第二章 细胞生物学研究方法

01_04_Early microscopes.jpg 1880 drawing Living cell photographed thru light microscope

01_05_Cells form tissues.jpg Stains help to see cell parts, but kill cell

01_09_Scale.jpg

01_07_Internal structures.jpg Internal structures of cell can be seen with light microscope

01_08_TEM.jpg Fine structures of cell viewed with transmission EM (TEM, vs SEM p. 9)

Visualizing Cells Cells are small and complex Typical cell μm in diameter (1/5 th size of smallest particle seen by naked eye) Resolution of cells is achieved by microscopy

Cells Under the Microscope 1) Light microscopes use visible light R = 2) Electron microscopes use beams of electrons as the source of illumination 0.61 N Sin α/2

Microscopy technologies Light microscopy(1600 ’ s) Bright field Phase contrast Nomarski Electron microscopy (1930 ’ s) Scanning EM Transmission EM Fluorescence microscopy (1911) Fluorescence Deconvolution Confocal 01_06_What can we see.jpg Size range of typical cells?? Typical molecule?

一、 Light Microscope Resolution limit– 0.2 μm defined as the limiting separation at which two objects can be seen as distinct bacteria and mitochondria ~ 0.5 μm (smallest objects discernible) Resolution of light microscope is limited by the wavelength of light –Why?

Smaller details obscured by the wave nature of light Light travels in waves that pursue different routes and interfere with one another * Light waves in phase reinforce one another * Light waves out of phase interfere and cancel each other partially or completely (see Fig 9.4)

Light waves are reinforced Resolution of structural details is possible Light waves interfere and Cancel each other out No resolution of structural details

1. Light field mucroscope

Tissues can be fixed, sectioned and stained –Fixatives (e.g. formaldehyde) makes cell permeable to stains and cross-links macromolecules –Stains selectively depict subcellular components e.g. hematoxylin stains DNA and RNA (see Fig. 9-11)

Stained tissue section showing urine- collecting Ducts in the kidney Stained with hematoxylin and eosin Duct composed of closely packed cells Nuclei stained red Extracellular matrix stained blue

切片流程图

2. Dark field microscope

Can view cells while they are still alive without fixation Phase of light changed as it passes through a cell –light passing through thick nucleus is retarded »phase shifted relative to light passed through adjacent thinner cytoplasm »Interference effects produced when the two sets of waves recombine creates an image of structure (see Fig. 9.7 ) 3. Phase-contrast microscopy

Light passing through unstained cells undergoes very little change in in amplitude But it does undergo a phase change Phase alterations can by made visible by a phase contrast microscope Contrast is obtained and structure is visualized

Stained portions of the cell reduce the amplitude of particular wavelengths passing through them This gives rise to a coloured image of the cell visible in a normal light microscope

Phase-contrast microscopy For unstained specimens such as a living cells. One basis upon which intracellular organelles differ is their refractive index resulting in the difference of light distance( phase position). The Phase- contrast microscopy converts differences in the later into differences in intensity (amplitude, brighter or darker) on the basis on interference of light (the background light of the field) from the light diffracted by the object, and causes these types of waves to be approximately 1/2 wavelength out of phase with one another so that they can interact (interference) and cause changes in intensity.

4. Differential interference contrast microscopy (DIC) Nomarski system To minimize the optical artifacts by achieving a complete separation of direct and diffracted beams using complex light paths (pass through by polarized light) and prisms. It delivers an image being an apparent three- dimensional quality, which depends on the rate of change of refractive index across a specimen particularly in the edges of structure. 偏振光经合成后，使样品中厚度上的微小区别转化 成明暗区别，增加了样品反差且具有立体感。适于研 究活细胞中较大的细胞器

Bright field and phase contrast

Nomarski and darkfield

二、 Fluorescence Microscope Based on the detection of fluorescent molecules Absorb light at one wavelength (the excitation wavelength) and emit at another (excitation wavelength) Viewed through a filter that only allows emitted light through See this against a dark background

