1. Lecture 2: Microscopy: Introduction to Microscopy
Cell Biology, BIOL350
Spring 2016
Sulaiman Al-Hashmi
Thursday, 4th February 2016

2. History Greeks & Romans Hans and Janssen
Used “lenses” to magnify objects > 1000 years ago
Hans and Janssen
Created the “first” compound microscope (1590’s)
Antonie van Leeuwenhoek and Robert Hooke
Made improvements by working on the lenses
Antonie van Leeuwenhoek
First to describe bacteria in 1676

3. Human: 390 to 700 nm.

4. Eye power in Nature Goats have rectangular pupils
Wide field of vision
Dragonfly eyes are made up of 30,000 visual units, each one containing a lens and a series of light sensitive cells
Assisting them with motion detection
Pupil بؤبؤ العين
Dragonfly يعسوب

5. Geckos can see colors around 350 times better than a human
The eyes of a chameleon are independent from each other
Look in two different directions at once
Chameleon حرباء
Geckos سحلية

6. Cat’s eyes have around 285 degrees of sight in three dimensions
Cuttlefish can perceive light polarization
Enhances their contrast
A moth’s eyes are covered with a water-repellant
Anti-reflective coating
Pigeons الحمام
Cuttlefish الحبار
Moth فراشة
Predators مفترس

7. Ant has two eyes, but each eye contains lots of smaller eyes
Giving it a “compound eye.”
Eagles have 1 million light-sensitive cells per square millimeter of the retina
humans only have 200,000
A honeybee’s eye is made of thousands of small lenses.

8. Microscope parts

10. Chromatic (color)

12. Terminology Magnification Resolution Contrast
Apparent enlargement of an object
The ratio of image size to actual size
100x magnification = 100 times bigger than the actual object
Resolution
The ability of a microscope to show two very close points separately
Clarity
sharpness
Contrast
Difference that makes an object distinguishable.
Contrast is determined by the difference in the color and brightness

13. Steps to use the microscope:
Rotate the low power objective into place
make sure the stage is all the way down.
Place slide on stage
make sure that the object to be viewed is centered over the hole in the stage
Use the stage clips to hold the slide in place.
Turn light on.
Focus first with the coarse adjustment knob.
Once in focus on low power, turn the nosepiece until the next higher lens is in place.
At the final adjustment, use FINE adjustment knob ONLY and focus the object

14. Magnification
Qs?
A) Ocular power = 10x low power objective = 20x high power objective = 50x
What is the highest magnification you could get using this microscope ?
Ocular x high power = 10 x 50 = 500
B) If the diameter of the low power field is 2 mm, what is the diameter of the high power field of view in mm ?
The ratio of low to high power is 20/50
So at high power you will see 2/5 of the low power field of view (2 mm)
2/5 x 2 = 4/5 = 0.8 mm

15. C) If 10 cells can be seen in the low power field of view, how many cells would you expect to see under high power ?
At high power we would see 2/5 of the low field
2/5 x 10 cells = 4 cells would be seen under high power

16. Types of Optical Microscopes
Reflected (bright field)
Transmitted
Polarized
Dark Field
Differential Interference Contrast
Confocal
Fluorescence

17. Light microscopy
Upright microscope
Inverted microscope
Stereomicroscope
Basic concepts of light microscope
Magnification
Resolution power and Numerical aperture
Types of light path
transmitted light
reflected light

18. Types of light microscopes
Upright Microscope Inverted Microscope Stereomicroscope

19. The light microscope Compound microscope
Common light microscope (Labs)
Contains two types of lenses
Ocular
Objective
Most microscopes have on their base an apparatus called a condenser
Condenses light rays to a strong beam
A diaphragm located on the condenser
Controls the amount of light coming through it.

20. Light pathway
To magnify an object, light is projected through an opening in the stage, where it hits the object and then enters the objective. An image is created, and this image becomes an object for the ocular lens, which remagnifies the image

22. Upright microscopy Designed for slide sample High magnification
High resolution in transmitted light sample

23. Inverted microscopy Lower resolution in transmitted light sample
Long working distance
Suitable for culture evaluation (flask, Petri dish)

24. Stereomicroscopes Long working distance Low magnification
Observation with stereo perception

25. Fluorescence Microscopy
Uses ultraviolet or laser as light source.
When ultraviolet light hits an object, it excites the electrons of the object, and they give off light in various shades of color.
Since ultraviolet light is used, the resolution of the object increases
Sheep Tongue

26. Confocal microscopy Laser light source
Scan across sample and optically section
Useful on live samples (monitor real time events)
Examine the organization/distribution of biomolecules within cells
embryonic mouse brain
Anatomy and Embryology 204: , 2001 

28. Electron Microscopy Electron beam as source (l ~ 0.005 nm)
Operate under vacuum (not all)
Electromagnets instead of glass lenses
Detect by fluorescent screen or photographic emulsion
Two major types
Transmission (TEM) and scanning (SEM)
TEM: Cellular ultrastructure
SEM: Surface ultrastructure
Electron microscopy provides the highest resolution of subcellular structures. Many electron microscopes are operated under vacuum, so they are not suitable for examining living specimens. The principles of EM are similar to light microscopy except that – EM uses an electrons as its source instead of light and electromagnets to focus the electron beam instead of glass lenses used to focus light in a typical microscope.

29. TEM
It has been exploited most widely in the examination of the internal structure of cells.
Works like a light microscope, it transmits a beam of electrons through a thin specimen
Then focusing the electrons to form an image on a screen
most common form of electron microscope
gives good resolution

30. SEM It is utilized primarily to examine the surfaces of objects
This scans a fine beam of electron onto specimen and collects electrons scattered by surface
This has poor resolution but gives good 3-D images

32. Blood
The erythrocytes (red blood cells)
SEM
Magnification 2200:1
Blood and bacteria
blood with staphylococci in an ultrathin section. The dark spots are staphylococci, erythrocytes are red and two cells of the immune system are colored in green. TEM
Magnification 3000:1