1. Cell Biology 3 - Tools of the cell biologist
Sung Youn Lee, PhD. Student
Veterinary collage, Room 320 ,

3. Microscope
Because cells and organelles are too small to be examined by naked eye, the backbone of the cell biology is the microscope.

6. Microscope Resolving power & Wavelength d= 0.61 λ
NA objective + NA condenser
NA; the numerical aperture
NA= sineθ (θ=1/2 angle) * refractive index (air=1.0, immersion oil=1.4)

7. Microscope Resolving power of light microscope 0.1μm (theoretical)
0.2μm (in real) ; x1,000 better than the unaided human eye

8. Microscope Phase-contrast microscope
Differential interference contrast (DIC) microscope
Dark-field microscope
Image processing

16. Daphnia, Bright Field
Daphnia, Dark Field

20. Microscope
Tissue usually are fixed and stained for histologic examination.
Individual proteins can be identified in cells using selective-staining procedures.
Samples for electron microscopy require special methods of preparation.
Cell surface morphology can be studied with modified electron-microscopic procedures.

21. Fixation
10% formalin
Formalin : Tap water = 1:9

23. Tissue Processing Washing (over 3hrs) – remove formalin
Dehydration ( % EtOH)
Clearance (Xylene)
Infiltration (Tissue prep-Tissue prep)
Embedding
Sectioning
Staining

37. Cell fraction Blender(homogenizer) : intact state
Ultracentrifugtion : size & density
Nuclei – very low centrifugation speed
Mitochondria and lysosomes – higher speed
Membranous structure – very high speed
Modified forms : buoyant density centrifugation

40. Protein purification
Column chromatography
Electrophoresis (SDS-PAGE)

46. Cell culture
Most cells, whether plant or animal, will grow in vitro if supplied with a growth medium of composition similar to the environment in which that particular cell type normally flourishes.
Cell culture media are designed to mimic the normal physiologic environment. These media are isoosmotically correct mixtures of amino acids, salts, vitamins, and other components such as glucose. In addition, most mammalian cell culture media contain between 5~10% horse or calf serum. Serum contains low levels of protein growth factors, such as platelet-derived growth factor, which are responsible for stimulating cell division.
Most types of human cells have a limited life span in culture and usually will die out after cell division.
Cells in primary culture usually show most of the differentiated properties that they exhibited in vivo.

48. Cell culture
Microelectrodes – patch clamping
Microinjection
Others

53. Pulse chase experiments
Radioisotope

55. Thank you for your attention ~