Learn about Cell biology techniques

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

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
– theoretically 0.005 nm; practically 0.1 nm –1 nm
(2000x better than LM)
– High – velocity electron beam passes through the
sample
– 50-100 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