In the late 16th century several Dutch lens makers designed devices that magnified objects, but in 1609 Galileo Galilei perfected the first device known as a microscope. Dutch spectacle makers Zaccharias Janssen and Hans Lipperhey are noted as the first men to develop the concept of the compound microscope.

1. History of Microscopy-I
Md. Saiful Islam
B.Pharm, M.Pharm (PCP)
North South University
Fb Group: Pharmacy Universe

2. The History
• Hans and Zacharias Janssen of Holland in the 1590’s
created the “first” compound microscope
• Anthony van Leeuwenhoek and Robert Hooke made
improvements by working on the lenses
Anthony van Leeuwenhoek
1632-1723
Robert Hooke
1635-1703
Hooke Microscope

3. Two Dutch eye glass makers, Zaccharias Janssen and son
Hans Janssen experimented with multiple lenses placed
in a tube.
The Janssens observed that viewed objects in front of
the tube appeared greatly enlarged, creating both the
forerunner of the compound microscope and the
telescope.
History Sequence…… 1590

4. English physicist, Robert Hooke looked at a sliver of
cork through a microscope lens and noticed some
"pores" or "cells" in it.
History Sequence…… 1665

5. Anton van Leeuwenhoek built a simple microscope with
only one lens to examine blood, yeast, insects and many
other tiny objects.
Leeuwenhoek was the first person to describe bacteria,
and he invented new methods for grinding and polishing
microscope lenses that allowed for curvatures providing
magnifications of up to 270 diameters, the best available
lenses at that time.
History Sequence…… 1674

6. Technical innovations improved microscopes,
leading to microscopy becoming popular among
scientists.
Lenses combining two types of glass reduced
the "chromatic effect" the disturbing halos
resulting from differences in refraction of light.
History Sequence…… 18th century

7. Joseph Jackson Lister reduces spherical
aberration or the "chromatic effect" by showing that
several weak lenses used together at certain
distances gave good magnification without blurring
the image.
This was the prototype for the compound
microscope.
History Sequence…… 1830

8. Ernst Abbe, then research director of the Zeiss Optical
Works, wrote a mathematical formula called the "Abbe
Sine Condition".
His formula provided calculations that allowed for the
maximum resolution in microscopes possible.
History Sequence…… 1872

9. Richard Zsigmondy developed the ultramicroscope that
could study objects below the wavelength of light.
He won the Nobel Prize in Chemistry in 1925.
History Sequence…… 1903

10. Ernst Ruska co-invented the electron microscope for
which he won the Nobel Prize in Physics in 1986.
An electron microscope depends on electrons rather
than light to view an object, electrons are speeded
up in a vacuum until their wavelength is extremely
short, only one hundred-thousandth that of white
light.
History Sequence…… 1931

11. it possible to view objects as
small as the diameter of an
atom.
Ernst Ruska…… 1931

12. Frits Zernike invented the
phase-contrast microscope that
allowed for the study of
colorless and transparent
biological materials for which he
won the Nobel Prize in Physics
in 1953.
History Sequence…… 1932

13. Gerd Binnig and Heinrich Rohrer invented the
scanning tunneling microscope that gives three-
dimensional images of objects down to the atomic
level.
Binnig and Rohrer won the Nobel Prize in Physics in
1986.
The powerful scanning tunneling microscope is the
strongest microscope to date.
History Sequence…… 1981

14. MICROSCOPY
• Microscopes are essential for microbiological studies
• Light microscopes: cellular resolution
– bright-field (stains)
– dark-field
– phase contrast
– fluorescence (stains)
• Electron microscopes: subcellular resolution

15. Light Microscopy: Optics
• Visualization depends on magnification (lenses) and resolution
(physical properties of light)
• The limit of resolution for a light microscope is about 0.2 m
(or 200 nm)
– Objects closer than 0.2 m cannot be resolved
• Total magnification is product of the magnification of its ocular
and its objective lenses

17. How a Microscope Works
Ocular Lens
(Magnifies Image)
Objective Lens
(Gathers Light,
Magnifies
And Focuses Image
Inside Body Tube)Body Tube
(Image Focuses)
•Bending Light: The objective (bottom) convex lens magnifies
and focuses (bends) the image inside the body tube and the
ocular convex (top) lens of a microscope magnifies it (again).

