion of left-right symmetry is called perversion. Thus, a plane mirror produces a virtual, perverted, true image.
The Concave Spherical Mirror
A spherical mirror is a reflection surface, whose radius of curvature is the radius of the sphere from which the mirror is formed.
Eve C is the center of curvature of the mirror and R is its radius of curvature. The line going through the center of the mirror, the vertex, is called the principal axis, or optical axis, of the mirror. Light rays that are parallel and close to the principal axis of the concave mirror converge to a point called the principal focus F of the mirror.
Focal Length of a Concave Spherical Mirror
The distance VF from the vertex of the mirror to the principal focus F is called the focal length f of the mirror.
It was proved that
The Lensmaker’s formula
Where n is the index of regraction of the glass, radius of curvature of the first surface of the lens R1 and second surface of the lens R2 ; f is the focal length of the lens.
Lens equation .
Is the lens equation and gives the relation beetwen the object distance p , the image distance f , and focal length f.
The linear magnification M= - ;
Linear magnification M of the mirror is the radio of the size of the image to the size of the object .
That is, .
Thus, the magnification tells how much larger the image is than the object. We can rewrite this as
Why Diffraction Occurs. Diffraction is another instance in which waves do something that is unique to their nature. The phenomenon of diffraction is easily explained qualitatively in terms of Huygen's theory. As a wavefront propagates along, it produces point sources which emit spherical waves. As long as all these point sources start at the same time and are allowed to emit waves without interruption, the wavefront along the direction of propagation is a straight line. If an obstruction or a barrier with a slit intrudes, however, then some of the spherical waves are blocked and cannot contribute to the wavefront. The response is that the wavefront becomes curved. To see this, consider the static images below that show a plane wave moving into a barrier with a slit or a just a barrier. As long as the obstruction or opening is much larger than the wavelength as shown in figure 1, the behavior of the waves is what we expect, namely the part of the wavefront that is allowed to continue does so with the wavefront remaining straight.
If, however, the size of the opening becomes comparable to the wavelength, the waves proceed to "bend through" or around the opening or obstruction as shown in figure 2.
Figure : Now the plane wavefronts impinge on a barrier with an opening or an obstruction which is not much larger than the wavelenth. The wavefront is not allowed to propagate freely through the opening or past the obstruction but experiences some retardation of some parts of the wavefront. The result is that the wavefront experiences significant curvature upon emerging from the opening or the obstruction.
Finally, as the obstruction or opening equals the wavelength, the diffraction becomes quite pronounced as shown in figure . We refer to this phenomenon as diffraction.
Given the mathematical complexity, only a part of what constitutes the theory of diffraction can be discussed in detail in the text. Just to introduce the nomenclature though, note that cases in which the source of radiation or the screen are close to the obstruction causing the diffraction are termed Fresnel diffraction. Cases in which the source and screen are far from the obstruction are termed Fraunhofer diffraction. The text describes only Fraunhofer diffraction in quantitative detail as Fresnel diffraction is beyond the mathematical scope of the text. To see the effect of Fraunhofer diffraction in action for visible light.
As described in the text, the minima of such diffraction is given by
where a is the size
of the obstruction and is
the angle relative to the horizontal as shown in figure .
Figure: If the distance from the aperture to the screen, x, is much greater than the size of the aperture, a, then the distance from the top half of the aperture to point P on the screen is less than the distance from the bottom half of the aperture to point P by approximately (a/2)sin.
So, we expect dark fringes at values of which satisfy
since we could divide the slit into quarters, eighths, 16th's, etc. and repeat the argument of having interference minima for each adjacent pair of intervals.
This is the formula for single slit diffraction minima. Note that there is no central minimum. The center of a diffraction pattern is always a maximum just as it is for interference in Young's experiment. The first minimum for diffraction therefore corresponds to m = 1 rather than zero as in Young's experiment. The vertical position of these minima is given approximately as
Initialize a healthy eye by clicking on the link at the bottom of the applet. Then add a far source of light using the link at the top of the applet. Notice how the parallel rays of light from the far away source converge at the back of the eye on the retina. The retina is to the eye what film is to a camera. The retina is made up of nerves that convert the light energy into an electrical signal that is sent to the brain. So in order for an object to be "seen" its image must be FOCUSED on the back of the retina.
Now remove the far source and add a near source. Notice that the light from the near by source is focused behind the retina. In this case the person would see a blurry image. As evolution would have it, our eyes have the ability to accommodate. You can change the focal length of your eye by using the muscles of your eye to change the curvature of the lens. Try looking at a far away object and then at something close by, such as your finger. You will feel the muscles in your eye respond as you change your focus. In the applet accommodation is accomplished by using the slider at the bottom to vary the focal length of the lens. Vary the focal length of the lens, using the slider, until the image of the light source is focused on the retina.
Viewing Far Away Objects
People with normal vision focus on far away objects with their eyes relaxed. Notice that the far source in the applet was focused when the focal length was at its maximum, 1 unit. As you use your muscles to accommodate you shorten the focal length of your eye.
Near Point and Far Point
Put your finger in front of your eyes about an arm's length away. You should be able to see a clear image of your finger. Now slowly bring your finger toward you. At some point, you will no longer be able to focus on your finger and it will become blurry. This is your near point. It is the closest distance at which you can focus on an object. If you have not already done so, initialize a healthy eye with a near source of light focused on the retina. Now move the source of light toward the eye. At some point you will no longer be able to accommodate (using the slider) to focus the source. That is the near point for the eye in the applet. Notice that the eye in the applet is not to scale relative to a real eye. If we had made it to scale you would need a MUCH wider computer screen.
The far point is just like the near point except it is the furthest point an eye can focus on. For people with normal vision, the far point is at infinity.
Nearsightedness and Farsightedness
Initialize a nearsighted eye and add a far source. Notice that the light does not focus on the retina when the eye is relaxed. Instead, it focuses in front of the retina. Use the slider to try and focus the light. Notice that accommodation does not help in this situation. Now remove the far source and add a near source. Notice that the nearsighted person has no trouble focusing on the near by source. A person who is nearsighted can clearly see near objects but not far away objects.
Now initialize a farsighted eye and investigate it as you did with the nearsighted eye. Notice that a farsighted person can see far away objects but has difficulty focusing on near by objects.
Initialize a nearsighted eye with a far source. Unaided, this eye can not focus on the far source. Now add an eyeglass lens. Notice that you can change the focal length (power) of the eyeglass lens by clicking on it and then dragging on the hotspots. You can make the lens either converging or diverging.
Since light is focused in front of the retina in a nearsighted eye, nearsightedness is corrected using a diverging lens. Can you find the correct focal length to correct this eye? In the same way, farsightedness is corrected using a converging lens.