Fluorescence Microscopy Fluorescence microscope similar to an ordinary light microscope, except: –Illuminating light is passed through 2 filters 1 st filter only passes wavelengths that excite the flurophore 2 nd filter blocks out the excitation wavelenghts and only passes those wavelengths that are emitted by the fluorphore (Fig. 9-12)

Fluorescence Microscopy Two commonly used fluorescent dyes that are covalently bound to antibodies: –Fluorescein Emits an intense green fluorescence when excited with blue light –Rhodamine Emits a deep red fluorescence when excited with green-yellow light In fact, there are a # of such dyes (Fig 9-13)

Fluorescent Dyes Excitation and emission wavelengths Photon emitted is at a lower energy ( longer wavelength) than the photon absorbed Thus the difference between excitation & emission peaks

Fluorescence microscope often used to detect specific proteins or other molecules in cells e.g. use of antibodies to which specific fluorescent dyes have been covalently attached

Fluorescent Image of a Cell in Mitosis Spindle microtubules revealed with a green fluorescent antibody Centromeres – red fluorescent antibody DNA – blue fluorescent dye

三、 Confocal Microscopy Emitted light from regions out of the plane of focus is out of focus at the pinhole and largely excluded

Confocal Microscopy For ordinary light microscopy, the tissue sliced into sections –Sectioning results in loss of information in the 3 rd dimension As well, optical microscope focussed on a specific focal plane –Parts above and below the focal plane are illuminated, but out of focus

Confocal Microscopy Confocal microscope allows 3 dimensional imaging by manipulation of light before it is measured –Uses fluorscence optics Does not illuminate the whole specimen simultaneously. Rather, uses a laser to focus a spot of light onto a single point at a specific depth in the specimen This is possible because of the power of a laser beam

Confocal Microscopy Emitted fluorescence collected and brought to an image on the detector –Pinhole aperture placed in front of the detector at a position confocal with the illuminating pinhole i.e. precisely where the rays from the illuminated point in the specimen come into focus (Fig. 9-18)

Fluorescein -excited by blue light

Fluroescein -emitted light is green

Confocal microscopy

Actin filaments in Drosophila embryo A) fluorescence microscope B) confocal microscope

A mitotic fertilized egg from sea urchin, anti-tubulin antibody

DNA: blue; microtubles: green; actin microfilaments: red

四、 Detection with Antibodies 1.Nature of Antibodies –Bind to specific antigens, usually 5 to 6 amino acid sequence on proteins (Fig )

Composed of 4 polypeptide chains -2 light and 2 heavy Two antigenic binding sites identical (formed by the N termini of light & heavy chains) Hinge region formed by the 2 heavy chains Typical Antibody

Detection with Antibodies 2. Types of Antibodies –Polyclonal Made by injecting antigen into rabbit (goat) Antiserum contains polyclonal antibodies –Each produced by a different antibody- secreting cell (B lymphocyte) (Fig ) »Each recognizes a certain part (epitope) of the antigen (Fig )

Naïve or memory B cells activated by the antigen They proliferate & differentiate into effector B cells Effector cells produce & secrete antibodies with a unique antigen-binding site The unique antigen-binding site is the same as that of the original membrane-bound antibody of the B cell that served as the antigen receptor

Globular Protein with a number of different antigenic determinants (epitopes) When the protein folds, antigenic determinants are formed on its surface (usually 5 to 6 amino acid residues) Monoclonal antibodies only recognize one epitope

Detection with Antibodies 2. Types of Antibodies –Monoclonal antibodies are epitope-specific (Fig ) –Because they are epitope-specific- Can be made against molecules that are only a minor component of a complex mixture –Proportion of polyclonal antibodies against this minor component would be too small to be useful

Detection with Antibodies Monoclonal Antibodies –Produced using hybridoma cell lines Fusion of a single antibody-secreting B lymphocyte from mouse with a mouse B lymphocyte tumor cell –Results in a hybridoma that can be propagated as a clone to produce monoclonal antibodies (Fig. 8-5)

Detection with Antibodies Monoclonal Antibodies –Hybridoma overcomes a problem B lymphocytes have a limited life span in culture and can ’ t be used as ongoing source of antibody The fusion with a tumor cell confers upon the lymphocyte the ability to multiply indefinitely in culture (Fig. 8-6)

