i am HAFIZ M WASEEM from mailsi vehari BSc in science college Multan Pakistan MSC university of education Lahore Pakistan I love Pakistan and my teachers

1. HAFIZ
MUHAM
MAD
WASEEM
BA BA G
LAHORE
BIOLOGICAL TECNIQUES
MICROSCOPY INTRODUCTION
 Micro refers to tiny while scope refers to view

2.  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
 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

3. 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
BIOLOGICAL TECHNIQUES
MICROSCOPY PRINCIPLE
 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
RESOLUTION
 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
MAGNIFICATION
 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

4.  Compound microscope has magnification of about 400 times
 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
ILLUMINATION
 An essential factor in producing a good image is obtaining good level oflight in the
specimen
 It involves the use ofcondenser
 Two apertures in the illumination system regulate the diameter ofthe illumination beam by
closing or opening iris diaphragms
 Illuminator is used to reflect light from an external light source to the specimen
BIOLOGICAL TECHNIQUES
Microscopy- Components
 Microscope is designed to emit light onto or through the objects and magnify the
transmitted or reflected light with the objective and ocular lenses

5.  A general biological microscope consists of
 A light source
 Stand
 Stage
 Objective lens
 Eyepiece lens
 Diaphragm
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

6.  As a result an image is created that can be interpreted by the human eye or recorded on a
photographic detector
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
Specimen stage

7.  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
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)
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
BIOLOGICAL TECHNIQUES
MICROSCOPY TYPE
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

8. • Phase -contrast microscope
• Fluorescence microscopes
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
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
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
SCANNING MICROSCOPE
 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

9.  A powerful tool for material characterization
 Since wavelength is smaller, the resolution is better than the normal light microscope
ELECTRON MICROSCOPE
 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
BIOLOGICAL TECHNIQUES
MICROSCOPY APPLICATIONS
Molecularimaging
 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
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 study of cell based screening for biological or chemical compounds at
translational level
 It is also possible to study various cellular processes at molecular level
Live cell imaging

10.  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
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
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
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