The document discusses microscopy techniques. It begins by defining microscopy as the technique of viewing tiny objects that are too small to see with the naked eye. It then covers the basic principles of microscopy including resolution, magnification, and illumination. The major components of light microscopes are described as the light source, stand, stage, objective lens, and eyepiece lens. Different types of light microscopes are outlined including brightfield, darkfield, phase contrast, and fluorescence microscopes. Applications of microscopy in various fields like molecular imaging, cellular imaging, and biomedical engineering are also summarized.

1. HAFIZ
MUHAM
MAD
WASEEM
BA BA G
LAHORE

2. Biological Techniques
UNIVESITY OF
EDUCATION
LAHORE

3. Introduction
Microscopy?
 Micro refers to tiny while scope refers to view
 So, microscopy is a technique to view the
specimens that are too small to be seen with the
naked human eye
 Our eyes cannot focus on the objects nearer than
about 25 cm or 10 inches
 This limitation is overcome by using a convex lens
as a simple magnifier (or microscope)
 It is a technical field to reveal greater details of the
samples

4. Introduction
 Robert Hooke was the first to use a microscope in
1665 and publish his work
 Beginnings of cell biology is traced to the invention
of the light microscope
 It became possible for the scientists to examine
enlarged images of the cells and analyze their
structure and functions
 Most images produced by microscopes are
recorded electronically by using digital cameras
and digital image acquisition software

5. Introduction
 Microscopy is used to study microorganisms,
cells, crystalline structures and the molecular
structures
 Light microscopes are the most commonly
used and the oldest types of microscopes
 Light passes through the specimen to
examine and magnify it
 Many modifications like fluorescent, electron
and confocal microscopy are used these days
 Backbone in biology

6. Biological Techniques

7. Principle of Microscopy
 Principle of microscopy is important to determine how
small an object can be observed and analyzed
 Three factors are needed to form an image regardless
of the type of microscope
• Resolution
• Magnification
• Illumination
 Microscopes differ in their magnification and
resolution and thus give different types of images of
the observed specimens

8. Principle of Microscopy
 It is ability to distinguish two very small and closely
spaced objects as separate entities
 Magnification of small things is a necessary facet of
biological research
 Resolution is the best when the distance separating the
two tiny objects is small
 Resolution is determined by parameters that include
the wavelength of light and the light gathering power
of the objective and lenses
Resolution

9. Principle of Microscopy
 It is a measure of how much larger a microscope
causes an object to appear
 In compound microscope final image is produced
by the objective and a ocular lens
 Magnification is dependent on the magnifying
power of the objective lens times the magnifying
power of the ocular lens
 Compound microscope has magnification of about
400 times
Magnification

10. Principle of Microscopy
 Generally greater the magnification, the greater
is the resolution
 Image may be magnified but may have low
resolution if not properly clear
 Resolution may be changed by changing the lens
 It is possible to see two objects which are about
0.1 mm apart and are held 10 inches away from
the face
 If two objects are 0.01 mm apart we magnify
them by 10X
Magnification

11. Principle of Microscopy

12. Principle of Microscopy
 An essential factor in producing a good image is
obtaining good level of light in the specimen
 It involves the use of condenser
 Two apertures in the illumination system regulate the
diameter of the illumination beam by closing or
opening iris diaphragms
 Illuminator is used to reflect light from an external
light source to the specimen
Illumination
?

13. Biological Techniques

14. Components of Microscopy
 Microscope is designed to emit light onto or through the
objects and magnify the transmitted or reflected light with the
objective and ocular lenses
 A general biological microscope consists of
• A light source
• Stand
• Stage
• Objective lens
• Eyepiece lens
• Diaphragm

15. Light source
 Source of illumination in a light microscope is visible
light (in the wavelength range of 400-700 nm)
 It may be natural or artificial
 Artificial sources may include UV, halogen lamps, LEDs
and others
 Specimen is illuminated for viewing and imaging
 As a result an image is created that can be interpreted
by the human eye or recorded on a photographic
detector
Components of Microscopy

16. Components of Microscopy

17. Stand
 This part of microscope holds all the components firmly in
position
 Microscopic stands may be upright or an inverted one
 Condenser and light source can often be swung out of the light
path
 This allows additional room for manipulating the specimen on
the stage
 Important part in handling and transportation of the microscope
Components of Microscopy

18. Specimen stage
 A mechanical device that is finely engineered to hold
the specimen firmly in its place
 It enables the specimen to be moved and positioned in
fine and smooth increments, both horizontally and
transversely, in the X and Y directions
 Specimen stage can either be moved manually or by a
stepper motor
 Clips are used to hold the specimen or the slide at its
position
Components of Microscopy

