Microscopy

Microscopy  An important tool used to study microbes  The human eye can resolve objects down to about 0.2 mm  The Microscope is used to magnify small objects, those below the 0.2 mm range  Bacteria and cells are measured in µ m or 1 x 10-6 meters  Viruses are even smaller, measured in nm or 1 x 10 –9 meters  There are several different types of microscopes and many manufacturers  We will use Binocular, compound, light microscopes  All have the same basic features

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Condenser/ Iris-Diaphragm Fine-focus Adjustment Knob Coarse-focus Adjustment Knob

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Arm

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Mechanical Stage Adjustment knobs

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Fine-focus Adjustment Knob

• Ocular lenses

• Nose piece

• Slide Clip

• Mechanical stage

• Light source

• Base

Fine-focus vs. Coarse-focus • Condenser/Iris-Diaphragm • Course-focus knob • Fine-focus knob

Objective Lenses • Objective lenses – 4x or Scanning lens – 10x or Low Power lens – 40x or High Power lens – 100x or Oil immersion lens

• Lens Power • Lens Numerical Aperture

Resolving Power 

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Resolving Power – the ability of an optical system to distinguish or separate two small, distinct objects or points from one another Resolving power is a function of the wavelength of light being used (λ), in nm, and another factor called “the numerical aperture” (NA) of the objective lens Numerical Aperture – a mathematical constant that describes the relative efficiency of a lens in bending light rays In general, the shorter the wavelength of light being used (λ), the better the resolving power

λ in nm Re solving power (R.P.) = 2 x N.A.

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Depth of Focus – The range in distance between the lens and the specimen that can be changed without upsetting the sharpness or resolution of the image. Depth of focus decreases as magnification increases.

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Diameter of Field – The diameter of the total field of view through a microscope at a given magnification. Diameter of field decreases as magnification increases.

Oil Immersion Lenses  

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Normal maximum magnification with the light microscope is about 400x To achieve the maximum possible magnification and resolution with the light microscope immersion oil must be used Immersion oil has the same optical density as glass The light rays do not bend when passing from glass to immersion oil Allows the maximum amount of light to enter the objective lens Effectively increases the Numerical Aperture (NA) of the lens Can achieve magnification up to 1000 x with the light microscope Immersion oil must always be used with an oil immersion lens (100x)! Conversely, immersion oil should never be used with the other objective lenses!

Metric Size Comparisons

Different Types of Light Microscopes   

There are several types of light microscopes Each with specific characteristics and functional uses Partial listing of types of microscopes:

Bright-field Microscopy  

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Most widely used Light passes through the specimen; some is transmitted to the objective lens some is absorbed or reflected by structures in the specimen with different optical densities Most cells are opaque or nearly clear Require different types of stains to visualize internal structures

Dark-field Microscopy 

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Uses a special disc called a “stop” with the condenser. Blocks all light from entering the objective lens except for peripheral light that is reflected off the edges of the specimen The background is dark and the specimen is illuminated Used to visualize living cells and rapidly moving cells, like certain protozoans

Light and Dark-field Photomicrographs

Phase Contrast / Interference Microscopy    

Uses the differences in optical densities of structures inside the cell Increases the amount of internal detail Useful when observing live specimens Stains are not required

Electron Microscopy There are two basic types of electron microscopes: 1. 2.

Transmission Electron Microscope (TEM) Scanning Electron Microscope (SEM)

Transmission Electron Microscope – – – – –

Transmission electron micrograph of a bacterial virus attached to the cell wall of a target bacterial cell.

Gives a two-dimensional image Basically a section through a specimen Requires thin slices of the specimen Prepared using a “Microtome” Magnification up to about 500,000X

Scanning Electron Microscope – – –

Produces a three-dimensional image Requires special metal coating to produce the image Magnification up to about 1,000,000X Scanning electron micrograph of a marine radiolarian. Shows the fine detail of the calcium carbonate shell.

Handling and Storage of Microscopes   

Expensive instruments Require care while using and handling Always carry using two hands, one hand supporting the base, and one hand grasping the arm

Steps using a compound Light Microscope             

Plug in the power cord . Turn the power on and adjust the light intensity as necessary. Use the Course Adjustment Knob to obtain maximum working distance. Place your slide on the stage in the slide clip. Adjust the light intensity using the iris-diaphragm. Begin observations using scanning power (4X) and use the coarse adjustment knob to obtain the minimum working distance until the specimen comes into focus. Adjust the slide position on the stage until the specimen is centered in the field of view. You may now use the stage adjustment knobs to scan the specimen. Use the Fine-focus knob to focus and obtain the sharpest possible image. You may now rotate the Low Power objective into position. Since these microscopes are parfocal the image should be just out of focus with this new objective lens. Use the Fine focus knob, moving in small increments in both directions until a clear, focused image is obtained. You may now move the High Power Objective lens into position. Do not use the coarse focus knob for focus adjustments with high power or oil immersion lenses. Adjust focus using the fine-focus knob only.

Using the Oil Immersion Lens 1.

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Once a clear focused image is obtained at high power (40X) rotate the objective lens halfway between the high power and oil immersion objective lens. Place a drop of immersion oil on the slide where the light passes through the glass slide. Then rotate the oil immersion lens (100X) into position. The lens should just contact the immersion oil. Moving only the fine focus knob in small increments, and in both directions, bring the specimen into sharp focus. Adjust the light intensity using the iris-diaphragm as necessary.

Cleaning and Storage 

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When finished viewing the slide use the coarse adjustment knob to maximize the working distance and remove the slide from the stage. If you want to view another slide start the process from the beginning and follow the steps previously listed. If you are finished with the microscope, clean the stage and lenses using only lens paper and lens cleaning fluid if necessary. Clean the ocular lenses, scanning objective lens, low power, high power, and finally the oil immersion lens, in that order. Rotate the scanning objective lens into position and move the stage to minimum working distance for storage. Turn the light off and unplug the power cord. Store the power cord as directed and replace the dust cover. Store in the appropriate space as directed.