Conjugate Planes in Optical Microscopy
In a properly focused and aligned optical microscope, a review of the geometrical properties of the optical train demonstrates that there are two sets of principal conjugate focal planes that occur along the optical pathway through the microscope. One set consists of four field planes and is referred to as the field or image-forming conjugate set, while the other consists of four aperture planes and is referred to as the illumination conjugate set. Each plane within a set is said to be conjugate with the others in that set because they are simultaneously in focus and can be viewed superimposed upon one another when observing specimens through the microscope.
Presented in Figure 1 is a cutaway diagram of a modern microscope (a Nikon Eclipse E600), which illustrates the strategic location of optical components comprising the two sets of conjugate planes in the optical pathways for both transmitted and incident (reflected or epi) illumination modes. Components that reside in the field set of conjugate planes are described in black text, while those comprising the aperture set are described in red text. Note that conjugate planes are illustrated for both observation and digital imaging (or photomicrography) modes. Table 1 lists the elements that make up each set of conjugate planes, including alternate nomenclature (listed in parentheses) that has often been employed and may be encountered in the literature. A minor difference exists in the relative location of the field and condenser apertures between the incident and transmitted modes of illumination, which will be explained later.
Numerical Aperture - The numerical aperture of a microscope objective is a measure of its ability to gather light and resolve fine specimen detail at a fixed object distance. All modern microscope objectives have the numerical aperture value inscribed on the lens barrel, which allows determination of the smallest specimen detail resolvable by the objective and an approximate indication of the depth of field.
Resolution - The resolving power of a microscope is the most important feature of the optical system and influences the ability to distinguish between fine details of a particular specimen. As discussed in this section, the primary factor in determining resolution is the objective numerical aperture, but resolution is also dependent upon the type of specimen, coherence of illumination, degree of aberration correction, and other factors such as contrast enhancing methodology either in the optical system of the microscope or in the specimen itself.
The resolution of an optical microscope is defined as the shortest distance between two points on a specimen that can still be distinguished by the observer or camera system as separate entities. An example of this important concept is presented in the figure below (Figure 1), where point sources of light from a specimen appear as Airy diffraction patterns at the microscope intermediate image plane.
The limit of resolution of a microscope objective refers to its ability to distinguish between two closely spaced Airy disks in the diffraction pattern (noted in the figure). Three-dimensional representations of the diffraction pattern near the intermediate image plane are known as the point spread function, and are illustrated in the lower portion of Figure 1. The specimen image is represented by a series of closely spaced point light sources that form Airy patterns and is illustrated in both two and three dimensions.
Resolution is a somewhat subjective value in optical microscopy because at high magnification, an image may appear unsharp but still be resolved to the maximum ability of the objective. Numerical aperture determines the resolving power of an objective, but the total resolution of the entire microscope optical train is also dependent upon the numerical aperture of the substage condenser. The higher the numerical aperture of the total system, the better the resolution.
Useful Magnification Range - The range of useful magnification for an objective/eyepiece combination is defined by the numerical aperture of the system. There is a minimum magnification necessary for the detail present in an image to be resolved, and this value is usually rather arbitrarily set to a value between 500 and 1000 times the numerical aperture (500 or 1000 x NA) of the objective.
Working Distance and Parfocal Length - Microscope objectives are generally designed with a short free working distance, which is defined as the distance from the front lens element of the objective to the closest surface of the coverslip when the specimen is in sharp focus. The parfocal length represents the distance between the specimen plane and the shoulder of the flange by which the objective is supported on the revolving nosepiece.
Image Brightness - Regardless of the imaging mode utilized in optical microscopy, image brightness is governed by the light-gathering power of the objective, which is a function of numerical aperture. Just as the brightness of illumination is determined by the square of the condenser working numerical aperture, the image brightness is proportional to the square of the objective numerical aperture.
Coverslip Correction - Non-immersion high-dry microscope objectives having a numerical aperture exceeding 0.75 are prone to introduction of aberration when imaging through coverslips that deviate from standard thickness and refractive index. To prevent artifacts, many objectives are equipped with correction collars that help compensate for coverslip thickness variations.
Adjustment of Objective Correction Collars - Most microscope objectives are designed to be used with a cover glass that has a standard thickness of 0.17 millimeters and a refractive index of 1.515, which is satisfactory when the objective numerical aperture is 0.4 or less. However, when using high numerical aperture dry objectives (numerical aperture of 0.8 or greater), cover glass thickness variations of only a few micrometers result in dramatic image degradation due to aberration, which grows worse with increasing cover glass thickness. To compensate for this error, the more highly corrected objectives are equipped with a correction collar to allow adjustment of the central lens group position to coincide with fluctuations in cover glass thickness. This interactive tutorial explores how a correction collar is adjusted to achieve maximum image quality.
Focusing and Alignment of Arc Lamps - Mercury and xenon arc lamps are now widely utilized as illumination sources for a large number of investigations in widefield fluorescence microscopy. Visitors can gain practice aligning and focusing the arc lamp in a Mercury or Xenon Burner with this interactive tutorial, which simulates how the lamp is adjusted in a fluorescence microscope.
Linear Measurements (Micrometry) - Performing measurements at high magnifications in compound optical microscopy is generally conducted by the application of eyepiece reticles in combination with stage micrometers. A majority of measurements made with compound microscopes fall into the size range of 0.2 micrometers to 25 millimeters (the average field diameter of widefield eyepieces). Horizontal distances below 0.2 micrometers are beneath the resolving power of the microscope, and lengths larger than the field of view of a widefield eyepiece are usually (and far more conveniently) measured with a stereomicroscope.
Linear Measurements (Micrometry)
All measurements of length are based on a comparison of the object under scrutiny with another of known dimensions, or with a standardized, calibrated scale. In order to determine the length or width of a wooden board, for example, a ruler or measuring tape is placed in contact with the board and the dimensions are noted by direct comparison to the graduated numerical markings on the ruler.
This basic principle is applicable to the measurement of specimens observed in the microscope, but in practice, it is often not possible with a compound microscope to place a ruler in direct contact with the specimen (although this is often done in low-magnification stereomicroscopy). Alternative mechanisms for performing measurements at high magnifications in compound optical microscopy must be employed, and the most common of these is the application of eyepiece reticles in combination with stage micrometers. A majority of measurements made with compound microscopes fall into the size range of 0.2 micrometers to 25 millimeters (the average field diameter of widefield eyepieces). Horizontal distances below 0.2 micrometers are beneath the resolving power of the microscope, and lengths larger than the field of view of a widefield eyepiece are usually (and far more conveniently) measured with a stereomicroscope.
Illustrated in Figure 1 is a modern microscope eyepiece (often termed an ocular) equipped with an internal reticle scale. Also presented in the figure is a stage micrometer, which contains a small metallized millimeter ruler that is subdivided into increments of 10 and 100 micrometers. Juxtaposing the graduations on the eyepiece reticle with those on the stage micrometer enables the microscopist to calibrate the reticle gauge and perform linear measurements on specimens.