Preparation of Hybridomas for Production of Monoclonal Antibodies HAT medium ： Hypoxanthine 次黄嘌呤 Aminopterin 氨基蝶呤 Thymine 胸腺嘧啶 HGPRT 磷酸核糖转移酶 Purine salvage pathway of nucleotide synthesis

Detection with Antibodies Fluorescently labelled antibodies can be used simultaneously to depict distributions of different molecules or structures (Fig )

Fluorescence Microscopy

Complex network of the cell Nucleus Actin Microtubules

Deconvolution

Detection with Antibodies Amplification of the signal 1.Unlabelled primary antibody and group of labeled secondary antibodies (Fig. 9-16)

Amplification: primary antibody recognized by many molecules of the secondary antibody

Detection with Antibodies Amplification of the signal 2. Alkaline phosphatase is linked to the secondary antibody –Produces localized accumulaion of coloured precipitate upon addition of suitable substrate »This can be detected by measuring absorbance on a Plate Reader

Detection with Antibodies Amplification of the signal 2. Alkaline phosphatase is linked to the secondary antibody This amplification is the basis for Elisa –Elisa (enzyme-linked immunosorbent assay) –Medical applications e.g tests for pregnancy; infection

Green Fluorescent Protein (GFP) Very powerful experimental tool when used in conjunction with confocal microscopy

Green Fluorescent Protein (GFP) Fluorescent dyes (e.g. fluorescently labelled antibodies) have to be introduced into the cell GFP can be used to tag individual proteins in living cells –Reason: this protein is naturally fluorescent

Green Fluorescent Protein (GFP) Gene encoding GFP isolated from the jellyfish Aequoria victoria –GFP can be cloned and introduced into cells of other species

Use of Green Fluorescent Protein (GFP) As a reporter molecule to monitor gene expression –Transgenic organism made with the GFP-coding sequence under the transcriptional control of the promoter belonging to the gene of interest

Gene A Promoter Coding region GFP-reporter gene construct Promoter for Gene A Coding region for GFP Can be used to visualize the expression of Gene A Promoter for Gene A regulates the expression of GFP

Use of Green Fluorescent Protein (GFP) As a tag to localize proteins –The GFP-encoding sequence is placed at the beginning or end of the gene for another protein This yields a chimeric protein consisting of the protein of interest with a GFP domain attached –GFP-fusion protein often behaves like the original protein, directly revealing its subcellular location (Fig. 9-44)

Gene A Promoter Coding region GFP-fusion protein construct Promoter for Gene A Coding region for GFP Coding region For Gene A Can be used to visualize the subcellular location of the protein encoded by Gene A

The single-cell hair of Arabidopsis leaf surface Talin （ actin-binding protein ） -GFP SEMConfocal

五、 Electron Microscope Resolves fine structure of the cell –Relationship between limit of resolution and wavelength applies for any form of radiation Wavelength of electron decreases as its velocity increases

Electron Microscope With an accelerating voltage of 100,000 V, wavelength of an electron is 0.004nm –In theory, resolution is ~0.002 nm 10,000 X that of light microscope –However, aberrations of electron lens more difficult to correct than those of light microscope Practical resolving power is 0.1 nm

1.Transmission electron microscope

Electron Microscope Design of transmission electron microscope (TEM) similar to light microscope, except: –Much larger –Upside down (Fig. 9-22)

Electron Microscopy Source of illumination is a filament (cathode) that emits electrons at the top of the column –Since electrons are scattered by collisions with air molecules, column must be under a vacuum

Under vacuum

Electron Microscopy Electrons are accelerated by a nearby anode –Then passed through a tiny hole to form an electron beam Magnetic coils focus the beam

Glass Magnetic coils Anode

Electron Microscopy How is contrast achieved in the electron microscope? Specimen is stained with an electron dense material –Some of the electrons passing through the specimen are scattered by structures stained with electron dense material Others pass through parts of the cell not stained to form an image on a phosphorescent screen

Electron Microscopy Because the scattered electrons are lost from the beam, the stained regions show up as dark –Thus the image is a montage of light (non stained) and dark (stained) regions

