Journey To Optical Light Microscopy

Magnifying lenses have been known for as long as taped history, but it was not until the awaited arrival of the modern compound optical light microscope that the device was used in biology. Lets begin our journey to optical light microscopy.

LENS :

  • Lens focus light rays at a specific place called the focal point.
  • The distance between center of lens and focal point is the focal length.
  • The strength of lens related to focal length i.e.  short focal length, more magnification.
light focus and magnification

Converging Lens

Field of View :

The field of view (also field of vision, abbreviated FOV) is the extent of the observable world that is seen at any given moment. In case of optical instruments or sensors it is a solid angle through which a detector is sensitive to electromagnetic radiation.

Vertical and Horizontal Field of View

Vertical and Horizontal FOV

Difference between Magnification and Resolution :

The function of any microscope is to enhance resolution(i.e. least distance between two points in object that can be distinguished). The microscope is used to create an enlarged view of an object such that we can observe details not otherwise possible with the human eye.

Because of the enlargement, resolution is often confused with magnification, which refers to the size of an image. In general, the greater the magnification, the greater the resolution, but this is not always true.

There are several practical limitations of lens design which can result in increased magnification without increased resolution. Without resolution, no matter how much the image is magnified, the amount of observable detail is fixed, and regardless of how much you increase the size of the image, no more detail can be seen. At this point, you will have reached the limit of resolution or the resolving power of the lens. This property of the lens is fixed by the design and construction of the lens. To change the resolution, a different lens is often the only answer.

The formula for resolution d, is given below,

resolution formula

where NA is Numerical aperture = refractive index(η) x angle of incidence(cone angle), while λ is wavelength of light used.

The numerical aperture of a lens is dependent upon two parameters, the angle of the incidence of light onto the lens, and the refractive index(a measure of how greatly a substance slows the velocity of light) of the glass of which the lens is composed.

The angle of incidence is also known as the cone angle and 1/2 of this value is designated by the symbol θ. Half the cone angle is used to calculate the angle the light subtends relative to the light axis.

The cone angle and thus θ can be altered by inclusion of a substage condenser. If the condenser is moveable, the cone angle can be varied; the closer the substage condenser is to the object, the greater is the cone angle. This is a relatively inexpensive means of effecting the resolution of the microscope and thus nearly all microscopes are equipped with substage condensers.

The refractive index is a function of the bending of light from air through glass and back again. In a microscope, the glass of the lens is specially formulated to increase its refractive index. Once manufactured, however, this property can not be changed. The media around the lens can be altered, however, by removing air from between the objective and the slide, and replacing it with immersion oil.

Cone Angle and Numerical Aperture vs magnification

Cone Angle and Numerical Aperture

Optical Light Microscopy:

There are two different types of microscopes: light and electron.  In this article we will restrict our discussion to light microscope.

Light microscopes have glass lenses which magnify objects, and use light to illuminate the objects being examined. If the beam of light is replaced by an electron beam, the microscope becomes a transmission electron microscope. If light is bounced off of the object instead of passing through, the light microscope becomes a dissecting scope. If electrons are bounced off of the object in a scanned pattern, the instrument becomes a scanning electron microscope.

Size Scale in microscopy

Size Scale from macroscopic to microscopic

For routine bright field microscopy, it is more convenient to work in the visible light range, and the shortest wavelength of visible light is blue. Thus, even inexpensive microscopes have incorporated a blue filter into their design, which is often referred to as a daylight filter. As a rule, the cheaper the microscope the thicker and darker this filter.

More expensive and higher quality lenses manipulate the light source to enhance the quality of the light and to correct for lens aberrations inherent in their design.

Resolution can be enhanced by reducing the wavelength to the ultraviolet range and yet again by levels of magnitude to the wavelengths electrons have in motion. The use of electrons as the light source gives rise to the electron microscope.

UV light can not be seen directly by the human eye (it will injure the retina of the eye) nor can we see electron beams. Thus, these forms of microscopy rely on photography, or upon fluorescent screens.

The Compound Microscope :

A compound microscope is composed of two elements; a primary magnifying lens and a secondary lens system, similar to a telescope. Light is caused to pass through an object and is then focused by the primary and secondary lens. Compound microscopes are used to examine objects in two dimensions. Very small organisms or cross-sections of organisms are placed on clear glass slides; these objects are viewed as light passes through them.