The first reported measurements performed with an optical microscope were undertaken in the late 1600s by the Dutch scientist Antonie van Leeuwenhoek, who used fine grains of sand as a gauge to determine the size of human erythrocytes. Since then, countless approaches have been employed for measuring linear, area, and volume specimen dimensions with the microscope (a practice known as micrometry or morphometrics), and a wide variety of useful techniques have emerged over the past few hundred years. Many of these methods are of practical use, and can be segregated into several generalized categories, as outlined below:
Measurements obtained by direct comparison of the specimen dimensions to a micrometer scale in the x-y plane of the microscope (for example, the use of calibrated mechanical stages and specialized measuring microscopes). Mechanical stages enable movement in both the x and y axes, and often employ a vernier scale that allows reading of the stage displacement with an accuracy of 0.1 millimeter (the accuracy of the method).
Techniques that utilize projected real images and those made by means of a traditional or digital camera system combined with a stage micrometer. Because the micrometer scale is not viewed simultaneously with the specimen, an image of the micrometer must be recorded by means of a photomicrograph or a digital camera system. This technique is very reproducible, often yielding results that are accurate to a micrometer or less.
Linear comparisons obtained by projecting a measuring scale into the field of view or by inclusion of objects having a known size with the specimen. Often, homogeneous preparations of polystyrene or glass beads can be included with specimens, such as erythrocytes, to provide a size reference. Measurements are then performed utilizing a photomicrograph or digital image. The accuracy of this method is variable and depends on the homogeneity of the comparison objects.
Direct specimen measurements made by means of graduated scales located within the microscope, such as eyepieces containing fixed or moveable reticles (the most common method). Reticles must be calibrated together with a stage micrometer, but provide an accuracy of approximately 2-10 micrometers (3 to 5 percent, depending on magnification and the resolution of the stage micrometer).
Calibrated microscope slides and counting chambers are utilized for direct linear measurements or for counting the density of specimen particles. Accuracy depends on the separation distance between ruled lines, but averages between 10 and 50 micrometers.
Fixed dimensions of the microscope can be employed to produce a very rough estimate of specimen dimensions. By measuring or calculating the viewfield size, the relative linear dimensions of a mounted specimen can be determined.
Determination of vertical distances along the microscope optical axis (z-direction) by utilization of a calibrated fine focus adjustment on the microscope. This technique is often complicated by refraction artifacts and spherical aberration, but can provide an average accuracy level of several micrometers.
A number of the techniques that are commonly employed for the measurement of objects (specimens), both in the microscope and in everyday surroundings, involve the principle of a transfer scale. Direct comparison measurements require access to the object under scrutiny, and an accurate ruler or graduated scale. If a measurement is necessary and no suitable ruler is available for comparison, a transfer scale can often be employed to determine critical dimensions. The transfer scale may be any suitable substitute that can be placed in contact with the object, allowing the length (or width) of the object to be directly compared, or transferred, onto the transfer scale. The absolute dimension of the object is later determined by comparison to a calibrated scale (or ruler). If the transfer scale itself is marked with graduations in arbitrary units, then the graduations must be referenced to absolute units by comparison to a standard.
Introduction to Fluorescence Microscopy
The absorption and subsequent re-radiation of light by organic and inorganic specimens is typically the result of well-established physical phenomena described as being either fluorescence or phosphorescence. The emission of light through the fluorescence process is nearly simultaneous with the absorption of the excitation light due to a relatively short time delay between photon absorption and emission, ranging usually less than a microsecond in duration. When emission persists longer after the excitation light has been extinguished, the phenomenon is referred to as phosphorescence.
Properties of Microscope Objectives
Three critical design characteristics of the objective set the ultimate resolution limit of the microscope. These include the wavelength of light used to illuminate the specimen, the angular aperture of the light cone captured by the objective, and the refractive index in the object space between the objective front lens and the specimen.
Presented in Figure 1 is a cut-away diagram of a microscope objective being illuminated by a simple two-lens Abbe condenser. Light passing through the condenser is organized into a cone of illumination that emanates onto the specimen and is then transmitted into the objective front lens element as a reversed cone. The size and shape of the illumination cone is a function of the combined numerical apertures of the objective and condenser. The objective angular aperture is denoted by the Greek letter and will be discussed in detail below.
Introduction to Stereomicroscopy
The first stereoscopic-style microscope having twin eyepieces and matching objectives was designed and built by Cherubin d'Orleans in 1671, but the instrument was actually a pseudostereoscopic system that achieved image erection only by the application of supplemental lenses.
Introduction to Confocal Microscopy
Confocal microscopy offers several advantages over conventional optical microscopy, including controllable depth of field, the elimination of image degrading out-of-focus information, and the ability to collect serial optical sections from thick specimens. The key to the confocal approach is the use of spatial filtering to eliminate out-of-focus light or flare in specimens that are thicker than the plane of focus. There has been a tremendous explosion in the popularity of confocal microscopy in recent years, due in part to the relative ease with which extremely high-quality images can be obtained from specimens prepared for conventional optical microscopy, and in its great number of applications in many areas of current research interest.
Confocal microscopy offers several advantages over conventional optical microscopy, including shallow depth of field, elimination of out-of-focus glare, and the ability to collect serial optical sections from thick specimens. In the biomedical sciences, a major application of confocal microscopy involves imaging either fixed or living cells and tissues that have usually been labeled with one or more fluorescent probes.
When fluorescent specimens are imaged using a conventional widefield optical microscope, secondary fluorescence emitted by the specimen that appears away from the region of interest often interferes with the resolution of those features that are in focus. This situation is especially problematic for specimens having a thickness greater than about 2 micrometers. The confocal imaging approach provides a marginal improvement in both axial and lateral resolution, but it is the ability of the instrument to exclude from the image the "out-of focus" flare that occurs in thick fluorescently labeled specimens, which has caused the recent explosion in popularity of the technique. Most current confocal microscopes are relatively easy to operate and have become part of the basic instrumentation of many multi-user imaging facilities. Because the resolution possible in the laser scanning confocal microscope (LSCM) is somewhat better than in the conventional widefield optical microscope, but still considerably less than that of the transmission electron microscope, it has in some ways bridged the gap between the two more commonly used techniques. Figure 1 illustrates the principal light pathways in a basic confocal microscope configuration.
Anatomy & Physiology of a Flexible Scope: Anatomical Structure
The anatomical structure of the flexible endoscope is the outer shell, composed of five basic sections: light guide connector, light guide tube, control body, insertion tube and bending section. Each section is constructed differently but each has a protective outer layer and hollow inner area that contains the delicate internal systems.
