This document discusses various cell biology techniques used to visualize and study cells. It describes microscopy techniques like light microscopy, fluorescence microscopy, confocal microscopy, electron microscopy, and flow cytometry that are used to visualize cells and specific cellular structures. It also discusses techniques used to isolate and purify cellular organelles like fractionation and density gradient centrifugation. Finally, it covers cell culture methods like culturing primary cells, cell strains, and cell lines on solid growth media.

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