A short introduction about light and electron microscopy has discussed.

1. Raveendra Kumar Vidyarthi
(Ph.D Scholar)
Supervisor- Dr. Rajeshwar K.K. Arya
Department of Pharmaceutical Sciences
Bhimtal Campus, K.U. Nainital

2. Contents
 Introduction to microscopy
 Microscope
 Variables used in microscopy
 Principle of microscopy
 Types of microscopy
 Types of Microscope
 Uses

3. Introduction to Microscopy
 The science of investigating small objects using a
microscope is called microscopy
 Anton van Leeuwenhoek is generally
credited with bringing the microscope
to the attention of biologists
 1661 - He discovered bacteria, free-living
parasitic microbes, sperm cells, blood cells, microscopic
nematodes etc.

4. Microscope
 A microscope (Greek: micron = small and scopos = aim)
 A device that transmits light through several lenses to
produce an enlarged image of a microscopic specimen
 MICROSCOPE - An instrument for viewing
objects that are too small to be seen by the
naked or unaided eye

5. Variables used in Microscopy
 MAGNIFICATION
 RESOLUTION
 LIMIT OF RESOLUTION (LR)
 REFRACTIVE INDEX
 NUMERICAL APERTURE(NA)
 ABERRATION
 AMPLITUDE (i.e. brightness)
 ENERGY LEVEL

6. Magnification
 Degree of enlargement
 No of times the length, breadth or
diameter, of an object is multiplied
 Also known as Resolving power
 The capacity of a microscope to distinguish images of
two pointed objects lying very close to each other.
Resolution

7. Limit of Resolution
 Limit of Resolution is described mathematically by an
equation developed in the 1870s by Ernst Abbe,
 Where, d= minimal distance (d) between two objects
λ= wavelength of light
(n sin ѳ)= numerical aperture of the lens
Resolving power of std LM=200nm

8. Numerical aperture
 Ratio of diameter of lens to its focal length
NA = n Sin θ
n = refractive index,
θ = angle of aperture
 The numerical aperture with respect to a point P
depends on the half-angle θ of the maximum cone of
light that can enter or exit the lens.

9. Aberration
 Chromatic aberration
 Correction of aberration – Achromatic
objective and Apochromatic objectives

10. Principle of Microscopy
 The principle of Microscopy is Refraction.
 Refractive index is the light bending ability of
a medium
 The light may bend in air so much
that it misses the small high-magnification
lens.
 Immersion oil is used to keep light
away from bending.
RI of Air- 1.0
RI of Water -1.3
RI of Glass(avg)- 1.5
RI of Oil- 1.52

11. TYPES OF LIGHT MICROSCOPY
 Bright Field Microscopy
 Dark Field Microscopy
 Phase Contrast Microscopy
 Fluorescent Microscopy
 Confocal Microscopy

12. A. Bright field microscopy

13. B. Dark field microscopy

14. This method used for unstained preparation

15.  Contrast i.e. structures with differing degrees of
brightness or darkness
 Denser parts of cell appear Bright
 While those have density close to water will appear
Dark
C. Phase contrast microscopy

16. D. Fluorescence microscopy

17. Most commonly used fluorescent dyes-
 Acridine orange Auramine/ rhodamine

18. Types of Microscope
 Light microscope (simple and Compound)
 Electron microscope ( SEM, TEM)
 Scanning Probe microscope
e.g. Scanning tunneling microscope &
Atomic force microscope

19. Compound Microscope
 Lets light pass through an object and then
through two or more lenses.
 PARTS OF COMPOUND MICROSCOPE


20. Transmission Electron Microscopy
 A beam of electrons is transmitted through the specimen
for a 2D view
 Provides for detailed study of the internal
ultrastructure of cells
 Allows the magnification
of objects in the order of
100, 000’s.

21. TEM Photographs
H1 N1 virus

22. Scanning Electron Microscope
 Use a beam of electrons
instead of a beam of light
to magnify the image
 Can achieve 3D images using
electrons on specimen’s surface
 Types of specimens:
-Whole organisms
-Natural tissue surfaces
-Exposed tissue structure

23. Images from SEM
Head of a butterfly A flea magnified 50 000 X spider

24. USES OF MICROSCOPE
 1. Drawing Sketches With Camera Lucida
It is used to draw clear, simple and exactly proportionate
outline sketches of the objects under study.
 2. Micrometry - the field of science in which microscopic
objects like chromosomes, cells, etc, are measured with the help
of the compound microscope with the help of micrometer.
 In pharmaceutical science- evaluation of nanoparticles,
microemulsions, microspheres, powder microscopy, etc

25. References
 http://nobelprize.org/educational/physics/microscop
es/1.html
 www.googlescholars.com
 J. M. Willey, L.M. Sherwood, C. J. Woolverton, “ Prescott’s
Microbiology” 7th edition, Mcgraw Hill publication. 2014 p.p 22-41.