The Dissecting Microscope :

Dissecting microscopes are used to observe material that is either too thick or too large to be viewed with the compound light microscope. With these microscopes, you see the surface of things that reflect the light. While the magnification and depth of field are smaller in the dissecting scope, the field of view is much larger. As its name implies, the dissecting scope is often used to look at plants as you dissect them, since it allows for manipulation of material. Since most of the parts of the dissecting microscope are the same as the compound microscope, they will not be reviewed here.

Components of compound microscope:

1.  The microscope has two magnifying lenses: the eyepiece or ocular lens and the objective lenses on a turret which revolves above the stage. The eyepiece lenses are usually 10X and are moveable so that they can be adjusted to the distance between the pupils of each viewer. The objective lenses (there are four: 4X, 10X, 40X and 100X) rotate on the nosepiece. By changing the objectives the effective power of magnification is changed. The total magnification observed is the product of the power of magnification of the eyepiece and the objective. Only the 100X objective is used immersed in a drop of special oil (between the lens and the slide; all others are designed to be used with air between the object and lens surface. The 100X objective will not be used in this course. The power of magnification is clearly indicated on each lens along with the numerical aperture of each lens. Depending upon their design and quality, different objectives have different resolving distances. The latter is the smallest distance between two points that allows both points to be viewed as separate. This resolving distance is dependent upon the wavelength of light used as well as the construction of the lens.

2. Microscopes contain elements designed to project parallel beams of light through the specimen and into the objective. These include the projection lens which focuses light onto the condenser lens. The condenser lens focuses light onto the object. To get the condenser lens in focus, place a slide containing a wax pencil mark on the stage. Focus on it with the lowest power objective lens and turn the iris diaphragm to the smallest opening. Then focus the condenser up and down until the edges of the iris diaphragm come into sharp focus without using the objective focusing adjustments. The condenser is now in focus.

3. The focusing knobs move the lens assembly up and down to bring the object in focus. The coarse adjustment should only be used with the shortest, low power objective lens. The fine adjustment (smaller knob) brings the object into critical focus. Notice that all objects are projected upside down in the microscope field. It takes a little practice in using the mechanical stage to move the slide where you want it.

light microscope details

Light Microscope

USING THE COMPOUND MICROSCOPE :

1. Use both hands to carry the microscope to your seat. Place the microscope on the table in front of you and position yourself so that you are comfortably seated while looking through the microscope.

2. If necessary, clean the lenses with lens paper only. Do not use anything else, like KimWipes or your shirt to clean the lenses–this will damage the microscope.

3. Place a slide of the ‘letter e’ on the stage. If your microscope has a built in light, plug in the scope and turn the light on. If not, bring a lamp to your table and position it so that the light shines above the object being viewed.

4. Turn the nosepiece so that you are using the lowest power objective lens. You should always use the lowest power objective when you begin viewing an object. While looking through the ocular lenses with both eyes, begin to focus on the object by turning the focus adjustment on the side of the microscope arm. If you see two images of the object or the reflection of your own eye/eyelashes, you probably need to adjust the ocular lenses. These lenses can be moved together or apart to better match the distance between your eyes.

5. Once the object is in focus, increase the magnification by rotating the nosepiece. Adjust the focus by using the fine adjustment knob only. Make sure that the objective lens does not come in contact with the slide.

6. Examine different parts of the object by moving it around the stage. Notice the direction that the image moves when the object is moved from left to right. Change the light level and observe differences in the way the image appears.

References and Further reading:

  1. Gustavus Adolphus College Resources:http://homepages.gac.edu/~cellab/chpts/chpt1/intro1.html
  2. Florida International University Resources:http://www2.fiu.edu/~kopturs/BOT1010/Lab/Microscopes_Periphyton_Prokaryotes_Fungi.pdf
  3. University of California – Los Angeles  Resources:http://www.sciencedaily.com/releases/2010/05/100504095216.htm
  4. https://en.wikipedia.org/wiki/Optical_microscope
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Healthtard

Healthtard

Pradeep completed his Masters in Clinical Engineering from IIT Madras and he likes to share the care! To know about him and his projects click here!

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