18. Body Tube
Nose Piece
Objective
Lenses
Stage
Clips
Diaphragm
Light Source
Ocular Lens
Arm
Stage
Coarse Adj.
Fine Adjustment
Base

19. Parts of a microscope
• Body : The Body tube holds the objective lenses and
the ocular lens at the proper distance.
• Nose Piece : The Nose Piece holds the objective
lenses and can be turned to increase the
magnification.
• Objective Lenses: The Objective Lenses increase
magnification (usually from 10x to 40x).
• Stage clips: These 2 clips hold the slide/specimen in
place on the stage.
• Diaphragm : The Diaphragm controls the amount of
light on the slide/specimen.

20. Parts of a microscope
• Light source : Projects light upwards through the
diaphragm, the specimen and the lenses. Some have
lights, others have mirrors where you must move the
mirror to reflect light.
• Ocular lens/ eye pieces: Magnifies the specimen
image.
• Arm: Used to support the microscope when carried.
Holds the body tube, nose piece and objective lenses.
• Stage : Supports the slide/specimen.
• Coarse Adjustment Knob: Moves the stage up and
down (quickly) for focusing your image.
• Base: Supports the microscope.

21. •The proper way to focus a microscope is to start
with the lowest power objective lens first and while
looking from the side, crank the lens down as close
to the specimen as possible without touching it.
•Now, look through the eyepiece lens and focus
upward only until the image is sharp.
• If you can't get it in focus, repeat the process
again.
How to use a microscope properly

22. •Once the image is sharp with the low power lens,
you should be able to simply click in the next power
lens and do minor adjustments with the focus knob.
• If your microscope has a fine focus adjustment,
turning it a bit should be all that's necessary.
•Continue with subsequent objective lenses and fine
focus each time.
How to use a microscope properly

23. Using a Microscope…..cautions
• Start on the lowest magnification
• Don’t use the coarse adjustment knob on high
magnification…can break the slide!!!
• Place slide on stage and lock clips
• Adjust light source (if it’s a mirror…don’t stand in
front of it!)
• Use fine adjustment to focus

24. • Always carry with 2 hands
• Never touch the lenses with your fingers.
• Only use lens paper for cleaning
• Do not force knobs
• Keep objects clear of desk and cords
• When you are finished with your "scope", rotate
the nosepiece so that it's on the low power
objective, roll the stage down to lowest level,
rubber band the cord, then replace the dust
cover.
Using a Microscope……cautions

25. Magnification
• To determine your magnification…you just
multiply the ocular lens by the objective lens
• Ocular 10x Objective 40x:10 x 40 = 400
Objective Lens have
their magnification
written on them.
Ocular lenses usually magnifies by 10x
So the object is 400 times “larger”

26. Microscopy: Stains
• Staining is used to increase contrast in bright-field
microscopy
– Simple: one dye stains all cells
– Differential: combination of dyes allows
differential staining of different populations

27. Simple Stain

28. Differential Stain

29. Microscopy: Dark Field
• Greater resolution
• Light reaches specimens only from
the side
• Only the specimen itself is
illuminated
Candida sp.
Treponema pallidum

30. Microscopy: Phase Contrast
• May be used to visualize live samples
and avoid distortion from cell stain
• Image contrast is derived from the
differential refractive index of cell
structures.

31. Microscopy: Fluorescence
• Visualization of autofluorescent cell structures (e.g.,
chlorophyll) or fluorescent stains
• Can greatly increase the resolution of cells and cell
structures
• Many functional probes available

32. Example for Differential Fluorescence Stain
Psuedomonas (green) Bacillus (orange)

33. Microscopy: Electron Microscopy
• Electron microscopes have far greater resolving
power than light microscopes, with limits of
resolution of about 0.2 nm
• Two major types of electron microscopes
– Transmission electron microscopy (TEM) for
observing internal cell structure down to the
molecular level
– Scanning electron microscopy (SEM) for 3-D
imaging and examining surfaces

34. Electron Microscopy
Pseudomonas Mycobacterium
TEM SEM

35. Secondary Electron Imaging
(SEI)
Transmitted Electron Imaging
(TEI)
Backscattered Imaging
(BSI)
• Surface Topography,
Morphology, Particle
Sizes, etc.
• Compositional Contrast
• Internal ultrastructure Energy-Dispersive
X-ray Spectrometry
(EDS)
• Elemental composition,
mapping and linescans
• Crystallographic Info
Electron Backscattered Electron
Diffraction
(EBSD)
SEM Capabilities
Scanning Electron Microscope
(SEM)

36. cockroach antenna
pigeon blood
Images by SEM

37. Microscope objectives