Light Guide Connector-The light guide connector is the solid end of the scope that connects to the light source or video cart system. It is the scope section most distant from the patient. On most models it is constructed of molded plastic and metal components with no moving parts. In some models, circuit boards may also be housed. The suction, air, water, light, video system and electrical ground all connect to the scope here. Damage to this part of the endoscope is most often associated with impact and least often with typical day to day use.
Light Guide Tube - Sometimes called the universal cord, this hollow tube connects the light guide connector to the control body. Some videoscope models may have two light guide tubes as the scopes may have separate connections to the light source and video processor. Multiple layers of steel coil, fiber mesh and vulcanized rubber are bonded together to make up this tube. A thin layer of clear polyurethane is applied to the tube for chemical and fluid resistance.
The light guide tube provides protection to the delicate internal components such as suction channels and fiber optics, and it is designed to accept some bending and twisting. However, over-twisting or tight coiling can result in tube damage as well as to the internal components. The diameter and length of the light guide tube is similar on most GI endoscope models.
Medical uses of Endoscopes and Lasers
The operation of optical fibres
Optical fibres are narrow tubes of glass fibres with a plastic coating that carry light from one end to the other. The light bounces off the walls of the fibre and can even bounce around corners. The properties of optical fibres make them useful for a wide range of applications including:
- Medical - to transmit pictures of organs and arteries
- Industrial - to transmit pictures of the inside of complex machinery
- Communications - to transmit data over long distances without transmission loss
Light rays use total internal reflection to travel along the fibres. In order for this to be achieved, the light ray must hit the walls of the fibre at a minimum angle of 82°, which is the critical angle for light travelling from glass to plastic. Since the fibres are very narrow, this is usually not a problem.
The parts of an endoscope
The shaft is only 10mm in diameter and can be up to 2 metres long. It is flexible and coated in steel and plastic in order to make it waterproof, prevent chemical damage and to make it easy to manoeuvre through the body. It has contains:
· Fibre optic bundles
Light is guided to the area under investigation by non-coherent fibre optic bundles (bundles where the optical fibres are not lined up at both ends). However, the image must be transmitted back by a coherent fibre optic bundle (a bundle where the optical fibres are lined up at both ends of the fibre so that an image can be transmitted). In order to produce a clear image, the shaft contains up to 10 000 fibres!
· Water Pipes
Carry water to wash the lens and keep the view clear.
· Operations channel
Carries accessories to the distil end for surgery.
· Control cables
Controls which way the distil end is bent.
· Aditional optional channel
Carries air or carbon dioxide to and from the distil end
The distil end is inserted into the patient's body. There are controls on the viewing end to make it bend in the desired direction. The image is focused by a lens on the end.
Uses of endoscopes
Five medical procedures carried out using an endoscope:
The endoscope is inserted through an incision in the skin near a joint under investigation. This can be used to look at the joint and preform operations such as removing torn tissues.
The endoscope is inserted through bronchial tubes within the lungs in order to look at the airway and to remove any objects blocking the airway.
· Endoscope Biopsy
The endoscope is inserted through an incision or opening in the body that leads to the area under investigation. Biopsy forceps are then used to take a sample of tissue that can then be analysed by a pathologist.
· Gastroscopy (Also called Oesophagogastroduodenoscopy)
The endoscope is inserted down the throat to look for problems with the oesophagus, stomach and duodenum such as bleeding or ulcers.
The endoscope is inserted through an incision in the abdominal in order to look at abdominal organs and preform minor surgery.
Advantages of using endoscopes
The use of endoscopes is much less invasive than open surgery because only a small incision in the body is required where as open surgery requires deep incisions. This also means that recovery is quicker and there is less swelling, scarring and risk of infection. Endoscopes can be used by an outpatients department and does not need to be done by a hospital. This reduces costs.
Keyhole surgery involves the use of lasers with endoscopes. Lasers are useful for such surgery because:
- They can shine high intensity light down an endoscope that can be focussed for cutting or destroying tissue.
- They produce heat that causes the tissue around the cut to seal and prevents bleeding.
- The beam is narrow and can therefore make very precise and accurate cuts.
- Different frequency lasers can be used depending on the area being targeted.
Other medical uses for lasers
Due to their high intensity, narrow beam and high precision, lasers can be used for surgery that was previously extremely difficult and dangerous. They are now used in cosmetic surgery for removal of scars, wrinkles, birthmarks, blood vessels and hair and can also be used in laser eye surgery to alter the surface of the cornea on a microscopic scale in order to correct sight.
Images of internal organs obtained using an endoscope
This image was taken of a patient with hematemesis. This image revealed that it was due to a bleeding ulcer in the oesophagus.
This image shows an elastic hair tie that was swallowed by a patient.
This is an image of a sessile polyp in a patient's duodenum. By using biopsy, it was found that it was a tubulovillous adenoma.
An endoscopy involves examining the inside of a person's body using an endoscope. An endoscope is a medical device consisting of a long, thin, flexible (or rigid) tube which has a light and a video camera. Images of the inside of the patient's body can be seen on a screen. The whole endoscopy is recorded so that doctors can check it again. Endoscopy is a minimally invasive diagnostic medical procedure. It is used to examine the interior surfaces of an organ or tissue.
The endoscope can also be used for enabling biopsies and retrieving foreign objects. Endoscopy is a noninvasive alternative to surgery for foreign object removal from the gastrointestinal tract.
When is an endoscopy used?
To confirm a diagnosis
An endoscopy is often used to confirm a diagnosis when other devices, such as an MRI, X-ray, or CT scan are considered inappropriate.
An endoscopy is often carried out to find out the degree of problems a known condition may have caused. The endoscopy, in these cases, may significantly contribute towards the doctor's decision on the best treatment for the patient.
The following conditions and illnesses are most commonly investigated or diagnosed with an endoscopy:
- Breathing disorders
- Chronic diarrhea
- Internal bleeding
- Irritable bowel syndrome
- Stomach ulcers
- Urinary tract infections
Endoscopies are commonly used for the diagnosis of cancer. They are used for biopsies - taking samples of tissue to find out whether it is cancerous. Thanks to an endoscope, biopsies of the intestines or lungs can be done without the need for major surgery. This study explains that colonoscopy is the most effective screening option for colorectal cancer.
Some surgical procedures can be carried out with a modified endoscope, such as the removal of the gallbladder, tying and sealing the fallopian tubes, and taking out small tumors and foreign objects from the lungs or digestive system. A study found that the removal through endoscopy of tumors that affect only the superficial layers of the esophagus can avoid complete extirpation of this part of the digestive tract.
A laparoscope is a type of endoscope which is used for keyhole surgery or laparoscopic surgery. Laparoscopic surgery requires only a small incision and is commonly used today for appendectomies, hysterectomies, and prostatectomies. Patients lose much less blood during and after surgery and recover much faster, compared to other surgical procedures.
