BIOL 2021 Lecture 1: Chapter 9

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6 Aug 2016
Department
Course
Visualizing*cells*and*microscopes
Cell:*fundamental*unit*of*life,*functions*independently*but*may*interact*with*other*
cells*(in*eukaryotes)
Robert*Hooke*-very*simple*light*microscope,*observed*dead*cells*of*a*leaf
All*organisms*made*up*of*cells
1.
Cell*is*fundamental*unit*of*life
2.
Cell*doctrine;*Schleiden*and*Schwann
Eyesight*limited*to*very*big*structures
Conventional*light*microscopes*can*resolve*details*0.2*micrometer*apart*so*individual*
cells
Super-resolution*and*electron*microscope*can*even*go*down*to*atomic*level.*
Figure*9-2
Bright*field*(same*as*the*one*Robert*Hooke*used)*we*can*alter*light*intensity
-
Phase*contrast*-more*of*a*3D
-
Differential*interference*-better*3D
-
Dark*field*-switch*the*light
-
Different*types*of*imaging
We*can*get*good*detail*about*features*and*structures
BUT
Problem:*difficult*to*distinguish*and*identify*specific*parts*of*the*cell.
->*can*be*solved*by*using*chemical*dyes*or*fluorescent*probes
Figure*9-10
We*can*see*better*detail*of*different*parts*of*the*cell.*Tissue*was*stained*with*H&E:
Hematoxylin:*dark*blue,*affinity*for*negatively*charged*molecules*such*as*DNA*RNA*
and*acidic*proteins
Eosin:*red*dye,*affinity*for*basic*molecules*such*as*proteins*(amine*groups)
BUT*
Still*limited,*we*have*some*information*but*not*specific.
->*Specific*molecules*can*be*located*in*cells*by*fluorescent*microscopy.
Figure*9-11
Identification*of*different*RNA*in*the*developing*Drosophila*embryo.
Figure*9-12
We*use*specific*wavelengths*of*white*not*just*a*random*white*light
Object*is*the*cell*that*has*been*treated*with*a*specific*fluorescent*probe.*The*blue*
excites*the*probe*and*green*is*emitted.*Blue*is*absorbed*and*excite* the*cell,*electrons*
switch*and*emit*a*photon*a*usually*longer*wavelength.*
Excitation*and*emission*-stoke*shift
Figure*9-13
It*can*be*a
ny*colour*but*there*are*sometimes*limits.*They*have*now*created*dyes*for*the*colours*
of*the*spectrum.
Some*of*them*are*better*than*others*because*they*start*and*end*at*very*different*
colours.*We*can*narrow*down*the*filter*to*a*few*nanometers*in*order*to*get*very*
specific*information*on*the*wavelength*of*the*light*absorbed*and*the*one*transmitted.*
(don't*need*to*remember*the*names*of*the*probes)
Figure*9-14
With*a*good*microscope,*you*can*see*every*molecule*present*in*the*cell.
Blue*is*condensed*chromosomes*of*DNA.
Red*represents*centromeres.
Green*is*for*mitotic*spindles*(microtubules).
You*can*add*other*colour*probes*to*see*other*proteins*present*in*hat*cell.*
It*is*important*to*examine*temporal*and*spatial*events*in*the*cell.
Fixed*cells*can*be*chemically*prepared*to*provide*only*a*snapshot*of*what*is*going*in*
and*out*of*the*cell.
->*sometimes*you*can*guess*what's*happening,*other*times*we*can't,*we*can*get*some*
information*but*we*want*to*get*more*information.*
We*want*to*use*probes*on*a*living*cell* to*see*what*is*going*on.*
Individual*proteins*can*be*fluorescently*tagged*in*living*cells*and*organisms.
Figure*9-23
Green*fluorescent*protein*(GFP)*is*a*bioluminescent*protein*isolated*from*jellyfish*
(Aequorea*victoria).
GFP*can*be*expressed*in*prokaryotic*and*eukaryotic*cells*and*that*gene*be*fused*with*
other*genes*to*study*the*expression*and*localization*of*selected*proteins.*
We*can*replace*regular*gene*with*the*manipulated*gene*after*the*promoter.
Light-emitting*indicators*can*measure*rapidly*changing*intracellular* ion*concentration.
