The Microscope
Magnification and Resolution


The two main, essentially equivalent, abilities of a microscope are magnification and resolution. Magnification is increasing the viewing size of the organism. This is accomplished by using two magnifying lenses, the ocular lenses and the objective lenses. The ocular, also called the eyepiece, often has magnifications of 10x, but other magnifications such as 12x and 15x may also be found. The objective comes in a variety of magnifications, with the most common being 10x, 40x, 60x and 100x-oil.

Total magnification may be determined by multiplying the ocular magnification by the objective magnification.

Ex. Tm =   ocular  *   objective
      Tm =  (10x)    *      (40x)     =   400x
Therefore, the object under view has been magnified 400x.

The resolution of a microscope may be defined as the ability to see detail.  In essence, it means how close together two objects may be and still see them as two distinct objects, rather than one ”blurry” object. Resolution may be calculated by:

Rp =  (wavelength * 0.5) / numerical aperture

alternatively

Rp= wavelength / (2 * numerical aperture)

Rp =  Resolving power. Essentially, how far apart two objects may be and still be seen as two objects.  It is measured in nanometers. The smaller the Rp the better, i.e. the closer two objects may be and still be seen as two objects.

Wavelength =  Wavelength of light. This is the distance between the tops of two adjacent light waves. Different wavelengths are seen as different colors:  
white light = 550nm, green light = 513nm, blue light = 475nm.

Numerical aperture: This is the width of the cone of light that may enter the lens. The light leaving a specimen may scatter in any number of directions. The numerical aperture measures how far the light can scatter and still enter the lens. This is a property of the lens and is usually written on it. It can simply be thought of as a measure of the light gathering power of the lens. Typical values are 0.25 for the 10x lens, 0.65 for the 40x lens and 1.25 for the 100x lens.

Taken altogether, the formula indicates that the smaller the wavelength and/or the larger the numerical aperture, the better (smaller) the resolving power.

Example: How close may two flagella be to be seen as two flagella rather than one flagella using white light and a 100x objective.

Rp = (550 * 0.5)/ 1.25
Rp=  220 nm

Therefore, the flagella can be no closer than 220 nanometers to be seen as two flagella.


Additional comments:

Working distance:  This refers to the distance between the end of the objective and the tip of the specimen.  As the magnification increases the working distance decreases.

Parfocal: This term refers to the ability of the better microscopes to keep a specimen, which is in focus at one power, remains approximately in focus at all other powers

Parcentral: This term refers to the ability of the better microscopes to keep a specimen, which is in the center of the field at one power, remains approximately in the center of the field at all other powers.

Empty magnification: This refers to increasing magnification without increasing resolution.  A small blurry object has been magnified to a big blurry object. There is no increase in detail just magnification.

Condenser: The condenser usually consists of one to a series of lenses that “focuses” the light from the light source onto the specimen. The diaphragm lever has the crude effect of affecting the amount of light that reaches the specimen, but it especially plays a role in affecting the orientation of the light hitting the specimen and thereby affecting the quality of the view.

Tips
-Clean your slides with a paper towel by gently wiping them.

-Clean lenses with lens paper only. Do not use paper towels, kim wipes, tissues, or anything other than lens paper.

-Look for color as usually the specimens are stained a color.

-If it is difficult distinguishing the specimen from the background, wiggle the slide and look for movement.

-The course adjustment knob may be considered the 10x focus knob, as that is the only time it is used. The fine adjustment knob is used at all other magnifications. Each movement of the course adjustment knob noticeably moves the objective/stage a significant distance while a turn of the fine adjustment knob barely moves the objective/stage.  Remembering this is important because as one increases in magnification, the working distance decreases, to the point where the objective and specimen are almost touching. If the course adjustment knob is used at the higher magnifications, it is very easy for the objective to strike the slide resulting in the breaking of the slide or the objective.  Any cracked slides in the slide box were the result of someone using the course adjustment at higher magnifications.