Electron micrograph of a cell in a root tip

Electron Microscopy Preparation of Specimens –Preserved by fixation 1 st, glutaraldehyde –Covalently cross-links proteins 2 nd, osmium tetroxide –Binds to and stabilizes lipid bilayers and proteins –Tissue dehydrated, permeated with a polymerizing resin & sectioned into ultra- thin sections 50 – 100 nm thick (1/200 thickness of a cell)

Electron Microscopy Sections stained with electron-dense material (e.g uranyl acetate) to achieve contrast How does this work? –Tissue composed of atoms of low atomic number (e.g. carbon, oxygen, nitrogen, hydrogen) –To make them visible impregnated with salts of heavy metals (Fig. 9-25)

Immunogold Electron Microscopy Used to visualize specific proteins –Incubate thin section with primary antibody Then incubate with secondary antibody to which colloidal gold has been attached –Gold is electron dense and shows up as black dots (Fig. 9-26)

Insulin-secreting cell in the pancreas ， in which a gold-labled anti-insulin antibody has revealed the subcellular location of the insulin

Localizing Proteins by Electron Microscopy Notes page Electron micrograph of a yeast mitotic spindle Spindle microtubules

Immunogold localization of 4 proteins of the spindle pole body

Electron Microscopy of Metal-Shadowed Samples The transmission electron microscope (TEM) can be used to resolve individual macromolecules on the surface of the specimen –Thin film of heavy metal (e.g. platinum) is evaporated onto the dried specimen

Preparation of a metal-shadowed Replica Note that the thickness of the metal reflects the surface contours of the original specimen Metal is sprayed from an oblique angle Lighter coating in region corresponding to the shadow with respect to the angle of coating Shadow effect gives image a 3-dimensional effect

Preparation of a metal-shadowed Replica Note that the thickness of the metal reflects the surface contours of the original specimen Carbon is not electron dense The electron beam will readily pass through the thin carbon film

Electron Microscopy of Metal-Shadowed Samples For thick samples (e.g. cells), the organic material is dissolved away after shadowing –Only the thin metal replica of the surface is left This is thin enough for the electron beam to penetrate (Fig. 9-32)

Preparation of a metal-shadowed Replica Note that the thickness of the metal reflects the surface contours of the original specimen

Freeze-Fracture Electron Microscopy Metal shadowing of replicas can be used in conjunction with freeze fracture electron microscopy Provides views of the inside surface of cell membranes –Cells are frozen in liquid nitrogen Frozen block is cracked with a knife blade –The fracture plane passes through the hydrophobic interior of membranes »Interior surfaces of the cell membrane are exposed Fracture faces are shadowed with platinum –Organic material is dissolved away »Replicas viewed under the electron microscope (Fig. 9-33)

Freeze-fracture electron micrograph of Thylakoid membranes

Transmission electron micrograph of a liver cell in cross section

2. Scanning electron microscope

01_30_protozoan eats.jpg Didinium ingesting another ciliated protozoan, Paramecium

Scanning electron microscopy Light microscope

六、 Flow cytometer ， FCM

10_02_cell_sorter.jpg Can also use to get fluorescently labeled chromosomes strong electric field

七、 Purification of Cells and Their parts Centrifugation Velocity sedimentation Differential centrifugation

Velocity sedimentation Subcellular componants sediment at different speeds according to their sizae when carefully layered over a dilute salt solution. continuous gradient uncontinuous gradient

Pouring a Sucrose gradient Stir Bar High Density Low Density

Differential centrifugation Repeated centrifugation at progressively higher speeds will fractionate cell homogenates into their components. size density

Column Chromatography

Liquid Chromatography

Protein electrophoresis

Isoelectric Focusing

10_05_gel.electrophor.jpg EtBr stain Autoradiography – 32 P

八、 Molecular Methods

10_13_hybridization.jpg Detection of allele for sickle- cell anemia

10_14_1_Southrn.blotting.jpg Southern blotting used to detect specific DNA

10_14_2_Southrn.blotting.jpg Southern blotting (continued) **Note: the same probe can detect fragments of different sizes, if complementary DNA is within it