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Objective Selection:

The objective is arguably the most important part of the microscope. The quality of many microscopes is judged by the image given by the objective. Objectives are also significant in determining the magnification of a specimen and the resolution under which fine details of the the specimen can be observed with the microscope.

Major microscope manufacturers offer a variety of objectives. Below are five common objectives.

  1. Achromatic: Lenses which bring parts of the color spectrum into the same plane of focus. Achromats are standard on many quality microscopes. They may be designated as flatfield objectives, but typically deliver between 60-70% flat field.

  2. Semi-Plan: Objectives that are corrected to attain a flatness of field of 80-85%.

  3. Plan Achromat: Objective lenses delivering nearly 100% flatness of field.

  4. Phase: Objectives that create the effect of "staining" by changing the optical light path without actually staining the slide. A major benefit of using phase objectives is that the specimen remains viable and intact.

Oil Immersion Lenses

When light rays from the objective lens to the specimen pass through air, they are distorted slightly. This is not a perceptual problem at a magnification of 400 or lower. However, at a magnification of 1000, the slight distortion is reduced significantly by placing a thin layer of very clear, but viscous, oil between the slide and the tip of the objective lens. It is important that you properly clean the objective lens after using oil to view a specimen, though, as the oil can get into the lens and ruin it.

Do not use a large excess of oil with the immersion objective; one small drop is sufficient for good lens contact.

The Care of Objectives

Never leave the eyepiece out of the eyepiece tube of the microscope for dust particles to settle on the back of the lens of the objective or on the binocular prisms.

The front lens surface of all dry objectives should be examined periodically with a 10X or higher aplanatic magnifier for freedom from grease and other foreign material. This examination applies especially to the high dry lens as a foggy image will result from any film of dried solution or immersion oil which may present on the front lens. When cleaning a lens surface, first brush lightly with a clean camel's hair brush and then breathe on the surface. Follow by wiping with a soft lens paper, using a light, circular motion. With any persistent dried film on a lens surface, try lens paper moistened with glass cleaner and dry with a soft absorbent cloth. Should the film still remain, try xylene. Do not use alcohol to clean lens surfaces.

Objectives higher in magnification than 10X should never be taken apart. Optical glass is softer than ordinary glass; consequently, the avoidance of scratches requires great care. Grease marks and mold left on a lens surface for any length of time may etch into the glass.


 

Illumination Selection:

There are several types of electric lighting for microscopes:

  • Tungsten is the least expensive illumination. It is hotter and less bright than the other kinds. 

  • Fluorescent illumination provides cooler and brighter light than tungsten. This is beneficial when viewing slides for long periods of time or observing live specimens, such as protozoa.

  • LED is a bright cool source of light with a bulb that is much longer lasting than a fluorescent bulb. Microscopes with LED lighting typically have a variable control allowing you to change the intensity of light.

  • Halogen provides the very brightest illumination. The best microscopes have halogen lighting and most stereo microscopes with top lighting also use halogen lighting. The intensity of halogen light can be adjusted with a variable control.


 

Basic Types of Microscopy:

Brightfield Microscopy:

Brightfield microscopy is the most elementary form of microscope illumination techniques and is generally used with compound microscopes. The name "brightfield" is derived from the fact that the specimen is dark and contrasted by the surrounding bright viewing field. Simple light microscopes are sometimes referred to as brightfield microscopes.

Phase Contrast Microscopy

Phase Contrast microscopy opened up an entire new world in the field of microscopy. Phase contrast is most useful in observing transparent, colorless and/or unstained specimens referred to as “phase objects”. By design the phase objectives optically stain a specimen creating a contrast with images not seen with a brightfield microscope.

Darkfield Microscopy

Darkfield microscopy is an illumination technique used to enhance the contrast in unstained samples. It works by illuminating the sample with light that will not be collected by the objective lens, and thus will not form part of the image. This produces the classic appearance of a dark, almost black, background with bright objects on it.

Polarizing or Petrographic Microscopy

This microscope differs from others because it contains the following components:

  • A polarizer and analyzer

  • A circular rotating stage

  • Special plates or filters placed between the object and light path.

  • Bertrand lens

A polarizer only allows certain light waves or vibrations to pass through it.

An analyzer, often a second polarizer located above the sample, determines the amount and direction of light that illuminates a sample.

At its most basic, the polarizer focuses the different wavelengths and vibrations of light onto a single plane. The relationship of the polarizer and analyzer, in addition to possible filters added, determines the amount of light absorbed, reflected, refracted and/or transmitted through the microscope.

Epi-Fluorescence Microscopy

Epi-Fluorescence microscopy illuminates specimens using a dichroic mirror that separates light waves illuminating different parts of a specimen within certain wavelengths of light. High intensity light is used instead of the standard illumination commonly found in compound microscopes. Using mercury and xenon arc lamps as high-intensity illumination sources causes a large amount of excitation energy in the specimen providing those details not visible using traditional light. For the specimen to be viewed sufficiently, image brightness is key and needs to be achieved using the correct wavelength of light. Selecting an efficient barrier filter (available from manufacturers) helps to allow for selected wavelengths of light, those that excite specific fluorophores, to enter the observation eyepiece while correctly blocking others.


 

Choosing the Right Microscope:

Simply speaking there are two types of microscopes. Either Compound, or high power and Stereo or low power.

Compound Microscope:

The Compound microscope is designed for smaller specimens such as blood samples, bacteria, pond water organisms, etc. These type of specimens require higher powers of magnification in order to see the detail. A compound microscope is also known as a high power microscope. Typically, a compound microscope has 3-5 objective lenses that range from 4x-100x. Assuming 10x eyepieces and 100x objectives, the total magnification would be 1,000 times.

Stereo Microscope:

The Stereo microscope is designed to view larger specimens such as insects, bugs, leaves, rocks, gems, etc. These specimens require lower power, magnification ranges from 6.5x-45x. A stereo microscope is also called a low power microscope. By definition, a stereo microscope has at least two eyepieces (binocular), and provides a three-dimensional image of the specimen. They are available in one of three configurations: fixed, dual power or zoom.  


 

Location

Capital Microscope Services, Inc.

P.O. Box 462, Marietta, GA. 30061

Mail to: info@microscopesandmore.com

Phone: 770-422-3314

Fax: 770-422-1833