COMMON TOOLS TO CELL BIOLOGY

Cytology: microscopic study of cell structure

Histology: tissue 

Cell physiology: cell fx

Cell biology = structure & fx of cells

Light microscopy

Magnification

Resolution: diffraction

Visibility: contrast, stains, Feulgen stain for DNA

Bright-field: suitable for high contrast

Whole mounts: intact, living or dead

Sections: fixative, embedding, sectioning, mounting, stain

Compound

Inverted

Phase-contrast

conversion of refractive indexes into differ intensities

intracellular

interference: sharp changes in refractive index, cause halos and shading

DIC or Nomaski more advance

Fluorescence

fluorochrome/phore: rhodamine, fluorescin, acridine orange, GFP, BFP, YFP, CFP

dynamic events

absorb UV radiation, release energy in visible wavelengths 

FRET

nanoscale distances btwn fluorochrome 

study changes in distance btwn 2 parts of a protein/close associated molecules

Laser scanning confocal

focusing a laser beam 

scans specimen at a single depth

use fluorescence

emitted light sharply focused thro pinhole aperture 

TEM

electron with shorter wavelength higher resolution

1k-250k X

phosphorescent screen 

low atomic no, poor scattering cell constituents require to fix & stain with heavy metals

Cryofixation - ultrarapid freeze with liquid propane or helium

Negative staining - virus, ribosome, protein complex - heavy metal deposits spray all over specimen grid except where particles are present 

Shadow casting - visualize particles - specimen grid seal with filament in vacuum chamber - heating filament cause deposition of metal - shadow behind particles 

Freeze fracture: examine integral membrane proteins 

Shadow staining done followed by carbon coating creates metal-carbon replica 

SEM: examine object surface 

Fixed specimen pass thro alcohols 

dry by critical-point drying ensures cells not expose to surface tension that may distort configuration when cell water vaporized 

dried specimen then coated with thin layer metal 

detection of backscattered electrons 

15-150k X

Radioisotopic and enzymatic tracer

Autoradiography - radioisotope tracers

Fluorescent/spectrofluorometry - fluorescent tracers 

Light microscopy/spectrophotometry - enzyme substrate tracers

Tissue culture

grow cell outside organism 

large qty homogenous cell populations 

cellular activities: endocytosis, cell movement, division, membrane trafficking, macromolecular synthesis 

cell used to test hormones, drugs, toxins, any active substances

low cost 

higher costs: investment, maintenance of sophisticated air filtration system

cell grown in plastic containers containing special media and serum

strict adherence to sterile technique

frozen existing cell = cell lines, secondary culture

primary culture: cells obtained from a living organism and processed to release cells from tissue matrix for propagation

normal cells 50-100 doublings then undergo senescence and death

most cells undergo genetic modification - immortal - frozen and regrown

Fractionation (subcellular)

cells lysed with homogenizer in isotonic buffer - membranous organelles are intact

lysate subject to differ centrifuge

hypertonic lysis for cytosol analysis 

large vesicles pellet under a relatively low centrifugal force 

microsomes derived from endomembrane system are smaller, require higher centrifugal forces at longer periods to separate from other organelles 

Density gradient equilibrium centrifugation

allows different organelles to separate based on density 

organelles move thro, form a band, in a dense solution under a very high centrifugal force, pipette out and wash for further analysis 

density of solution>organelles, organelles stop moving

Isolation - selective precipitation

Purification/fractionation - column chromatography, polyacrylamide gel electrophoresis

Structural elucidation - X-ray crystallography, NMR

Analysis DNA

purification, fractionation, hybridization, chemical synthesis, recombinant technology, amplification by PCR, sequencing, libraries, transferring into eukaryotic cells, gene elimination 

C1 ESSENTIAL CELL BIOLOGY