19. Objective lens
 It is a lens which is closer to the object
 There may be one or two lenses in a microscope
 This component is responsible for producing the
magnified image and can be the most expensive
component of the microscope
 Objectives are available in many different varieties
(4X, 10X, 20X, 40X, 60X and 100 X)
 May have immersion requirements (air, water or oil)
Components of Microscopy

20. Eyepiece lens
 It is a lens closer to the eye of the user
 It works in combination with the objective lens
to magnify the image
 It allows the image to be detected by eye for
direct viewing or for recording purposes
 Eyepieces usually magnify by 10X since an
eyepiece of higher magnification only enlarges
the image without improving the resolution
Components of Microscopy

21. Biological Techniques

22. Types of Microscopy
Light microscope
 The simplest form of light microscope is a magnifying lens
which consists of a glass lens mounted in a metal frame
 Contrast in images is usually produced by staining the sample
using dyes
 Compound microscopes use 2 lenses to produce the image
 Main types are
• Bright -field microscope
• Dark -field microscope
• Phase -contrast microscope
• Fluorescence microscopes

23. Types of Microscopy
Bright Field
 These are the simplest of all types of light microscopes
 Specimen is dark and contrasted by the surrounding
bright viewing field
 Sample illumination is via transmitted white light, i.e.
illuminated from below and observed from above
 Limitations include low contrast and low resolution
 Minimum preparation required is the significant
advantage

24. Types of Microscopy
Dark Field
 Dark field microscopes are used to improve the
contrast of unstained and transparent
specimens
 Light scattered by the specimen enters the
objective lens to produce a bright image against
the dark background
 It has low resolution
 Used in microbiology and autoradiography
 Many variations are available for better results

25. Types of Microscopy
Phase contrast
 Phase contrast is a widely used technique
where differences in light intensity alter the
path of light more than the less dense regions
 Interference with the diffracted light results in
the phase contrast image
 It is not used to study thick objects
 Used to study changes during cell division,
pseudopodia formation, exocytosis, etc. in
unstained cells

26. Types of Microscopy
 It uses electron beam to create an image
 Electrons interact with the samples to produce various
types of signals
 Informations are used to create topography and
composition
 A powerful tool for material characterization
 Since wavelength is smaller, the resolution is better
than the normal light microscope
Scanning microscope

27. Types of Microscopy
 A beam of accelerated electrons is used
 Uses electron optical lens system as compared with
lenses
 Investigate ultrastructure of the biological or inorganic
structures
 Involves the use of very thin sections (100 nm thick)
 Resolution is much better than a compound
microscope
 Recent modifications involves the use of digital
cameras, etc
Electron microscope

28. Biological Techniques

29. Molecular imaging
 It is an interplay between molecular biology and in
vivo imaging
 Optical molecular imaging is used as a powerful tool
for studying the temporal as well as spatial dynamics
of the bio-molecules and their interactions
 Single molecule imaging in living cells is also possible
 It is used to reveal interaction and functions of
molecules in different cells
Applications of Microscopy

30. Cellular imaging
 It is used for the imaging of biophysical processes at
the cellullar scale
 It requires intensive sample preparation whereas the
imaging of higher scale processes is also possible
 Used in the tudy of cell based screening for biological
or chemical compounds at translational level
 It is also possible to study various cellular processes at
molecular level
Applications of Microscopy

31. Live cell imaging
 It is used to study imaging of illuminated live cells
 Study of fluorescent cells can lead to photobleaching
which may result in cellular damages
 Confocal microscopy may be helpful which focuses
only on the point being imaged
 This decreases excess exposure to the rest of the cells
and thus the damages
 It is possible to study cell to cell interactions
Applications of Microscopy

32. In pharmaceuticals
 Used in the characterization of many pharmaceutical
products including tablets, film coatings and colloidal
systems
 It has also been used to study the interaction at
biological barriers of the skin, eyes and intestinal
epithelia
 It also gives information about the effectiveness of
dosage forms at delivering the drugs through these
barriers
Applications of Microscopy

33. Clinical applications
 Used in the hematological investigations of various
diseases
 It has also been used in immunological and histological
studies to examine behavior and interaction of
molecules, organelles and cells
 Urine analysis reveals kidney and other body functions
 Different surgical procedures may also involve the
related techniques in health and diseases
Applications of Microscopy

34. Biomedical engineering
 Biomedical optics are the future of our health care
industry
 It provides a safe, non-invasive, and non-destructive
method of analysis for a variety of medical needs
 Instrumentation is durable and portable
 It is possible to monitor blood oxygen levels and
hemoglobin saturation
 Used in various types of surgical procedures
Applications of Microscopy