1. Visualizing Prokaryote Cells Chapter 3 - Black

2. Light

3. Key Words - Visible light Visible light Ultraviolet light Ultraviolet light Reflection Reflection Transmission Transmission Refraction Refraction Absorption Absorption

4. Light Microscopy

5. Resolution Resolution is the ability to see two images as separate and discrete. Resolution is the ability to see two images as separate and discrete. The wavelengths of visible light from 420 to 620 prevent resolution of two points closer than 220 nm The wavelengths of visible light from 420 to 620 prevent resolution of two points closer than 220 nm By using the light emitted from an electron it is possible to resolve two points that are.2nm apart By using the light emitted from an electron it is possible to resolve two points that are.2nm apart

6. Specialized Microscopy Dark field Dark field Phase Contrast Phase Contrast Differential interference Differential interference Fluorescence Fluorescence Confocal Confocal Digital Digital

7. Digital photomicrosopy Camera can be used to photograph images Camera can be used to photograph images Specimen can be viewed on TV screen as well as on computer screen Specimen can be viewed on TV screen as well as on computer screen

8. Classroom Microscopy

9. Phase Contrast Microscopy Accentuate small differences in the refractive index of the specimen Accentuate small differences in the refractive index of the specimen More detail is apparent in living cells More detail is apparent in living cells Assist in the visualization of cell structure in transparent cells Assist in the visualization of cell structure in transparent cells micro.magnet.fsu.ed u/.../dicphasecompar ison.html micro.magnet.fsu.ed u/.../dicphasecompar ison.html micro.magnet.fsu.ed u/.../dicphasecompar ison.html micro.magnet.fsu.ed u/.../dicphasecompar ison.html

10. Differential interference microscopy Produces higher resolution Produces higher resolution Depends on a gradient Depends on a gradient It can produce almost a three dimensional image It can produce almost a three dimensional image

11. Comparison of Phase Contrast and Differential Interference Contrast

12. Fluorescent Microscopy Organisms such as Mycobacterium tubercuolosis and Treponema pallidum are treated with a fluorochrome dye Organisms such as Mycobacterium tubercuolosis and Treponema pallidum are treated with a fluorochrome dye Ultra violet light is used to excite the fluorochrome molecules and produce a glowing image Ultra violet light is used to excite the fluorochrome molecules and produce a glowing image Used in clinical work Used in clinical work Also used with antigens and antibodies to identify the presence of molecules on the surface of a cell Also used with antigens and antibodies to identify the presence of molecules on the surface of a cell

13. Fluorescent antibody staining Used in immunology Used in immunology Fluorescent antibodies are used to detect antigens on the surface of cells Fluorescent antibodies are used to detect antigens on the surface of cells In the picture to the right are Bacillus anthracis cells tagged with a fluorescent antibody In the picture to the right are Bacillus anthracis cells tagged with a fluorescent antibody

14. Confocal Microscopy Utilizes beams of ultraviolet light to excite fluorescent dye molecules. Utilizes beams of ultraviolet light to excite fluorescent dye molecules. The exciting light is focused on the specimen with a thin optical fiber The exciting light is focused on the specimen with a thin optical fiber Resulting fluorescence is focused through a narrow aperture Resulting fluorescence is focused through a narrow aperture The light is detected and analyzed by a computer The light is detected and analyzed by a computer Very sharp focus Very sharp focus For thick specimens an image is constructed in layers For thick specimens an image is constructed in layers

15. Confocal images http://www.microscopyu.com/galleri es/confocal/applecedarrustaeciaand pycnia.html http://www.microscopyu.com/galleri es/confocal/applecedarrustaeciaand pycnia.html http://www.microscopyu.com/galleri es/confocal/applecedarrustaeciaand pycnia.html http://www.microscopyu.com/galleri es/confocal/applecedarrustaeciaand pycnia.html http://www.microscopyu.com/galleri es/confocal/chlamydomonas.html http://www.microscopyu.com/galleri es/confocal/chlamydomonas.html http://www.microscopyu.com/galleri es/confocal/chlamydomonas.html http://www.microscopyu.com/galleri es/confocal/chlamydomonas.html

16. Electron Microscopy Transmission electron microscopy Transmission electron microscopy Scanning electron microscopy Scanning electron microscopy

17. Transmission Electron Microscopy Electrons are used as the source of light Electrons are used as the source of light Produced by a high voltage current running through a tungsten filament Produced by a high voltage current running through a tungsten filament www.steve.gb.com /science/electron _microscopy.html www.steve.gb.com /science/electron _microscopy.html www.steve.gb.com /science/electron _microscopy.html www.steve.gb.com /science/electron _microscopy.html

18. Transmission Electron Microscope The lenses are electromagnetic The lenses are electromagnetic They act on the negatively charged electrons to focus them in a concentrated path through the specimen They act on the negatively charged electrons to focus them in a concentrated path through the specimen The image is magnified by additional lenses and visualized on a screen The image is magnified by additional lenses and visualized on a screen

19. TEM images Images produced display high resolution Images produced display high resolution Staining with heavy metals that interact with the electrons Staining with heavy metals that interact with the electrons Gradations of black, gray, and white contrast areas of greater density that absorb the stain Gradations of black, gray, and white contrast areas of greater density that absorb the stain

20. Scanning Electron Microscopy Electrons are reflected and collected off of the surface of a cell Electrons are reflected and collected off of the surface of a cell

21. SEM Images show surface contours Images show surface contours Three dimensional image Three dimensional image

22. Freeze Fracture Cells are quickly frozen in liquid nitrogen (196C), which immobilizes cell components instantly. 2. Block of frozen cells is fractured. This fracture is irregular and occures along lines of weakness like the plasma membrane or surfaces of organelles. 3. Surface ice is removed by a vacuum (freeze etching) 4. A thin layer of carbon is evaporated vertically onto the surface to produce a carbon replica. 5. Surface is shadowed with a platinum vapor. 6. Organic material is digested away by acid, leaving a replica. 7. Carbon-metal replica is put on a grid and examined by a transmission electron microscope. Cells are quickly frozen in liquid nitrogen (196C), which immobilizes cell components instantly. 2. Block of frozen cells is fractured. This fracture is irregular and occures along lines of weakness like the plasma membrane or surfaces of organelles. 3. Surface ice is removed by a vacuum (freeze etching) 4. A thin layer of carbon is evaporated vertically onto the surface to produce a carbon replica. 5. Surface is shadowed with a platinum vapor. 6. Organic material is digested away by acid, leaving a replica. 7. Carbon-metal replica is put on a grid and examined by a transmission electron microscope.

23. Freeze Fracture and Etching

24. Freeze Fracture

26. References http://micro.magnet.fsu.edu/primer http://micro.magnet.fsu.edu/primer http://micro.magnet.fsu.edu/primer http://www.mos.org/sln/SEM/ http://www.mos.org/sln/SEM/ http://www.mos.org/sln/SEM/