This document provides an overview of microscopy. It defines microscopy as investigating small objects using a microscope. The key parts of a microscope that provide magnification and resolution are described. Different types of microscopy are outlined, including bright field, dark field, phase contrast, and fluorescence microscopy. Examples of uses include drawing sketches, micrometry in pharmaceutical sciences, and evaluating nanoparticles. Electron microscopes like TEM and SEM are also introduced.

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.