Short history of endoscopy
Reports indicate that the first endoscope was devised in 1805. It consisted of a large tube and a candle. Because it was cumbersome and large it had very limited uses. Fiber optics, which appeared in the 1960s, was a major factor in the endoscopy revolution. With fiber optics it really became possible for the doctor to see and record the inside of the patient's body with a small and relatively painless device.
Endoscopy has many uses today
An endoscope can be fitted with surgical instruments; it can send pulses or heat and electricity and destroy small tumors or gallstones. Specialized endoscopes have their own names, such as:
- Bronchoscopes - they examine the air passages and the lungs.
- Colonoscopes - they examine the colon.
- Gastroscopes - they examine the small intestine, stomach and esophagus (throat).
- Arthroscopes - they examine the joints.
- Hysteroscopes - they examine a woman's uterus.
- Cystoscopes - they examine the urinary bladder.
Types of endoscopies
Here is a list of some types of endoscopies and their meanings:
- Amnioscopy - examination of the amniotic cavity and fetus.
- Arthroscopy - examination of the joints.
- Bronchoscopy - examination of the air passages and the lungs.
- Colonoscopy - examination of the colon.
- Colposcopy - examination of the cervix and the tissues of the vagina and vulva.
- Cystoscopy - examination of the urinary bladder.
- EGD (Esophageal Gastroduodenoscopy), also known as panendoscopy - examination of the esophagus, stomach and duodenum.
- ERCP (endoscopic retrograde cholangio-pancreatography) - examination of the liver, gallbladder, bile ducts, and pancreas.
- Fetoscopy - examination of the fetus.
- Laparoscopy - a small incision to examine the abdominal cavity.
- Laryngoscopy - examination of the back of the throat, including the voice box (larynx) and vocal cords.
- Proctoscopy - examination of the rectum and the end of the colon.
- Rhinoscopy - examination of the inside of the nose.
- Thoracoscopy - examination of the lungs or other structures in the chest cavity.
Endoscope the patient swallows - wireless capsule endoscopy
The patient swallows a capsule which wirelessly sends images of the inside of his/her stomach and digestive tract. Eventually the capsule will exit the patient when he/she has a bowel motion. Currently, capsule endoscopy is used to find out why a patient is bleeding in their digestive system, with no clear cause. It can also be used to diagnose GERD (Gastroesophageal reflux disease). A study indicated that capsule endoscopy is effective in diagnosing gastrointestinal bleeding and small bowel Crohn's disease in children. Another study showed how wireless capsule endoscopy is turning up Crohn's diseases diagnoses among patients whose illness had not been spotted for over a decade.
The capsule is about the size of a multi-vitamin and has a camera attached to it. As it moves through the digestive tract it takes pictures. The patient wears a small data recorder on his/her belt; this recorder receives the data from the capsule. The pictures are later downloaded and interpreted by a doctor.
Before swallowing the capsule the patient must have fasted overnight. The next day he/she will be fitted with the equipment, which includes a belt that contains a battery and a data recorder. Leads will be hooked up to the abdomen; this is painless and does not perforate the skin. As soon as the equipment is hooked up the patient swallows the capsule. Eight hours later he/she returns and the equipment is disconnected.
The main components of an endoscope
An endoscope consists of:
- A flexible or rigid tube.
- A light that illuminates what the doctor wants to examine. The light is delivered via an optical fiber system.
- A lens system that transmits an image to the viewer from the fiberscope.
- Another channel to allow the entry of medical instruments or manipulators.
What happens during an endoscopy?
The patient may be asked to fast (not eat) or drink for a period before the endoscopy if the instrument is going to go in through the anus. In some cases the patient may be given a laxative. Some patients are given antibiotics to prevent infection.
Patients on blood-thinning medications, such as warfarin, may be asked to stop taking them for a number of days before their endoscopy. There is a risk the blood thinner may cause excessive bleeding during the procedure. It is important the patient only does so if the doctor tells him/her. A study concluded that anti-inflammatory drugs, such as aspirin, do not increase the patient's risk of bleeding during an endoscopy.
In the UK most endoscopies are done in hospital, or some large GP (general practice) clinics.
The vast majority of endoscopies do not require a general anesthetic. Some patients may receive a local anesthetic. A study found that administering a lidocaine lollipop as a single-agent anesthetic to patients undergoing an upper gastrointestinal endoscopy procedure eliminated the need for sedation in the majority of patients. Patients describe the procedure as possibly 'uncomfortable', but hardly ever 'painful'. This study explains that the use of an evidence-based sedation protocol for endoscopic procedures improves the quality of practice and reduces the incidence of sedation-related adverse events.
Most endoscopes will enter the patient via the:
- Urethra (urine exits the body through the urethra)
- A small incision made in the skin
In most cases endoscopies will last from 15 to 60 minutes. The patient rarely has to spend the night in hospital. Some patients may notice some blood in their urine after a cystoscopy (bladder examination) or when they pass a stool after a prostate biopsy, for example - this is normal for a few days.
Most patients can get up within an hour of their endoscopy. It is advisable that the patient does not drive out of the hospital after an endoscopy.
What are the complications of an endoscopy?
According to the National Health Service (NHS), UK, less than 1% of endoscopies have complications. When they do occur, they may include:
- An infection, possibly somewhere along the path of the endoscope.
- Piercing or tearing of an organ. This may require subsequent surgery. This article explains how tears and perforations caused by endoscopy can be fixed without invasive surgery.
- Bleeding more than normally expected. This may require subsequent surgery.
- An allergy to the anesthetic. Antihistamines may be used to treat this.
The following signs may indicate an infection has developed after the endoscopy:
Any patient who experiences these signs after an endoscopy should contact their doctor. A course of antibiotics should clear up the infection.
Hand held refractometers are one of the most popular analytical devices. They are used in many places - like in wine growing and wine making, both by professionals and amateurs, in beer brewing, in garages to check battery electrolyte and cooling liquid quality, and so on. They are very simple in use and give almost instant result, without tedious and costly laboratory procedures to follow.
Hand held refractometer - working principle. Note that real devices usually contain additional optical elements, like lenses and optical wedges, that help to obtain sharp shadow boundary.
Instead of having an illuminating prism, hand held reflractometers have an illuminator flap which produces a diffused light at a grazing angle and helps to keep the sample in place. Light passes through the sample, enters the measuring prism and possibly other lenses, and finally falls on the measuring scale where it can be read. Depending on the reason for using the refractometer, its scale can be graduated in Brix degrees, percentage of alcohol or glycol percentage, etc.
