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34. Summary. Cells follow rules of chemistry; Water is the most abundant substance proteins constitutes most of a cell’s dry mass; Four major classes of small organic molecules make macromolecules; Living cells undergo metabolism; A reaction will happen if it can result in lower

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34

Summary

Cells follow rules of chemistry;

Water is the most abundant substance

proteins constitutes most of a cell’s dry mass;

Four major classes of small organic molecules

make macromolecules;

Living cells undergo metabolism;

A reaction will happen if it can result in lower

free energy in the system;

Proteins and protein complexes execute almost

all cell functions.


Lecture 3 Microscopy


Birth of Cell Biology

1838 Schleiden and

Schwann “cell

doctrine”


A typical cell is

10-20 um

Light microscope

sees 0.2 um

1 mm=10-3 m

1 um=10-6 m

1 nm=10-9 m

1 A=Angstrom

=10-10 m


Resolution is limited

by the wavelength of

radiation

Nondestructive

Live imaging

Challenge: sample preparation

Computerized 3-D reconstruction


Four types of light microscopy

Normaski DIC

Bright-field

Phase-contrast

Dark-field


Images can be digitally enhanced

Eyes have

limitation in

seeing dim signals

and resolve bright images

Camera (the same kind

as in night surveillance)

2. Digitally extract info (contrast)

Improves interpretation, resolution (0.025 um),

but appears 0.2 um (can’t tell whether it is single or

double MT)


Sample preparation: sectioning


Sample preparation: staining

to visualize cellular contents

Hematoxylin

Eosin: HE


Fluorescence microscope


Fluorescent dyes


Immunofluorescence imaging does not show actual sizes


3-D imaging (esp fluorescence)

Image deconvolution: a computational approach to remove

blur from a stack of images taken in different focal planes

(digital technique)

Needs a fast computer, less bleaching, very sensitive


3-D imaging (esp fluorescence)

Confocal microscope produces optical sections by

excluding out-of-focus light (analog technique)

Expensive scope, more bleaching, limitations in depth


Two photon


Fast electrons has short waves: 100,000V and 0.004 nm

In theory 0.002 nm resolution: 10,000 of light microscope

In practice, 0.1 nM (1A)

Difficulties:

Specimen preparation

Contrast

Radiation damage

Therefore, effective 2 nM (20 A)

100 better than light microscope

Gold atoms (bright spots)

0.2 nm apart


Transmission EM (TEM)


Immunogold EM


3D reconstruction

Distorions of immuno EM:

Large depth of field

deep structures in the same layer

Ab and Gold-Ab don’t

penetrate too deep

Label before

imbedding


Scanning EM for surface imaging

(SEM)

Smaller

cheaper

10 nM

resolution


Imaging surface

DIC

TEM

SEM


Metal shadowing (platinum) and observe under TEM

Individual marcomolecules

can been seen


Freeze-fracture electron microscopy

Intramembrane particles (large TM proteins)

Chloroplast


Freeze-etch electron microscopy

Interior of cells

Protein filaments in muscle cells

Crack the frozen block, lower ice level by subliming ice

In a vacuum (freeze drying), shadow the exposed parts of cells

and observe replica


Negative staining of actin filaments

Helical chain of acitin monomers

8 nm

Diameter


EM tomography for 3-D reconstruction

TEM

Hepatitis B virus

See

details

of macro-

molecular

Complexes!!!

Resolution rivals X-ray crytallography

0.3 nm for crystalline arrays

0.8 nm for single particle reconstruction

(subunits, domains, 2nd structures)


Calcium imaging

Ion-sensitive indicators

Brighter

better

resolution

0.5-10 um

Not bright

Fluorescent Ca indicator:fura2

Aequorin, a luminescent protein


Introducing membrane-impermeable substance into a cell


Caged molecules

Ca++ can be caged too

photolysis

Tubulin labeled

With caged fluoresent dyes


Jellyfish GFP

Live imaging Cajal bodies


Pulse-chase experiments using

radioisotopes


Summary

Cell doctrine;

Two major types of microscopes: light and electron;

Limitation of resolution: wavelength of radiation;

Advantage and disadvantage of light and electron MS

Different types of light microscopes: bright field,

phase contrast, DIC, dark field,fluorescent, confocal

Image processing: digital enhancement

Two major types of EM: TEM and SEM

Additional tricks: shadowing, freeze-fracture, freeze

etching, negative staining, tomography;

Live imaging, calcium indicators, caged compounds,

GFP, pulse chasing


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