Experimental Techniques in Cell Biology Part 1 Whole Cells a c Outline a Microscopy beginning of cell biology essential tool in the field b Cell Growth separating cells in tissues growing cells in culture c Perturbing Cellular Functions Organelles d d Cell and Organelle Fractionation e Studying Macromolecules Macromolecules e DNA RNA protein 10 30 m Microscopy 10 30 m Animal Cell 5X smaller than smallest particle visible to the naked eye Cell doctrine proposed by Schleiden and Schwann 1838 ALL PLANT AND ANIMAL TISSUES ARE AGGREGATES OF INDIVIDUAL CELLS What is the most difficult aspect of observing small cells cellular structures 1 Magnification 2 Resolution 3 Sample Preparation 1 Magnification 10X 100X Final Magnification 1000X Lodish 7e Fig 9 8 a 2 Resolution Resolution ability to distinguish between 2 objects Beam of radiation ex light or electrons wavelength beam of any type of radiation cannot be used to probe structures much smaller than its limits of light microscopy set by of visible light visible light 0 45 m violet to 0 7 m deep red 2 Resolution Resolution D minimum distance between 2 distinguishable objects Ex If D 0 5 m then one can never resolve objects that are less than 0 5 m apart such objects will appear as one 2 Resolution 2 D 0 61 Nsin Nsin numerical aperture N refractive index of the air or fluid medium between the specimen and objective lens air water or oil angular aperture 2 Resolution What is the best resolution one can obtain with a simple bright field light microscope Maximum 70o sin 70o 0 94 Shortest 0 45 m Best medium immersion oil N 1 5 D 0 61 Nsin Simple light microscopy can distinguish objects separated by approx 0 2 m or more D 0 61 0 45 194 m 1 5 0 94 Bright Field Microscopy Live Macrophages Lodish 7e Fig 9 9 Bright Field Microscopy Advantages simple inexpensive no staining required live cell imaging Limitations Very low contrast of most biological samples Low apparent optical resolution due the blur of out of focus material OPTICAL and PHYSICAL AND BIOCHEMICAL ways to improve these limitations Optical Methods to Improve Images Phase Contrast and Differential Interference Contrast DIC Microscopy Nomarski Microscopy Live Macrophages Bright Field Phase Contrast DIC Lodish 7e Fig 9 9 Physical and Bio Chemical Methods For Improving Images 3 Sample Preparation for Staining B Embed Section thick specimens only A Fix C Stain 3 Sample Preparation for Staining A Fixation 1 Cross links macromolecules cross linking agents glutaraldehyde and formaldehyde form covalent bonds with free amino groups 2 Partially permeabilizes cells for staining 3 Sample Preparation for Staining B Embedding and Sectioning thick specimens only i Embedding using wax or resins Liquid embedding medium Cooling or Polymerization Tissue sample 3 Sample Preparation for Staining B Embedding and Sectioning thick specimens only ii Sectioning Microtom e Lodish 7e Fig 9 10 Cell Staining Some Common Stains hematoxylin nuclear stain blue violet base that binds acids e g DNA eosin acid that binds base cytoplasm proteins pink U of Washington Dept of Pathology Hematoxylin and eosin H E stained section of a tumor found in brain biopsy Bright Field Cell Staining Some Common Stains benzidine binds heme containing proteins Stage 35 Xenopus Embryo Bright Field Hemmatti Brivanlou Thomsen 1995 VBI ventral blood island Experimental Techniques in Cell Biology End Part 1 Experimental Techniques in Cell Biology Part 2 Fluorescence Microscopy Fluorescent Molecule fluor phore chrome absorbs light at one wavelength excitation and emits light at a specific longer wavelength emission Fluorescein Excitation 492 nm Emission 525nm excitation ex 492 nm Stain cell with fluorescent compound Filter that blocks all light except emission ex 525 nm Fluorescence Microscopy Lodish 7e Fig 9 8 b d Fluorescence Cell Staining Ex 350 nm Em 461 nm Adenocarcinoma breast cancer cells MCF7 cell line Nuclei were stained with Hoescht 33342 http www asylumresearch com Variation 1 Immunofluorescence Microscopy Inject Protein X Antigen Antibodies proteins produced by B cells that recognize and bind to foreign antigen protein 8 10 weeks Serum contains Ab to protein X Specific Binding X Immunofluorescence Microscopy Protein X Fix Permeablize Section Fluorescent labeled Second Antibody Add primary 1o antibody recognizing Protein X View by Fluorescence Microscopy Add fluorescent 2o Ab Why use secondary antibody Immunofluorescence Microscopy Entamoeba histolytica Immunostained with an antibody to a vesicle associated protein Immunofluorescence Microscopy Human Skin Fibroblast anti actin Antibody CHO cell anti tubulin antibody Lodish 5e Fig 5 33 Immunofluorescence Microscopy Cultured Nerve Cells Cytokine red Nuclei blue Hoechst Cytoskeleton green http www kumc edu cic image gallery htm Variation 2 Localization of Proteins in Living Cells Ex 492 nm Em 525 nm Aequorea Green Fluorescent Protein GFP Variants EGFP CFP cyan YFP yellow RFP red ECFP EYFP ERFP Localization of Proteins in Living Cells Gene XRecombinant DNA Techniques GFP Gene X Transcription and Translation GFP Protein X Localization of Proteins in Living Cells Dictyostelium discoideum expressing GFP Rab8 Localization of Proteins in Living Cells Actin Localizes to Forming Pinosomes GFP ABD 11 2 1 3 4 Fluorescence Phase Contrast Fluorescence Phase Contrast Jellyfish Zebra Fish Tobacco Mouse Variation 3 Confocal Laser Scanning Microscopy CLSM ordinary light and fluorescence microscopy requires sectioning thick specimens therefore 3D information is lost if thick specimens are use for conventional light microscopy the image is blurred above and below the plane of focus CLSM makes it possible to focus on a chosen plane of a thick specimen while rejecting the light that comes from out of focus areas generally used with fluorescence optics Confocal Laser Scanning Microscopy CLSM See also Lodish 7e Fig 9 17 Confocal Laser Scanning Microscopy CLSM Conventional Fluorescence Microscopy CLSM Lodish 7e Fig 9 18 Sea Urchin Egg Anti tubulin antibody Experimental Techniques in Cell Biology End Part 2 Experimental Techniques in Cell Biology Part 3 FRET Fluorescence resonance energy transfer Acceptor Donor FRET no signal Distance 10 100 angstroms See also Lodish 7e Fig 9 23a FRET fluorphores are close enough together such that YFP can absorb light from CFP emission Distance 10 100 angstroms FRET Fluorescence resonance energy transfer Following Ras Activation in Real Time Ras is a monomeric G protein FRET