Light*microscopy*can*be*used*to*measure*the*dynamic*of*temporal*changes*of*
intracellular* ions*(calcium…),*cyclic*AMP
Figure*9-30:*sear*urchin*fertilization
Aequorin*injected*in*a*fish*egg.*When*egg*is*fertilized*by*sperm,*there*is*a*rapid*rise*in*
intracellular* calcium*which*occurs*as*a*wave*away*from*the*site*of*sperm*entry.
Superresolution*Fluorescence*Techniques*can*overcome*Diffraction-limited*
resolution.*
Conventional*light*microscopy*limited*to*200*nm.
Superresolution*fluorescence*imaging*of*microtubules*show*a*clear*image*of*their*true*
size*with*a*more*precise*representation*of*25nm.*
Transmission*Electron*Microscope*(TEM)*resolves*the*fine*structure*of*the*cell.*
Figure*9-44
Fixed*image*for*the*electrons*to*be*able*to*get*through*-much*detailed*
Different*views*of*a*single*object*can*be*combined*to*give*a*3D*reconstruction.*
Figure*9-47
Multiple*thick*sections*of*the*Golgi*apparatus*which*have*been*digitally*
reconstruction.*
Images*of*surfaces*can*be*obtained*by*scanning*electron*microscopy.
Scanning*electron*microscopy*(SEM)*can**provide*a*high*resolution*3D*surface*image.*
However,*the*resolution*is*not*as*great*as*TEM
.*
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Visualizing*cells*and*microscopes
Cell:*fundamental*unit*of*life,*functions*independently*but*may*interact*with*other*
cells*(in*eukaryotes)
Robert*Hooke*-very*simple*light*microscope,*observed*dead*cells*of*a*leaf
All*organisms*made*up*of*cells1.
Cell*is*fundamental*unit*of*life2.
Cell*doctrine;*Schleiden*and*Schwann
Eyesight*limited*to*very*big*structures
Conventional*light*microscopes*can*resolve*details*0.2*micrometer*apart*so*individual*
cells
Higher*resolution*microscopes*allow*one*to*see*inside*the*cells* as*well*as*organelles,*
protein*structures…
Super-resolution*and*electron*microscope*can*even*go*down*to*atomic*level.*
Figure*9-2
Bright*field*(same*as*the*one*Robert*Hooke*used)*we*can*alter*light*intensity
-
Phase*contrast*-more*of*a*3D
-
Differential*interference*-better*3D
-
Dark*field*-switch*the*light
-
Different*types*of*imaging
We*can*get*good*detail*about*features*and*structures
BUT
Problem:*difficult*to*distinguish*and*identify*specific*parts*of*the*cell.
->*can*be*solved*by*using*chemical*dyes*or*fluorescent*probes
Figure*9-10
We*can*see*better*detail*of*different*parts*of*the*cell.*Tissue*was*stained*with*H&E:
Hematoxylin:*dark*blue,*affinity*for*negatively*charged*molecules*such*as*DNA*RNA*
and*acidic*proteins
Eosin:*red*dye,*affinity*for*basic*molecules*such*as*proteins*(amine*groups)
BUT*
Still*limited,*we*have*some*information*but*not*specific.
->*Specific*molecules*can*be*located*in*cells*by*fluorescent*microscopy.
Figure*9-11
Identification*of*different*RNA*in*the*developing*Drosophila*embryo.
Figure*9-12
We*use*specific*wavelengths*of*white*not*just*a*random*white*light
Object*is*the*cell*that*has*been*treated*with*a*specific*fluorescent*probe.*The*blue*
excites*the*probe*and*green*is*emitted.*Blue*is*absorbed*and*excite* the*cell,*electrons*
switch*and*emit*a*photon*a*usually*longer*wavelength.*
Excitation*and*emission*-stoke*shift
Figure*9-13
It*can*be*a
ny*colour*but*there*are*sometimes*limits.*They*have*now*created*dyes*for*the*colours*
of*the*spectrum.
Some*of*them*are*better*than*others*because*they*start*and*end*at*very*different*
colours.*We*can*narrow*down*the*filter*to*a*few*nanometers*in*order*to*get*very*
specific*information*on*the*wavelength*of*the*light*absorbed*and*the*one*transmitted.*
(don't*need*to*remember*the*names*of*the*probes)
Figure*9-14
With*a*good*microscope,*you*can*see*every*molecule*present*in*the*cell.
