Microscopy .

i am HAFIZ M WASEEM from mailsi vehari
bsc in science college multan pakistan
msc univesity of education lahore pakistan
i love pakistan and my teachers

1. HAFIZ MUHAMMAD WASEEM BA BA G LAHORE

2. Microscopy-Introduction 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. Microscopy-Principle 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. Microscopy- Components 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. Microscopy- Types 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. Microscopy -Applications 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