1. CELL BIOLOGY TECHNIQUES
Visualize cells - Microscopy
Organelles – Fractionation of subcellular components
Culturing cells

2. Light Microscopy

3. Light Microscopy Resolution of 0.2µm
Magnification – objective and projection lens
Resolution
D = 0.61λ/N sin α
Resolution is improved by using shorter wavelengths or increasing either N or α.

4. BRIGHT FIELD PATH MICROSCOPY

5. Visualize unstained living cells
Phase Contrast microscopy
Thin layers of cells but not thick tissues
Differential Interference contrast
Suited for extremely small details and thick objects
Thin optical section through the object

7. Microscopy of Live cells

8. Fluorescence Microscopy
Major Function: Localization of specific cellular molecules – example proteins
Major Advantages:
Sensitivity:“glow” against dark background
Specificity: immunofluorescence
Cells may be fixed or living
Fluorescent dyes or proteins (Flurochromes)
flurochromes may be indirectly or directly associated with the cellular molecule
Multiple flurochromes may be used simultaneously

9. Absorb light at one wavelength and emit light at a specific and longer wavelength

10. HYDRA EXPRESSING GFP
Fluorescent protein in live cells
Fura-2

11. FIX
EMBED
SECTION
STAIN

12. Immunofluorescence Microscopy and Specific Proteins
Fluorescently tagged primary anti body
Fluorescently tagged secondary antibody
Fluorescently labelled antibody to tagged proteins such as myc or FLAG

13. RAT INTESTINAL CELL WALL – GLUT 2

14. CONFOCAL AND DECONVOLUTION MICROSCOPY
This overcomes the limitations of Fluorescence microscopy
Blurrred images
Thick specimens

15. REMOVES OUT OF FOCUS IMAGES

16. EXAMPLE OF IMAGE RECONSTRUCTED AFTER DECONVOLUTION MICROSCOPY

17. ELECTRON MICROSCOPY

18. Transmission EM Scanning EM
theoretically nm; practically 0.1 nm –1 nm (2000x better than LM)
High – velocity electron beam passes through the sample
nm thick sections
2-D sectional image – surface details are revelaed
Subcellular organelles
Scanning EM
Resolution about 10 nm
Secondary electrons released from the metal coated unsectioned specimen
3-D surface image

19. GOLD PARTICLES COATED WITH PROTEIN A ARE USED TO DETECT ANTIBODY BOUND TO PROTEIN

20. TEM IMAGE

21. CRYOELECTRON MICROSCOPY
HYDRATED, UNFIXED AND UNSTAINED SAMPLES
SAMPLES ARE OBSERVED IN ITS NATIVE HYDRATED STATE
METHOD - AN AQUEOUS SUSPENSION OF SAMPLE IS APLLIED ON A GRID AND HELP B Y A SPECIAL MOUNT
5 nm RESOLUTION

22. SURFACE DETAILS BY METAL SHADOWING

24. SEM OF EPITHELIUM LINING THE INTESTINAL LUIMEN

25. PURIFICATION OF CELL ORGANELLES
CELL DISRUPTION
SEPARATION OF DIFFERENT ORGANELLES USING CENTRIFUGATION
PREPARATION OF PURIFIED ORGANELLES USING SPECIFIC ANTIBODIES

26. BREAKING OPEN PLASMA MEMBRANES IN CELLS
CELLS ARE SUSPENDED IN ISOTONIC SUCROSE
SONICATION
HOMOGENIZATION
CELLS IN HYPOTONIC SOLUTION – RUPTURE OF CELL MEMBRANES

27. SEPERATING ORGANELLES
DIFFERENTIAL CENTRIFUGATION
DENSITY GRADIENT CENTRIFUGATION

29. DENSITY GRADIENT CENTRIFUGATION

30. ANTIBODIES ARE USED TO MAKE HIGHLY PURIFIED ORGANELLES

32. CELL SORTER – FLOW CYTOMETRY

33. CELL CULTURE REQUIREMENTS
SOLID MEDIA
Specially coated plastic dishes or flasks (CAMs’)
Agar as the medium
GROWTH MEDIA
Rich in nutrients- amino acids, vitamins, salts fatty acids, glucose, serum provides the different growth factors,

34. TYPES OF CULTURED CELLS
PRIMARY CELL CULTURES – DIFFERENTIATE IN CELL CULTURE
CELL STRAIN – ALSO HAVE A FINITE LIFE SPAN (FROM A PRIMARY CULTURE)
CELL LINE - INDEFINITE LIFE SPAN

35. PRIMARY CULTURES

36. STAGES IN CELL CULTURE

37. DIFFERNTIATION OF A CELL LINE – C2C12 IN CULTURE

38. HOMEWORK-1 CHAPTER 9 DUE NEXT WEEK IN THE WORKSHOPS
REVIEW CONCEPTS QUESTIONS -2,5,7,9
ANALYZE THE DATA
DUE NEXT WEEK IN THE WORKSHOPS