1. Microscopy

2. Scale

3. Lenses and the Bending of Light light is refracted (bent) when passing from one medium to another refractive index –a measure of how greatly a substance slows the velocity of light direction and magnitude of bending is determined by the refractive indexes of the two media forming the interface

4. Lenses focus light rays at a specific place called the focal point distance between center of lens and focal point is the focal length strength of lens related to focal length –short focal length  more magnification

6. The Light Microscope Types –Bright-field microscope –Dark-field microscope –Phase-contrast microscope –Fluorescence microscopes Modern –Compound microscopes –Image formed by action of  2 lenses

7. Image Quality Resolution –Ability of a lens to separate or distinguish small objects that are close together –Factors WavelengthWavelength –shorter wavelength  greater resolution (450-500nm) Numerical apertureNumerical aperture 0.61 λ n sinθ Working distance –distance between the front surface of lens and surface of cover glass or specimen

8. Brightness - How light or dark is the image? Focus - Is the image blurry or well-defined?

9. Resolution - How close can two points in the image be before they are no longer seen as two separate points? Contrast - What is the difference in lighting between adjacent areas of the specimen?

10. The Bright-Field Microscope Dark image against a brighter background Stained Specimen Several objective lenses (3-5) –Parfocal microscopes Total magnification – Product of the magnifications of the ocular lens and the objective lens –45x (objectice) X 10x (eye piece)= 450x

12. Figure 2.4

13. Phase Contrast Microscopy In phase contrast a phase plate is placed in the light path.In phase contrast a phase plate is placed in the light path. In a phase-contrast microscope, the annular rings in the objective lens and the condenser separate the light.In a phase-contrast microscope, the annular rings in the objective lens and the condenser separate the light. Barely refracted light passes through the center of the plate and is not retarded.Barely refracted light passes through the center of the plate and is not retarded.

14. Phase Contrast Microscopy Highly refracted light passes through the plate farther from center.Highly refracted light passes through the plate farther from center. The interference produced by these two paths produces images in which the dense structures appear darker than the background.The interference produced by these two paths produces images in which the dense structures appear darker than the background.

15. The Phase-Contrast Microscope

16. Phase Contrast Microscopy

18. The Differential Interference Contrast Microscope creates image by detecting differences in refractive indices and thickness of different parts of specimen

19. Differential Interference Contrast Microscopy

20. DIC works by separating a polarised light source into two beams which take slightly different paths through the sample.DIC works by separating a polarised light source into two beams which take slightly different paths through the sample. The beams interfere when they are recombined.The beams interfere when they are recombined.

21. Differential Interference Contrast Microscopy This gives the appearance of a three- dimensional physical relief corresponding to the variation of optical density of the sample, emphasizing lines and edges though not providing a topographically accurate image.This gives the appearance of a three- dimensional physical relief corresponding to the variation of optical density of the sample, emphasizing lines and edges though not providing a topographically accurate image.

23. The Fluorescence Microscope exposes specimen to ultraviolet, violet, or blue light specimens usually stained with fluorochromes shows a bright image of the object resulting from the fluorescent light emitted by the specimen

25. Dark Phase Microscopy

26. Opaque disc is placed underneath the condenser lensOpaque disc is placed underneath the condenser lens Only light that is scattered by objects on the slide can reach the eye.Only light that is scattered by objects on the slide can reach the eye. Light is reflected by specimen on the slide.Light is reflected by specimen on the slide. Bright white against a dark background.Bright white against a dark background. Pigmented objects-false colorsPigmented objects-false colors

27. Electron Microscopy Use of electrons (short wavelength)Use of electrons (short wavelength) 100,000 time smaller wavelenght than light100,000 time smaller wavelenght than light 0.0037nm (light 400-700nm)0.0037nm (light 400-700nm) 1000 time better resolution1000 time better resolution Magnetic field direct the path of electronMagnetic field direct the path of electron Increase in electron velocityIncrease in electron velocity Decrease in wavelengthDecrease in wavelength Resolution 0.1nm (100x more than light microscope)Resolution 0.1nm (100x more than light microscope) denser regions in specimen, scatter more electrons and appear darker

28. Types Transmission electron microscope –internal structure –electrons pass through thin specimens (50-1000 nm). scanning electron microscope –3d structure –In scanning electron microscopy signals emitted from the surface of thick specimens.

29. Transmission electron microscope

31. Electron micrograph of a cell in a root tip

32. Specimen preparation Vaccum to prevent colliding of electron –dead specimen –insufficient electron density –electron dense salt (gold, uranium) Embedded in a polymer for thin sections –microtome cut slices (micrometer thick) Sprayed onto copper grid –viruses and macromolecules Flash frozen (for cryo EM) Artifact