Blue*is*condensed*chromosomes*of*DNA.
Red*represents*centromeres.
Green*is*for*mitotic*spindles*(microtubules).
You*can*add*other*colour*probes*to*see*other*proteins*present*in*hat*cell.*
It*is*important*to*examine*temporal*and*spatial*events*in*the*cell.
Fixed*cells*can*be*chemically*prepared*to*provide*only*a*snapshot*of*what*is*going*in*
and*out*of*the*cell.
->*sometimes*you*can*guess*what's*happening,*other*times*we*can't,*we*can*get*some*
information*but*we*want*to*get*more*information.*
We*want*to*use*probes*on*a*living*cell* to*see*what*is*going*on.*
Individual*proteins*can*be*fluorescently*tagged*in*living*cells*and*organisms.
Figure*9-23
Green*fluorescent*protein*(GFP)*is*a*bioluminescent*protein*isolated*from*jellyfish*
(Aequorea*victoria).
GFP*can*be*expressed*in*prokaryotic*and*eukaryotic*cells*and*that*gene*be*fused*with*
other*genes*to*study*the*expression*and*localization*of*selected*proteins.*
We*can*replace*regular*gene*with*the*manipulated*gene*after*the*promoter.
Light-emitting*indicators*can*measure*rapidly*changing*intracellular* ion*concentration.
Light*microscopy*can*be*used*to*measure*the*dynamic*of*temporal*changes*of*
intracellular* ions*(calcium…),*cyclic*AMP
Figure*9-30:*sear*urchin*fertilization
Aequorin*injected*in*a*fish*egg.*When*egg*is*fertilized*by*sperm,*there*is*a*rapid*rise*in*
intracellular* calcium*which*occurs*as*a*wave*away*from*the*site*of*sperm*entry.
Superresolution*Fluorescence*Techniques*can*overcome*Diffraction-limited*
resolution.*
Conventional*light*microscopy*limited*to*200*nm.
Superresolution*fluorescence*imaging*of*microtubules*show*a*clear*image*of*their*true*
size*with*a*more*precise*representation*of*25nm.*
Transmission*Electron*Microscope*(TEM)*resolves*the*fine*structure*of*the*cell.*
Figure*9-44
Fixed*image*for*the*electrons*to*be*able*to*get*through*-much*detailed*
Different*views*of*a*single*object*can*be*combined*to*give*a*3D*reconstruction.*
Figure*9-47
Multiple*thick*sections*of*the*Golgi*apparatus*which*have*been*digitally*
reconstruction.*
Images*of*surfaces*can*be*obtained*by*scanning*electron*microscopy.
Scanning*electron*microscopy*(SEM)*can**provide*a*high*resolution*3D*surface*image.*
However,*the*resolution*is*not*as*great*as*TEM
.*
Chapter(9
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Visualizing*cells*and*microscopes
Cell:*fundamental*unit*of*life,*functions*independently*but*may*interact*with*other*
cells*(in*eukaryotes)
Robert*Hooke*-very*simple*light*microscope,*observed*dead*cells*of*a*leaf
All*organisms*made*up*of*cells1.
Cell*is*fundamental*unit*of*life2.
Cell*doctrine;*Schleiden*and*Schwann
Eyesight*limited*to*very*big*structures
Conventional*light*microscopes*can*resolve*details*0.2*micrometer*apart*so*individual*
cells
Higher*resolution*microscopes*allow*one*to*see*inside*the*cells* as*well*as*organelles,*
protein*structures…
Super-resolution*and*electron*microscope*can*even*go*down*to*atomic*level.*
Figure*9-2
Bright*field*(same*as*the*one*Robert*Hooke*used)*we*can*alter*light*intensity
-
Phase*contrast*-more*of*a*3D
-
Differential*interference*-better*3D
-
Dark*field*-switch*the*light
-
Different*types*of*imaging
We*can*get*good*detail*about*features*and*structures
BUT
Problem:*difficult*to*distinguish*and*identify*specific*parts*of*the*cell.
