Tools of the Biologist

History Anton Von LeeuwenhoekAnton 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"

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

Types of Microscopes Simple microscope – 1.Simple microscope – Hand lens (magnifying glass) 3 – 40 times magnification

2.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 »Total magnification on our scopes = times »Total magnification = Ocular (10X) x Objective (40X )

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

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

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

Microscope Principles  Magnification  Field of View  Inversion  Working Distance  Depth of Field  Resolution

Magnification Need light and lens Image formation Convex lens

Field of View

Inversion Microscope Image Original Object

Working Distance

Depth of Field/Focus

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

Rules for using the Microscope 1.Use only the assigned microscope 2.Carry & place the scope properly (3cm from edge of table) 3.Do not let the cords dangle or get into the sinks 4.Clean lens only with lens paper. NO FINGERS! 5.Do not reuse the same spot on your lens paper 6.Start on low (4x) power when you start your observations 7.Always focus (move the stage) away from the slide 8.Use the coarse adjustment first then the fine adjustment 9.Be careful when switching to high (40x) power to se that there is enough clearance between the objective and the slide 10.Do not use the coarse adjustment knob on high (40x) power 11.When you are done with the scope, turn off the light switch 12.Always put scope away with cord wrapped around it, cover on & the low power objective in place 13.Put scopes away with the numbers facing out into the proper slot 14.Clean and dry all slides and cover slips before putting them away

Making a Wet Mount

The Letter “e” Normal View 40X 400X 100X

Crossed Threads Total Magnification Gold Thread Blue Thread

Diameter = 3.75 mm or 3750 um 1mm Field of View Specimen = 4/3750um Length of Specimen =937.5um

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 Low Power Field of View Medium Power Field of View

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

Compound Microscope images Paramecium Vorticella Daphnia Amoeba Diatom Hydra budding

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

Microtome

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

Two types Transmission Electron Microscope (TEM) 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

Staphylococcus aureus E. coli bacteria Herpes simplex viruses

Scanning Electron Microscope (SEMScanning Electron Microscope (SEM) Scanning Electron Microscope (SEM) – 1935 (Germany) Scanning Electron Microscope (SEM 1.Gives a 3D image 2.Electrons scan around specimen 3.Shows only the outside of the specimen 4.Gives very clear surface details

Images Weevil Radiolarian Diatom Tick Side 2: 02255

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

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