Tools of the Biologist

1
Tools of the Biologist
2
History

• Anton Von Leeuwenhoek Born in Holland 1632
• First to observe living bacteria drew them.
• Also looked at protists, sperm, blood
• 1st simple scope
• Made over 500 "microscopes"

3
(No Transcript)
4

• Robert Hooke (1665)
• Used compound scope to examine cork.
• Coined the term cell referring to the many
  little boxes. Actually saw dead plant cells.

5
Types of Microscopes

• 1. Simple microscope Hand lens (magnifying
  glass)
• 3 40 times magnification

6

• Compound Light Microscope
• The type we use in our labs
• Most commonly used microscope
• Uses light and lenses to magnify view the
  specimen
• Has two sets of lenses Ocular (eye piece)
  Objective (near the object being viewed)
• Total magnification on our scopes 40-400 times
• Total magnification Ocular (10X) x Objective
  (40X)

7
Ocular Eye piece 10x Body Tube Supports the
eyepiece. Nosepiece rotates
objectives Objectives 40 400x total
magnification Arm Supports neck and
objectives. Carry by this Stage and clips
Holds slides in place Adjustments Coarse
Fine. Focuses image Diaphragm Controls the
amount of light coming through the stage Light
Electric light source Base Bottom of scope.
One hand goes underneath
8
The Diaphragm

• Use the Diaphragm to adjust the amount of light
• Image of pollen grain under good brightness
  (left) and poor brightness (right)

9
Focusing

• Use the Adjustment knobs to focus the image
• Coarse adjustment brings the image into near
  focus
• Fine adjustment (smaller knob) brings it into
  fine focus
• Use fine adjustment under 40x

10
Microscope Principles

• Magnification
• Field of View
• Inversion
• Working Distance
• Depth of Field
• Resolution

11
Magnification

• Need light and lens
• Image formation
• Convex lens

12
Field of View
13
Inversion
Original Object
Microscope Image
14
Working Distance
15
Depth of Field/Focus
16
Resolution

• Ability to clearly distinguish two objects that
  are close together.
• Image of pollen grain with good resolution (left)
  and poor resolution (right)
• 
  Resolving power of our scope 0.2um

17
Rules for using the Microscope

• Use only the assigned microscope
• Carry place the scope properly (3cm from edge
  of table)
• Do not let the cords dangle or get into the sinks
• Clean lens only with lens paper. NO FINGERS!
• Do not reuse the same spot on your lens paper
• Start on low (4x) power when you start your
  observations
• Always focus (move the stage) away from the slide
• Use the coarse adjustment first then the fine
  adjustment
• Be careful when switching to high (40x) power to
  se that there is enough clearance between the
  objective and the slide
• Do not use the coarse adjustment knob on high
  (40x) power
• When you are done with the scope, turn off the
  light switch
• Always put scope away with cord wrapped around
  it, cover on the low power objective in place
• Put scopes away with the numbers facing out into
  the proper slot
• Clean and dry all slides and cover slips before
  putting them away

18
Making a Wet Mount
19
The Letter e
40X
Normal View
400X
100X
20
Crossed Threads
Total Magnification
Blue Thread
Gold Thread
21
Field of View
1mm
1mm
1mm
1mm
Diameter 3.75 mm
Specimen 4/3750um
or 3750 um
Length of Specimen 937.5um
22
Calculating Fields of View

• Once you have your field of view for Low Power,
  you will no longer use the ruler GIVE BACK THE
  RULER
• For Medium Power
• Low Power Field of View (um) Medium
  Power Mag
• Medium Power Field of View (um) Low Power
  Mag
• For High Power
• Low Power Field of View (um High Power
  Mag
• High Power Field of View (um) Low Power Mag

Medium Power Field of View
Low Power Field of View
23

• 3. Binocular (Has two oculars)
• Gives a 3D image.
• Also called a Dissecting scope or Stereo scope
  
• Monocular (1 ocular) Light Microscope
• 2D image

24
Compound Microscope images
Diatom
Paramecium
Amoeba
Vorticella
Daphnia
Hydra budding
25
Since most of the specimens we observe will be
clear, what could be done to enhance the image we
view through the scope?

1. Adjust the diaphragm to allow less light to come
   through
2. Use a Stain to make transparent specimens
   visible. Ie. Iodine, methyl blue
3. Specimens must be sliced very thin. Use a
   Microtome to make thin slices

26
Microtome
27
Electron Microscopes

1. Uses electromagnets and streams of electrons to
   view a specimen
2. Limit of Resolution is 1000x finer than light
   microscope
3. 200,000 1,000,000x magnification

28
Two types

• Transmission Electron Microscope (TEM) 1931
  (Germany)
• Image is seen on a fluorescent screen
• Specimen must be thinly sliced and coated with Au
  or Ag.
• Gives a 2D image of specimen
• Specimen must be dead

29
Herpes simplex viruses


Staphylococcus aureus
E. coli bacteria
30

• Scanning Electron Microscope (SEM) 1935
  (Germany)
• Gives a 3D image
• Electrons scan around specimen
• Shows only the outside of the specimen
• Gives very clear surface details

31
Images
Diatom
Weevil
Side 2 02255


Tick
Radiolarian

32
(No Transcript)
33
Limitations of Electron Microscopes

1. Specimens must be very thin
2. Specimens must be stained or coated
3. Specimens must be dried out (Mounting chamber is
   vacuum sealed)
4. Specimens must be dead
5. Black and white images only! Any color you may
   see is added in

34
(No Transcript)
35

• Gold coater - 1,950 used
• Transmission Electron Microscopes (TEM)90,000 -
  2,000,000
• UsedScanning Electron Microscopes (SEM)45,000 -
  200,000 Used