Biology 2201Using a Microscope – Lab

Preparation and skills

Types of Microscopes

1. Electron Microscopes

2. Light Microscopes

3. Phase Contrast Microscopes

Electron Microscopes

1. Use electrons for illumination of the subject

SEM → Scanning Electron Microscope– Scans the surface of a specimen – Provides a 3D image of a specimen

TEM→ Transmission Electron Microscope– Shoots a beam of electrons through a specimen– Provides a 2D image of a specimen– Magnification of about 500,000 X

Note: Images are called “micrographs”

Electron microscopes are used to observe a wide range of biological and inorganic specimens including:

– microorganisms, cells, large molecules, biopsy samples, metals, and crystals.– Industrially, used for quality control and failure analysis in semiconductor device

fabrication (computer chips!).

Electron Microscopes

Transmission Electron Microscope

Scanning Electron Microscope

Light Microscopes

Light Microscope

1. Uses light for illumination

Simple → usually only one lens

Compound→ greater than one lens

• Magnification is limited to about 2000 X

• Resolving power of about 200 nm

Stereomicroscope

Uses light – bounces off the surface.

Consists of two eyepieces that gives a 3D picture of the surface of an organism.

• Magnification is very low ( 2 – 16 x)

• The stereo microscope is often used to study the surfaces of solid specimens or to carry out close work such as sorting, dissection, microsurgery, watch-making, small circuit board manufacture or inspection, and the like.

The Light Microscope

How to properly use a Compound Light Microscope

See P. 16 in text for function of parts! DOE!!!

Magnification of a Microscope

• Magnification is defined as the ratio of image size to actual size.

• Calculating Magnification:

Calculate magnification using the following formula:

Maximum magnification of a Light microscope is usually between 1500x and 2000x.

Resolving Power

• Resolving Power is the ability of a microscope to show two very close objects separately.

• Maximum Resolving power of a light microscope is 0.2 um or 200 nm.

• TEM = 0.2 nm of resolving power.

Field of View

• Field of View is defined as the area you see as you look through the ocular lens/eyepiece.

Field of View

Calculating the Field of View

• Low Power

– Usually done using a thin transparent ruler to measure the diameter.

• Medium/High Power

– Lens is usually too close to stage to measure so done using a calculation.

Objective low

Mag of low powerF.O.V. F.O.V. x

Mag of med/high power=

Calculating the Field of View continued

• Field of View on Low Power. • Field of View on Medium Power

3.5 mm across F.O.Vmed = 3.5mm x 4x

10x

F.O.Vmed = 3.5mm x 0.4

F.O.Vmed = 1.4 mm

Field of View calculations

1. The field of view of a light microscope on low power is 2.3 mm. What is the FOV on medium power?

2. FOV of light microscope on low power is 8 mm. What is FOV on high power in micrometers?

Field of View Practice

FOV on Low Power Medium Power High Power

3.4 mm

1.2 mm

1300 µm

2750 µm

9.3 mm

Specimen Size

• Specimen Size is referred to as the actual size of a specimen being viewed under the microscope.

– Usually found by calculation:

F.O.V.Specimen Size =

# of Specimens Across

Field of View on

Medium power = 3mm

Specimen Size practice

FOV # specimens fitting across

Specimen Size

1.2 mm 15

800 µm 1.5

1650 µm 11

0.85 mm 24

2.15 mm 6

Preparing a Wetmount Slide

• Steps in making a Wetmount Slide

1. Obtain clean slide and coverslip.

2. Place drop of water on slide using a dropper.

3. Place specimen in drop of water.

4. Place coverslip on 45degree angle on one side of drop of water. (do not touch the water)

5. Move coverslip until it just touches the drop of water.

6. Drop coverslip.

Preparing a Wetmount Slide

Creating Biological Drawings

• Used to show detail in specimens being viewed under a microscope.

• Follows the “contour drawing” mechanism.

Biological Drawings Rules + Activity

Guidelines for Biological Drawings

1. The diagram should be titled appropriately and the title should be underlined.2. The diagram should be done in pencil and should be at least ½ page in size.3. Magnification should be included and written like this: viewed at X100 magnification.4. Lines should be sharp and concise. No shading - only stippling should be used to show

detail.5. Do not draw the field of view. If a cluster of cells is drawn, each individual cell should

be large enough to label details.6. If drawing a cluster of cells, you need to label the details of only one of the cells in the

cluster.7. All label lines should be drawn horizontally using a ruler.8. All words (labels) should be written neatly and horizontally.9. At the bottom of your biological drawing make notes ie. Stain type, if it is a prepared

slide or wet mount.

– Create your own Biological drawing using one of the sample images to follow. • Assume your magnification is 400x.

Sample Drawing 1

Prepared slide

Sample Drawing 2

Wet mount

Sample Drawing 3

Sample Drawing 4

Making Biological Drawings Self Quiz

• Go to this website and complete the online quiz for biological drawings.

http://www.utas.edu.au/scienceskilling/exdesign/flash/drawing.swf

In text

Assignment and Quiz Prep

• https://www.youtube.com/watch?v=uuKMKNdlTQY (how to prepare a wet Mount slide Onion)… Quiz for wet Mount and focusing

Biological Drawing Assignment- View any slide(s) – complete 2 properly

labelled biological drawings! Be sure to place ALL workings on back of assignment.

- A) First under Medium Power- B) Second under High Power