To take care of temperature differences, simple hand held refractometers have to be either calibrated before taking measurements (using calibration screw and distilled water), or the result have to be converted using a temperature corrections table (which requires separate temperature measurement). However, many refractometers have built in temperature compensation - either scale or additional optical wedge are mounted on the bimetallic strip, which bends when the temperature changes, compensating for changes of refractive index. That makes them much easier to use.
Measurement technique is very simple. First, you open the illuminator flap (it is connected to the device by a small hinge) and put a sample on the measurement prism surface. To put the sample on th prism you can use a pipette, but when taking measurements in the field even squeezing a few drops of juice from the fruit will do. After the flap is closed, you look through the eyepiece, and read result from the scale. That's all. For easier reading it may be necessary to place the refractometer in the direction of some light source (like Sun or lamp), but during a day ambient light is usually strong enough. After finishing measurement, you should wipe dry prism and flap with a clean, soft cloth.
Nikon's binoculars have received high evaluation because of their excellent optical system. Nikon knows that a bright image and sharp details are the priority of binoculars, and makes utmost efforts to achieve this. Correcting lens aberration is vitally important.
Nikon's binoculars are designed to correct the aberration described below properly to realize the brightest and sharpest image.
A bundle of light rays coming from one point on the optical axis is focused at a different place than the focused point depending on the distance from the optical axis when the light incidents. This deviation is caused by variations in angles of each incident light ray, and is called spherical aberration.
Suppose a screen is placed at P' in the illustration above, the image of P will not be a focused point, but a blurred circle. Making the lens diameter smaller reduces such spherical aberration.
- *Photos are simulated viewfinder images.
This is caused by the difference in distance of the incident light from the optical axis. While spherical aberration is caused by a difference in focused point, coma is caused by a difference in magnification. A point image such as a star tails toward the exterior like a comet, which "coma" refers to. Coma strongly influences image quality in the periphery of the field.
- *Photos are simulated viewfinder images.
The meridional plane (which contains the optical axis
of the lens) and the sagittal plane (which is vertical to the meridional plane)
have different radii. Therefore, the meridional and sagittal rays have
different focal points.
This is called an astigmatism aberration. When a latticepatterned object is viewed using a lens with an astigmatism, horizontal stripes appear in focus and vertical stripes appear out of focus. Conversely, when horizontal stripes appear out of focus, vertical stripes are in focus.
Since astigmatism increases in proportion to the incident angle squared, astigmatism greatly influences image quality at the peripheral area of binoculars with a wide field of view.
Horizontal stripes appear out of focusVertical stripes appear out of focus
- *Photos are simulated viewfinder images.
Curvature of Field
In the case of a lens fully compensated for coma aberration and astigmatism, the light rays coming from a point apart from the optical axis are focused at one point. But this point is not always included in the vertical plane to the optical axis. This is called Curvature of Field. With a lens having this aberration, even if you focus around the center of the field, the periphery of the field appears out of focus. It can cause very bad effects especially on wide-field-type binoculars.
- *Photos are simulated viewfinder images.
Distortion is caused by variations in the magnification
of the image depending on the distance from the optical axis. There are two
types of distortion: positive and negative.
This image distortion, irrespective of image visibility, increases in proportion to the incident angle cubed.
- *Photos are simulated viewfinder images.
This is caused by a difference in light wavelength.
The focal point or magnification of a lens varies according to the wavelength
of each type of incident light. Therefore, if you look at an image through a
lens with chromatic aberration, color fringing may occur.
Because a single lens cannot compensate for chromatic aberration, two lenses of different optical characteristics are combined to correct this aberration.
Nikon's original ED (Extra-low-Dispersion) glass lenses effectively compensate for color fringing.
- *Photos are simulated viewfinder images.
ED glass and secondary spectrum
Visible light is composed of
lights of various wavelengths. Gathering up all of these lights to a point is
ideal for objective lenses.
With a single lens, because light is bent in the same way as with a prism, the focal lengths of lights with different wavelengths vary. As a result, not all light rays reach the same point, which causes chromatic aberration.
An achromatic lens made with conventional glass materials can match focal lengths of two different wavelengths. For red and blue colors, for example, that contain both ends of the wavelengths of visible light, chromatic aberration can be reduced to a certain extent by conforming their focal lengths. However, with more detailed examination, because light with other wavelengths such as green has different focal lengths, residual chromatic aberration results. This residual chromatic aberration is known as secondary spectrum.
Combinations of conventional glasses cannot solve this secondary spectrum problem, but particular optical materials which have a unique characteristic of dispersion are needed.
ED (Extra-low Dispersion) glass has this unique characteristic and when combined with other glasses minimizes the effects of the secondary spectrum. Comparing to achromatic lenses, ED glass reduces chromatic aberration to a remarkable degree.
A refractometer is a laboratory or field device for the measurement of an index of refraction (refractometry). The index of refraction is calculated from Snell's law and can be calculated from the composition of the material using the Gladstone–Dale relation.
Types of refractometers
There are four main types of refractometers: traditional handheld refractometers, digital handheld refractometers, laboratory or Abbe refractometers, and inline process refractometers. There is also the Rayleigh Refractometer used (typically) for measuring the refractive indices of gases.
In drug diagnostics, a refractometer is used to measure the specific gravity in human urine.
In gemology, a refractometer is used to help identify gem materials by measuring their refractive index.Gemstones are transparent minerals and can therefore be examined using optical methods. As the refractive index is a material constant dependent on the chemical composition of a substance, it provides information on the type and quality of a gemstone. Classification with a special gemstone refractometer is an easy-to-use method with which the authenticity and quality of a stone can be evaluated. The gemstone refractometer is therefore a piece of basic equipment in a gemological laboratory. Due to the dependence of the refractive index (dispersion) on the wavelength of the light used, the measurement is normally taken at the wavelength of the sodium-D-line (NaD) of 589 nm. This is either filtered out from daylight or generated with a monochromatic light-emitting diode (LED). Certain stones such as rubies, sapphires, tourmalines and topaz are optically anisotropic. They demonstrate birefringence based on the polarisation plane of the light. The two different refractive indexes are classified using a polarisation filter. Gemstone refractometers are available both as classic optical instruments and as electronic measurement devices with a digital display.
Gemology refractometer ER604 used to test light bending in gemstones; courtesy of A.KRÜSS Optronic GmbH
a wine grower with refractometer
density evaluation of abdominal fluid of a cat with FIP by a refractometer.
