Transcript The current state of Confocal Scanning Laser Microscopy

The current state of Confocal
Scanning Laser Microscopy
Hjalmar Brismar
Cell Physics, KTH
• What are we doing in Cell Physics
• Confocal microscopy
– History
– Present
• Applications
• Areas of development
– Excitation
– Detection
– Scanning
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
Instrumentation
• Microscopy (widefield,
confocal, multiphoton)
– Fluorescencent probes
– Fluorescent labels, antibodies
– Genetically engineered, GFP
• Electrophysiology
– Patch clamp
– MEA, multi electrode arrays
Confocal microscopy
• Marvin Minsky, 1955
– Laser (1958)1960
– Affordable computers with memory > 64kB
– CSLM 1986-87
Widefield
Confocal
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
Confocal industry
• Carl Zeiss (physiology, dynamic measurements)
• Leica (spectral sensitivity)
• Biorad (multiphoton)
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(olympus)
(nikon)
(EG&G Wallac)
…
Zeiss 510
Zeiss 510
Spectra Physics Millenia X - Tsunami
Leica TCS SP
Leica TCS SP
Spectra Physics 2017UV
Applications - Techniques
• GFP
– FRAP
– FRET
• Multiphoton excitation
GFP- Green Fluorescent Protein
Aequoria Victoria
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
Dynamics
GFP-Tubulin in Drosophila
Protein mobility – bleaching experiments
FRAP – Fluorescence recovery
after photbleaching
immobile
mobile
Bleach
Variants of FP
– Blue BFP
– Cyan CFP
– Green GFP
– Yellow YFP
– Red DsRed
HcRed
• GFP timer
CFP GFP
CFP YFP
Fluorescence Resonance Energy Transfer
FRET
Donor
Acceptor
•Spectral overlap
•Distance <10 nm
Interaction - FRET
(Fluorescence Resonance Energy Transfer)
Excitation
430-450 nm
Donor
ProteinA
CFP
< 5-10 nm
Acceptor
ProteinB
YFP
Emission
>570 nm
FRET: NKA – IP3R
NKA – IP3R
Before
After
Photobleaching of
acceptor removes FRET
detected as increased
donor signal
Distance < 12 nm
Donor
GFP-NKA
Donor diff
Acceptor
Cy3-IP3R
Ouabain binding to NKA
shortens the distance –
stronger interaction –
increased FRET efficiency
15-25%
FRET based Ca2+ sensor
YFP
YFP
CFP
CaM
CFP
+ 4 Ca2+
CaM
Multiphoton excitation
1-photon
2-photon
Builtin confocality
1-photon
2-photon
Konfokal
Multifoton
PMT
PMT
0
80 mm
20
Better penetration (2-400 mm)
Enables measurements from
intact cells in a proper physiological
environment.
40
60
Electrophysiology
80
1-photon
2-photon
FRET CFP-YFP multiphoton
CFP – YFP separated by a
6 aminoacid linker
Fluorochrome distance 5 nm
2-photon @ 790 nm
790
2-photon @ 790 nm
790
YFP – Calcyon
No excitation at 790 nm
YFP excited at 880 nm
2-photon
790 @ 790 nm
1-photon
880 @ 514 nm
Development - Excitation
Currently 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 lasers
370-700 nm !
Development - Detection
• Spectral separation
– Optical filters
– Prism or grating
• Detectors
– PMT
– Photon counting diodes
We need higher sensitivity, QE !
Development - Scanning
• Speed
• Flexibility
Ultrafast 3D spline scan
• Biological motivation
– Ca2+ signals
• Measurement approach
– Intracellular ion measurements
– Combined electrophysiology
Frequency modulated Ca2+ signals
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
Approach
• High resolution 3D recording of Ca2+
• High speed recording
• Combined CSLM - electrophysiology
• Big cells – hippocampal pyramidal
neurons
Scan speed
Confocal - line scan
• High time resolution (ms)
• Scan geometry  cell geometry
• 2D – cell cultures
2 s.
Arbitrary scan – 2D
(Patwardhan & Åslund 1994)
2D specimen
Tissue – 3D cells
3D arbitrary scan
z
y
x
Design criteria
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Z-axis precision >= optical resolution
Bidirectional scan (to gain speed)
Focusing distance 20-50+ um
>100 Hz
Nonharmonic
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 ?
Piezo focus with specimen protection
V/I
40X/0.9NA