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Microbiology Online Focus On: Theory and Practice in Light Microscopy (Part III)

Light microscopy is the most largely used microscopy technique in microbiology labs. In light (optic) microscopy, the objects studied look darkened and the adjacent area looks very well illuminated. The magnification power of an optic microscope is up to 2000 times. The main difficulty in light microscopy is to obtain a high magnification power along with clearness and image definition.

In light microscopy, the resolution power represents the size of the smaller object that can be seen on the microscope with total clearness. A good microscope shall combine magnification power with resolution power; otherwise an image could be amplified 2000 times, but it would show up distorted and without definition.

The lower the resolution power, smaller the size of the object that can be observed on the microscope. That said, a light microscope with a resolution power of 0.5 µm is able to magnify, with clearness, objects of 0.5 µm or higher.

In order to calculate the resolution power of an optic microscope, we need to know:

• The light wavelength;
• The objective lenses numeric aperture;
• The condenser lens numeric aperture.

The wavelength of the visible light is known and ranges from 400 to 700 nm, or 0.4 to 0.7 µm.

In light microscopy, the Numeric Aperture (NA) is calculated from the following equation:

NA = n sen?, where

n = the medium refraction index. n=1.56 when an immersion oil is used and n=1 when it is not used.

? = half angle of the cone light that enters the objective or condenser lenses.

In the modern microscopes, the NA value of the objective and condenser lenses is indicated at the microscope user's guide.

At last, the resolution power (RP) is calculated from the following equation:

RP = light wavelength/(NA objective + NA condenser)

At microbiology labs, the light microscopy technique is usually performed using immersion oil. The immersion oil has a refraction index higher than the air, what increases NA and decreases RP; in practical terms, it allows the observation of smaller objects, like cells and bacteria, for example.

About the Author

Fabio L. C. Pacheco invites you to visit Microbiology Online. If you liked this article, you will also enjoy Microbiology Online. It's all about great tutorials in industrial microbiology, focusing on pharmaceutical microbiology labs.

Bio statement matching question?

Use these to match with the alphabetical statements:
i. confocal technology
ii. staining techniques
iii. resolution
iv. electron microscopy
v. contrast
vi. fluorescence microscopy
vii. brightfield microscopy

i. confocal technology--> a. provides information about molecules on the cell surface

ii. staining techniques--> d. the ability to distinguish between two structures close together

iii. resolution
iv. electron microscopy-->b. uses a beam of electrons to producs images with fine detail

v. contrast-->c. provides contrast for observing individual structures, but kills cells

vi. fluorescence microscopy-->e. using laser beams, provides views of living cells viewed in three dimensions

vii. brightfield microscopy-->f. normal view under a light microscope

There is one extra.
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TO BE 100% confirmation
Check the answers from some link information about the types of microscopes and their functions and staining techniques
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2. Optical microscope - Wikipedia, the free encyclopedia
... chemical or ion staining of non-living samples, ... also: timeline of microscope technology. Compound microscope made by ... Other types of optical ...
http://en.wikipedia.org/wiki/Optical_microscope

3.AGRICULTURAL MICROBIOLOGY
Bacterial morphology- Staining of microorganisms – basic principles ... Nutritional types of bacteria-Autotrophs, heterotrophs, phototrophs and chemolithotrophs. ...
http://www.tnau.ac.in/micro/ug.htm

4.Electron microscope - Wikipedia, the free encyclopedia
3 Types. 3.1 Transmission Electron Microscope (TEM) 3.2 Scanning Electron ... (ultrathin sectioning) and increase their electron optical contrast (staining) ...
http://en.wikipedia.org/wiki/Electron_Microscope

5.Nikon | Instruments Products | Types of microscopes
... are used for viewing stained or unstained transparent biological ... Products > Technology > Introduction to Microscopes > Overview > Types of microscopes ...
http://www.nikon.com/products/instruments/tech/guide/overview/index_02.htm

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