Automatic benchtop refractometer
Schematic setup of an automatic refractometer: An LED light source is imaged under a wide range of angles onto a prism surface which is in contact with a sample. Depending on the difference in the refractive index between prism material and sample the light is partly transmitted or totally reflected. The critical angle of total reflection is determined by measuring the reflected light intensity as a function of the incident angle - Source of image: Anton Paar GmbH, www.anton-paar.com
Automatic refractometers automatically measure the refractive index of a sample. The automatic measurement of the refractive index of the sample is based on the determination of the critical angle of total reflection. A light source, usually a long-life LED, is focused onto a prism surface via a lens system. An interference filter guarantees the specified wavelength. Due to focusing light to a spot at the prism surface, a wide range of different angles is covered. As shown in the figure "Schematic setup of an automatic refractometer" the measured sample is in direct contact with the measuring prism. Depending on its refractive index, the incoming light below the critical angle of total reflection is partly transmitted into the sample, whereas for higher angles of incidence the light is totally reflected. This dependence of the reflected light intensity from the incident angle is measured with a high-resolution sensor array. From the video signal taken with the CCD sensor the refractive index of the sample can be calculated. This method of detecting the angle of total reflection is independent on the sample properties. It is even possible to measure the refractive index of optical dens strongly absorbing samples or samples containing air bubbles or solid particles . Furthermore, only a few microliters are required and the sample can be recovered. This determination of the refraction angle is independent of vibrations and other environmental disturbances.
Influence of Wavelength
The refractive index of a certain sample varies for nearly all materials for different wavelengths. This dispersion relation is characteristic for every material. In the visible wavelength range a decrease of the refractive index and nearly no absorption is observable. In the infrared wavelength range several absorption maxima and fluctuations in the refractive index appear. To guarantee a high quality measurement with an accuracy of up to 0.00002 in the refractive index the wavelength has to be determined correctly. Therefore, in modern refractometers the wavelength is tuned to a bandwidth of +/-0.2 nm to ensure correct results for samples with different dispersions.
Modern Automatic Refractometers - Source of image: Anton Paar GmbH, www.anton-paar.com
Influence of Temperature
Temperature has a very important influence on the refractive index measurement . Therefore, the temperature of the prism and the temperature of the sample have to be controlled with high precision. There are several subtly different designs for controlling the temperature but there are some key factors common to all such as high precision temperature sensors and Peltier devices to control the temperature of the sample and the prism. The temperature control accuracy of these devices should be designed so that the variation in sample temperature is small enough that it will not cause a detectable refractive index change.
External water baths were used in the past but are no longer needed.
Refractometer with funnel type sampler
- Flow cell with filling funnel for an automatic refractometer assures fast sample exchange, e.g. in quality control - Source of image: Rudolph Research Analytical - www.rudolphresearch.com
Extended possibilities of automatic refractometers
Automatic refractometers are microprocessor-controlled electronic devices. This means they can have a high degree of automation and also be combined with other measuring devices
There are different types of sample cells available, ranging from a flow cell for a few microliters to sample cells with a filling funnel for fast sample exchange without cleaning the measuring prism in between. The sample cells can also be used for the measurement of poisonous and toxic samples with minimum exposure to the sample. Micro cells require only a few microliters volume, assure good recovery of expensive samples and prevent evaporation of volatile samples or solvents. They can also be used in automated systems for automatic filling of the sample onto the refractometer prism. For convenient filling of the sample through a funnel, flow cells with a filling funnel are available. These are used for fast sample exchange in quality control applications.
Automatic Sample Feeding
Automatic refractometer with sample changer for automatic measurement of a large number of samples - Source of image: Anton Paar GmbH, www.anton-paar.com
Once an automatic refractometer is equipped with a flow cell, the sample can either be filled by means of a syringe or by using a peristaltic pump. Modern refractometers have the option of a built-in peristaltic pump. This is controlled via the instrument‘s software menu. A peristaltic pump opens the way to monitor batch processes in the laboratory or perform multiple measurements on one sample without any user interaction. This eliminates human error and assures a high sample throughput.
If an automated measurement of a large number of samples is required, modern automatic refractometers can be combined with an automatic sample changer. The sample changer is controlled by the refractometer and assures fully automated measurements of the samples placed in the vials of the sample changer for measurements.
Measuring combination of an automatic refractometer and a density meter as widely used in the flavors and fragrances industry - Source of image: Anton Paar GmbH, www.anton-paar.com
Today’s laboratories do not only want to measure the refractive index of samples, but several additional parameters like density or viscosity to perform efficient quality control. Due to the microprocessor control and a number of interfaces, automatic refractometers are able to communicate with computers or other measuring devices, e.g. density meters, pH meters or viscosity meters, to store refractive index data and density data (and other parameters) into one database.
Automatic refractometers do not only measure the refractive index, but offer a lot of additional software features, like
- Instrument settings and configuration via software menu
- Automatic data recording into a database
- User-configurable data output
- Export of measuring data into MS Excel data sheets
- Statistical functions
- Predefined methods for different kinds of applications
- Automatic checks and adjustments
- Check if sufficient amount of sample is on the prism
- Data recording only if the results are plausible
Pharma Documentation and Validation
Typical Pharma Validation and Qualification Folder - Source of image: Anton Paar GmbH, www.anton-paar.com
Refractometers are often used in pharmaceutical applications for quality control of raw intermediate and final products. The manufacturers of pharmaceuticals have to follow several international regulations like FDA 21 CFR Part 11, GMP, Gamp 5, USP<1058>, which require a lot of documentation work. The manufacturers of automatic refractometers support these users providing instrument software fulfills the requirements of 21 CFR Part 11, with user levels, electronic signature and audit trail. Furthermore, Pharma Validation and Qualification Packages are available containing
- Qualification Plan (QP)
- Design Qualification (DQ)
- Risk Analysis
- Installation Qualification (IQ)
- Operational Qualification (OQ)
- Check List 21 CFR Part 11 / SOP
- Performance Qualification (PQ)
When light travels from one medium to another it changes velocity and direction a bit. If you’ve ever looked at a spoon in a glass of water, the image of the spoon in water is displaced a bit from the image of the spoon in air, and the spoon looks broken. When the light rays travel from the spoon in water and break out into the air, they are refracted, or shifted (Fig. 102). If we take the ratio of the sine of the angles formed when a light ray travels from air to water, we get a single number, the index of refraction, or refractive index. Because we can measure the index of refraction to a few parts in 10,000, this is a very accurate physical constant for identification of a compound.
The refractive index is usually reported as nff, where the tiny 25 is the temperature at which the measurement was taken, and the tiny capital D means we’ve used light from a sodium lamp, specifically a single yellow frequency called the sodium D line. Fortunately, you don’t have to use a sodium lamp if you have an Abbe refractometer.
Fig. The refraction of light.
THE ABBE REFRACTOMETER
The refractometer looks a bit like a microscope. It has
1. An eyepiece. You look in here to make your adjustments and read the refractive index.
2. Compensation prism adjustment. Since the Abbe refractometer uses white light and not light of one wavelength (the sodium D line), the white light disperses as it goes through the optics and rainbow-like color fringing shows up when you examine your sample. By turning this control, you rotate some compensation prisms that eliminate this effect.
