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                   Summary


1. Cells follow rules of chemistry;
2. Water is the most abundant substance
   proteins constitutes most of a cell’s dry mass;
3. Four major classes of small organic molecules
   make macromolecules;
4. Living cells undergo metabolism;
5. A reaction will happen if it can result in lower
   free energy in the system;
6. 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

 Bright-field      Normaski DIC




Phase-contrast      Dark-field
          Images can be digitally enhanced

 Eyes have
 limitation in
 seeing dim signals
 and resolve bright images

1.   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:
              1. Large depth of field
                   deep structures in the same laye
              2. 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
Resolution rivals X-ray crytallography                details
                                                      of macro-
 0.3 nm for crystalline arrays                        molecular
 0.8 nm for single particle reconstruction            Complexes!!!

 (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


1.   Cell doctrine;
2.   Two major types of microscopes: light and electron;
3.   Limitation of resolution: wavelength of radiation;
4.   Advantage and disadvantage of light and electron MS
5.   Different types of light microscopes: bright field,
     phase contrast, DIC, dark field,fluorescent, confocal
6.   Image processing: digital enhancement
7.   Two major types of EM: TEM and SEM
8.   Additional tricks: shadowing, freeze-fracture, freeze
     etching, negative staining, tomography;
9.   Live imaging, calcium indicators, caged compounds,
     GFP, pulse chasing

								
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