the current state of confocal scanning laser microscopy hjalmar brismar cell physics, kth
TRANSCRIPT
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The current state of Confocal Scanning Laser Microscopy
Hjalmar Brismar
Cell Physics, KTH
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• What are we doing in Cell Physics• Confocal microscopy
– History– Present
• Applications
• Areas of development– Excitation– Detection– Scanning
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Cell Physics
• Study the biological cell from a physical perspective– Use tools and concepts from physics on biological problems– Develop methods and techniques– Describe biological functions and systems within a
physical/mathematical framework
• We focus on:– Cell volume
• Osmolyte transport• Water transport
– Cell mass• Measurement techniques• Cell cycle/cell mass regulation
– Intracellular signalling • Frequency modulated Ca2+ signals
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Instrumentation
• Microscopy (widefield, confocal, multiphoton)– Fluorescencent probes– Fluorescent labels, antibodies– Genetically engineered, GFP
• Electrophysiology– Patch clamp– MEA, multi electrode arrays
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Confocal microscopy
• Marvin Minsky, 1955
– Laser (1958)1960– Affordable computers with memory > 64kB
– CSLM 1986-87
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Widefield Confocal
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Confocal evolution• 1 st generation CSLM (1987)
– 1 channel fluorescence detection– 50 Hz line frequency
• 2nd generation (commercial systems ca1990) – 2-3 channel detection– >=100 Hz
• 3rd generation (1996)– 4 channel detection– 500 Hz
• 4th generation (2001)– 32 channels– 2.6 kHz– AOM, AOBS control
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Confocal industry
• Carl Zeiss (physiology, dynamic measurements)• Leica (spectral sensitivity)• Biorad (multiphoton)
• (olympus)• (nikon)• (EG&G Wallac)• …
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Zeiss 510
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Zeiss 510Spectra Physics Millenia X - Tsunami
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Leica TCS SP
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Leica TCS SPSpectra Physics 2017UV
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Applications - Techniques
• GFP– FRAP– FRET
• Multiphoton excitation
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GFP- Green Fluorescent Protein
Aequoria Victoria
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GFP• Discovered 1962 as companion
to aequorin• Cloned 1992, expression 1994• 238 Aminoacids• 27-30 kDa• Fluorophore made by 3 aminoacids
(65-67) ”protected” in a cylinder
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Dynamics
GFP-Tubulin in Drosophila
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Protein mobility – bleaching experiments
Bleach
mobile
immobile
FRAP – Fluorescence recovery after photbleaching
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Variants of FP
– Blue BFP– Cyan CFP– Green GFP– Yellow YFP– Red DsRed
HcRed
• GFP timer CFP YFPCFP GFP
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Fluorescence Resonance Energy Transfer FRET
•Spectral overlap•Distance <10 nm
Donor Acceptor
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Interaction - FRET(Fluorescence Resonance Energy Transfer)
ProteinA CFP
ProteinB YFP
< 5-10 nm
Excitation 430-450 nm
Emission>570 nm
Donor
Acceptor
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FRET: NKA – IP3R
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NKA – IP3R
DonorGFP-NKA
AcceptorCy3-IP3R
Before After
Donor diff
Photobleaching ofacceptor removes FRETdetected as increased donor signalDistance < 12 nm
Ouabain binding to NKAshortens the distance – stronger interaction –increased FRET efficiency15-25%
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FRET based Ca2+ sensor
YFP
CFP
CaM440 nm
480 nm
YFP
CFP
CaM
535 nm440 nm
+ 4 Ca2+
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Multiphoton excitation
1-photon 2-photon
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Builtin confocality
1-photon 2-photon
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PMT PMT
Konfokal Multifoton
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0
20
40
60
80
80 m
1-photon 2-photon
Better penetration (2-400 m)Enables measurements from intact cells in a proper physiologicalenvironment.
Electrophysiology
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FRET CFP-YFP multiphoton
2-photon @ 790 nm 2-photon @ 790 nm
2-photon @ 790 nm 1-photon @ 514 nm
CFP – YFP separated by a 6 aminoacid linkerFluorochrome distance 5 nm
YFP – CalcyonNo excitation at 790 nmYFP excited at 880 nm
790 790
790 880
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Development - ExcitationCurrently used lasers
– Ar ion, 458,488,514 nm– HeNe 543, 633 nm
– Ar ion 351,364 nm– ArKr 488,568 nm– HeCd 442 nm
– Diode 405 nm– HeNe 594 nm
– Multiphoton excitation, TiSa 700-1100
We need affordable, low noise, low power consumption lasers370-700 nm !
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Development - Detection
• Spectral separation– Optical filters
– Prism or grating
• Detectors– PMT
– Photon counting diodes
We need higher sensitivity, QE !
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Development - Scanning
• Speed• Flexibility
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Ultrafast 3D spline scan
• Biological motivation– Ca2+ signals
• Measurement approach– Intracellular ion measurements– Combined electrophysiology
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Frequency modulated Ca2+ signals
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Data from live cell experiments combined with biochemical data is used as input for
mathematical modeling-simulations
[Ca2+] Ca - wave
Models verified by experiments can provide new information and direct the further investigations
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Approach
• High resolution 3D recording of Ca2+
• High speed recording
• Combined CSLM - electrophysiology
• Big cells – hippocampal pyramidal neurons
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Scan speed
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Confocal - line scan
• High time resolution (ms)• Scan geometry cell geometry• 2D – cell cultures
2 s.
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Arbitrary scan – 2D
(Patwardhan & Åslund 1994)
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2D specimen
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Tissue – 3D cells
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3D arbitrary scan
x
y
z
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Design criteria
• Z-axis precision >= optical resolution
• Bidirectional scan (to gain speed)
• Focusing distance 20-50+ um
• >100 Hz
• Nonharmonic
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Ideas for ultrafast 3D scan
• Stage scan– High mass, impossible patch clamp
• Scan objective– Well defined mass, side effects in specimen ?
• Scan focusing lens inside objective– Tricky optics ?
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Piezo focus with specimen protection
40X/0.9NA
V/I
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