CELL BIOLOGY TECHNIQUES

Visualize cells - MicroscopyOrganelles – Fractionation of

subcellular componentsCulturing cells

Light Microscopy

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.

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

FIXEMBEDSECTIONSTAIN

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 DECONVOLUTIONMICROSCOPY

• 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 THEINTESTINAL 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 mediumGROWTH 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