CELL BIOLOGY TECHNIQUES

Visualize cells - Microscopy

Organelles Fractionation of
subcellular components
Culturing cells
Light Microscopy
 Light Microscopy
Resolution of 0.2m
Magnification objective and projection lens
Resolution
D = 0.61/N sin

Resolution is improved by using shorter

wavelengths or increasing either N or .
BRIGHT FIELD PATH MICROSCOPY
 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
Microscopy of Live cells
 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
Absorb light at one
wavelength and
emit light at a
specific and longer
wavelength
 HYDRA EXPRESSING GFP

Fluorescent
protein in live
cells
FIX
EMBED
SECTION
STAIN
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
RAT INTESTINAL CELL WALL GLUT 2
 CONFOCAL AND DECONVOLUTION
MICROSCOPY
This overcomes the limitations of
Fluorescence microscopy
Blurrred images
Thick specimens
REMOVES OUT OF FOCUS IMAGES
EXAMPLE OF IMAGE RECONSTRUCTED
AFTER DECONVOLUTION MICROSCOPY
 ELECTRON
MICROSCOPY
 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
GOLD PARTICLES COATED WITH PROTEIN A ARE
USED TO DETECT ANTIBODY BOUND TO PROTEIN
TEM IMAGE
 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
SURFACE DETAILS BY METAL
SHADOWING
SEM OF EPITHELIUM LINING THE
INTESTINAL LUIMEN
 PURIFICATION OF CELL ORGANELLES
CELL DISRUPTION
SEPARATION OF DIFFERENT ORGANELLES
USING CENTRIFUGATION
PREPARATION OF PURIFIED ORGANELLES
USING SPECIFIC ANTIBODIES
 BREAKING OPEN PLASMA
MEMBRANES IN CELLS
CELLS ARE SUSPENDED IN ISOTONIC SUCROSE
SONICATION
HOMOGENIZATION
CELLS IN HYPOTONIC SOLUTION RUPTURE
OF CELL MEMBRANES
 SEPERATING ORGANELLES
DIFFERENTIAL CENTRIFUGATION
DENSITY GRADIENT CENTRIFUGATION
DENSITY GRADIENT CENTRIFUGATION
ANTIBODIES ARE USED TO MAKE
HIGHLY PURIFIED ORGANELLES
CELL SORTER FLOW CYTOMETRY
 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,
 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
PRIMARY CULTURES
STAGES IN CELL CULTURE
DIFFERNTIATION OF A CELL LINE
C2C12 IN CULTURE
 HOMEWORK-1

CHAPTER 9
REVIEW CONCEPTS QUESTIONS -2,5,7,9
ANALYZE THE DATA

DUE NEXT WEEK IN THE WORKSHOPS