3. Hinged sample prisms. This is where you put your sample.
4. Light source• This provides light for your sample. It’s on a moveable arm, so you can swing it out of the way when you place your samples on the prisms.
5. Light source swivel arm lock. This is a large slotted nut that works itself loose as you move the light source up and down a few times. Always have a dime handy to help you tighten this locking nut when it gets loose.
6. Sample and scale image adjust. You use this knob to adjust the optics such that you see a split field in the eyepiece. The refractive index scale also moves when you turn this knob. The knob is often a dual control; use the outer knob for a coarse adjustment and the inner knob as a fine adjustment.
7. Scale/sample field switch. Press this switch, and the numbered refractive index scale appears in the eyepiece. Release this switch, and you see your sample in the eyepiece. Some models don’t have this type of switch. You have to change your angle of view (shift your head a bit) to see the field with the refractive index reading.
8. Line cord on – off switch. This turns the refractometer light source on and off.
9. Water inlet and outlet. These are often connected to temperature-controlled water recirculating baths. The prisms and your samples in the prisms can all be kept at the temperature of the water.
Absorption (electromagnetic radiation)
An overview of electromagnetic radiation absorption. This example discusses the general principle using visible light as specific example. A white light source — emitting light of multiple wavelengths — is focused on a sample (the pairs of complementary colors are indicated by the yellow dotted lines). Upon striking the sample, photons that match the energy gap of the molecules present (green light in this example) are absorbed, exciting the molecules. Other photons are transmitted unaffected and, if the radiation is in the visible region (400–700 nm), the transmitted light appears as the complementary color (here red). By recording the attenuation of light for various wavelengths, an absorption spectrum can be obtained.
In physics, absorption of electromagnetic radiation is the way in which the energy of a photon is taken up by matter, typically the electrons of an atom. Thus, the electromagnetic energy is transformed into internal energy of the absorber, for example thermal energy. The reduction in intensity of a light wave propagating through a medium by absorption of a part of its photons is often called attenuation. Usually, the absorption of waves does not depend on their intensity (linear absorption), although in certain conditions (usually, in optics), the medium changes its transparency dependently on the intensity of waves going through, and saturable absorption (or nonlinear absorption) occurs. Quantifying absorption
There are a number of ways to quantify how quickly and effectively radiation is absorbed in a certain medium, for example:
- The absorption coefficient, and some closely related derived quantities:
- The attenuation coefficient, which is sometimes but not always synonymous with the absorption coefficient
- Molar absorptivity, also called "molar extinction coefficient", which is the absorption coefficient divided by molarity (see also Beer–Lambert law).
- The mass attenuation coefficient, also called "mass extinction coefficient", which is the absorption coefficient divided by density (see also mass attenuation coefficient).
- The absorption cross section and scattering cross-section are closely related to the absorption and attenuation coefficients, respectively.
- "Extinction" in astronomy is equivalent to the attenuation coefficient.
- Penetration depth and skin effect,
- Propagation constant, attenuation constant, phase constant, and complex wavenumber,
- Complex refractive index and extinction coefficient,
- Complex dielectric constant,
- Electrical resistivity and conductivity.
- Absorbance (also called "optical density") and optical depth (also called "optical thickness") are two related measures of the total light-blocking power of a certain medium with a certain thickness.
- Percentage of the incoming light which gets absorbed.
All these quantities measure, at least to some extent, how well a medium absorbs radiation. However, practitioners of different fields and techniques tend to conventionally use different quantities drawn from the list above.
The absorbance of an object quantifies how much of the incident light is absorbed by it (instead of being reflected or refracted). This may be related to other properties of the object through the Beer–Lambert law.
Precise measurements of the absorbance at many wavelengths allow the identification of a substance via absorption spectroscopy, where a sample is illuminated from one side, and the intensity of the light that exits from the sample in every direction is measured. A few examples of absorption spectroscopy, in different parts of the spectrum, are ultraviolet–visible spectroscopy, infrared spectroscopy, and X-ray absorption spectroscopy.
Understanding and measuring the absorption of electromagnetic radiation has a variety of applications. Here are a few examples:
- In meteorology and climatology, global and local temperatures depend in part on the absorption of radiation by atmospheric gases (such as in the greenhouse effect) and the ground (see albedo).
- In medicine, X-rays are absorbed to different extents by different tissues (bone in particular), which is the basis for X-ray imaging.For example, see computation of radiowave attenuation in the atmosphere used in satellite link design.
- In chemistry and materials science because different materials and molecules will absorb radiation to different extents at different frequencies, which allows for material identification.
- In optics, sunglasses, colored filters, dyes, and other such materials are designed specifically with respect to which visible wavelengths they absorb, and in what proportions.
- In biology, photosynthetic organisms require that light of the appropriate wavelengths be absorbed within the active area of chloroplasts, so that the light energy can be converted into chemical energy within sugars and other molecules.
Fig. 103 One model of a refractometer.
USING THE ABBE REFRACTOMETER
1. Make sure the unit is plugged in. Then turn the on – off switch to ON. The light at the end of the moveable arm should come on.
2. Open the hinged sample prisms. NOT touching the prisms at all, place a few drops of your liquid on the lower prism. Then, swing the upper prism back over the lower one and gently close the prisms. Never touch the prisms with any hard object or you’ll scratch them.
3. Raise the light on the end of the moveable arm so that the light illuminates the upper prism. Get out your dime and, with permission of your instructor, tighten the light source swivel arm lock nut as it gets tired and lets the light drop.
Fig. 104 Your sample through the lens of the refractor.
4. Look in the eyepiece. Slowly, carefully, with very little force, turn the large scale and sample image adjust knob from one end of its rotation to the other. Do not FORCE! (If your sample is supposedly the same as that of the last person to use the refractometer, you shouldn’t have to adjust this much if at all.)
5. You are looking for a split optical field of light and dark (Fig. 104). This may not be very distinct. You may have to raise or lower the light source and scan the sample a few times.
6. If you see color fringing at the boundary between light and dark (usually red or blue), slowly turn the compensating prism adjust until the colors are at a minimum. You may now have to go back and readjust the sample image knob a bit after you do this.
7. Press and hold the scale/sample field switch. The refractive index scale should appear (Fig. 105). Read the uppermost scale, the refractive index, to four decimal places. (If your model has two fields, with the refractive index always visible, just read it.)
Fig. 105 A refractive index of l .4398.
1. The refractive index changes with temperature. If your reading is not the same as that of a handbook, check the temperatures before you despair.
2. Volatile samples require quick action. Cyclohexene, for example, has been known to evaporate from the prisms of unthermostatted refracto-meters more quickly than you can obtain the index. It may take several tries as you readjust the light, turn the sample and scale image adjust, and so on.
3. Make sure the instrument is level. Often organic liquids can seep out of the jaws before you are ready to make your measurement.