->*can*be*solved*by*using*chemical*dyes*or*fluorescent*probes
Figure*9-10
We*can*see*better*detail*of*different*parts*of*the*cell.*Tissue*was*stained*with*H&E:
Hematoxylin:*dark*blue,*affinity*for*negatively*charged*molecules*such*as*DNA*RNA*
and*acidic*proteins
Eosin:*red*dye,*affinity*for*basic*molecules*such*as*proteins*(amine*groups)
BUT*
Still*limited,*we*have*some*information*but*not*specific.
->*Specific*molecules*can*be*located*in*cells*by*fluorescent*microscopy.
Figure*9-11
Identification*of*different*RNA*in*the*developing*Drosophila*embryo.
Figure*9-12
We*use*specific*wavelengths*of*white*not*just*a*random*white*light
Object*is*the*cell*that*has*been*treated*with*a*specific*fluorescent*probe.*The*blue*
excites*the*probe*and*green*is*emitted.*Blue*is*absorbed*and*excite* the*cell,*electrons*
switch*and*emit*a*photon*a*usually*longer*wavelength.*
Excitation*and*emission*-stoke*shift
Figure*9-13
It*can*be*a
ny*colour*but*there*are*sometimes*limits.*They*have*now*created*dyes*for*the*colours*
of*the*spectrum.
Some*of*them*are*better*than*others*because*they*start*and*end*at*very*different*
colours.*We*can*narrow*down*the*filter*to*a*few*nanometers*in*order*to*get*very*
specific*information*on*the*wavelength*of*the*light*absorbed*and*the*one*transmitted.*
(don't*need*to*remember*the*names*of*the*probes)
Figure*9-14
With*a*good*microscope,*you*can*see*every*molecule*present*in*the*cell.
Blue*is*condensed*chromosomes*of*DNA.
Red*represents*centromeres.
Green*is*for*mitotic*spindles*(microtubules).
You*can*add*other*colour*probes*to*see*other*proteins*present*in*hat*cell.*
It*is*important*to*examine*temporal*and*spatial*events*in*the*cell.
Fixed*cells*can*be*chemically*prepared*to*provide*only*a*snapshot*of*what*is*going*in*
and*out*of*the*cell.
->*sometimes*you*can*guess*what's*happening,*other*times*we*can't,*we*can*get*some*
information*but*we*want*to*get*more*information.*
We*want*to*use*probes*on*a*living*cell* to*see*what*is*going*on.*
Individual*proteins*can*be*fluorescently*tagged*in*living*cells*and*organisms.
Figure*9-23
Green*fluorescent*protein*(GFP)*is*a*bioluminescent*protein*isolated*from*jellyfish*
(Aequorea*victoria).
GFP*can*be*expressed*in*prokaryotic*and*eukaryotic*cells*and*that*gene*be*fused*with*
other*genes*to*study*the*expression*and*localization*of*selected*proteins.*
We*can*replace*regular*gene*with*the*manipulated*gene*after*the*promoter.
Light-emitting*indicators*can*measure*rapidly*changing*intracellular* ion*concentration.
Light*microscopy*can*be*used*to*measure*the*dynamic*of*temporal*changes*of*
intracellular* ions*(calcium…),*cyclic*AMP
Figure*9-30:*sear*urchin*fertilization
Aequorin*injected*in*a*fish*egg.*When*egg*is*fertilized*by*sperm,*there*is*a*rapid*rise*in*
intracellular* calcium*which*occurs*as*a*wave*away*from*the*site*of*sperm*entry.
Superresolution*Fluorescence*Techniques*can*overcome*Diffraction-limited*
resolution.*
Conventional*light*microscopy*limited*to*200*nm.
Superresolution*fluorescence*imaging*of*microtubules*show*a*clear*image*of*their*true*
size*with*a*more*precise*representation*of*25nm.*
Transmission*Electron*Microscope*(TEM)*resolves*the*fine*structure*of*the*cell.*
Figure*9-44
Fixed*image*for*the*electrons*to*be*able*to*get*through*-much*detailed*
Different*views*of*a*single*object*can*be*combined*to*give*a*3D*reconstruction.*
Figure*9-47
Multiple*thick*sections*of*the*Golgi*apparatus*which*have*been*digitally*
reconstruction.*
Images*of*surfaces*can*be*obtained*by*scanning*electron*microscopy.
Scanning*electron*microscopy*(SEM)*can**provide*a*high*resolution*3D*surface*image.*
However,*the*resolution*is*not*as*great*as*TEM
.*
Chapter(9
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