第二章 细胞生物学研究方法

01_04_Early microscopes.jpg1880 drawing

Living cell photographed thru light microscope

01_05_Cells form tissues.jpgStains 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 10 -20 μm in diameter (1/5th 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 fieldPhase contrastNomarski

Electron microscopy (1930’s)Scanning EMTransmission EM

Fluorescence microscopy (1911)FluorescenceDeconvolutionConfocal

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 o

bjects 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 perme

able to stains and cross-links macromolecules

– Stains selectively depict subcellular component

s

• e.g. hematoxylin stains DNA and RNA (see F

ig. 9-11)

Stained tissue sectionshowing 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 throughunstained cells undergoesvery 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 micros

copy (DIC) Nomarski system To minimize the optical artifacts by achieving a comple

te 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 qu

ality, which depends on the rate of change of refractive ind

ex 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 mole

cules

Absorb light at one wavelength (the excitation w

avelength) and emit at another (excitation wavel

ength)

• Viewed through a filter that only allows emitte

d light through

• See this against a dark background

Fluorescence Microscopy

• Fluorescence microscope similar to an ordinary li

ght microscope, except:

– Illuminating light is passed through 2 filters

• 1st filter only passes wavelengths that excite th

e flurophore

• 2nd filter blocks out the excitation wavelenghts

and only passes those wavelengths that are e

mitted 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 differencebetween 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 – bluefluorescent 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 slic

ed into sections

– Sectioning results in loss of information in the 3rd dimension

• As well, optical microscope focussed on a sp

ecific focal plane

–Parts above and below the focal plane ar

e 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 pinho

le

• i.e. precisely where the rays from the illumina

ted point in the specimen come into focus (Fi

g. 9-18)

Fluorescein-excited by blue light

Fluroescein-emitted light is green

Confocal microscopy

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. 24-21)

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 formedby 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-se

creting cell (B lymphocyte) (Fig. 24-17)

» Each recognizes a certain part (epitop

e) of the antigen (Fig. 24-29)

Naïve or memory B cellsactivated 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 anumber 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 (Fi

g. 24-29)

– Because they are epitope-specific-

• Can be made against molecules that are onl

y a minor component of a complex mixture

– Proportion of polyclonal antibodies agains

t this minor component would be too smal

l to be useful

Detection with Antibodies

• Monoclonal Antibodies

– Produced using hybridoma cell lines

• Fusion of a single antibody-secreting B lymp

hocyte from mouse with a mouse B lymphoc

yte tumor cell

– Results in a hybridoma that can be propa

gated as a clone to produce monoclonal a

ntibodies (Fig. 8-5)

Detection with Antibodies

• Monoclonal Antibodies

– Hybridoma overcomes a problem

• B lymphocytes have a limited life span in cu

lture and can’t be used as ongoing source o

f antibody

• The fusion with a tumor cell confers upon th

e lymphocyte the ability to multiply indefinite

ly in culture (Fig. 8-6)

Preparation of Hybridomas forProduction 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 u

sed simultaneously to depict distributions

of different molecules or structures (Fig. 9-

14)

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 secon

dary antibody

– Produces localized accumulaion of colo

ured precipitate upon addition of suitable

substrate

» This can be detected by measuring a

bsorbance on a Plate Reader

Detection with Antibodies

• Amplification of the signal

2. Alkaline phosphatase is linked to the secon

dary antibody

• This amplification is the basis for Elisa

– Elisa (enzyme-linked immunosorbent ass

ay)

– Medical applications e.g tests for pregna

ncy; infection

Green Fluorescent Protein (GFP)

• Very powerful experimental tool when used i

n 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 jellyfis

h Aequoria victoria

– GFP can be cloned and introduced into cells of o

ther 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 t

he 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 ori

ginal 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 regionFor 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

SEM Confocal

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• 1st, glutaraldehyde

–Covalently cross-links proteins• 2nd, 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 cel

l)

Electron Microscopy• Sections stained with electron-dense materi

al (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 ofa 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 anoblique angle

Lighter coating in regioncorresponding to the shadow with respect

to the angle of coating

Shadow effect givesimage 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 willreadily pass throughthe thin carbon film

Electron Microscopy of Metal-ShadowedSamples

• 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

HighDensity

LowDensity

Differential centrifugation

Repeated centrifugation at progressively

higher speeds will fractionate cell

homogenates into their components.

• size

• density

Column Chromatography

Liquid Chromatography

Liquid Chromatography

Liquid Chromatography

Protein electrophoresis

Isoelectric Focusing

10_05_gel.electrophor.jpgEtBr stain

Autoradiography – 32P

八、 Molecular Methods

10_13_hybridization.jpgDetection of allele for sickle-cell anemia

10_14_1_Southrn.blotting.jpgSouthern blotting used to detect specific DNA

10_14_2_Southrn.blotting.jpgSouthern blotting (continued)

**Note: the same probe can detect fragments of different sizes, if complementary DNA is within it