The current state of Confocal Scanning Laser Microscopy

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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 gt 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
• gt100 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)
• (EGG Wallac)

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Zeiss 510
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Zeiss 510 Spectra Physics Millenia X - Tsunami
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Leica TCS SP
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Leica TCS SP Spectra 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
FRAP Fluorescence recovery after photbleaching
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Variants of FP

• Blue BFP
• Cyan CFP
• Green GFP
• Yellow YFP
• Red DsRedHcRed
• GFP timer

CFP YFP
CFP GFP
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Fluorescence Resonance Energy Transfer FRET
Donor
Acceptor

• Spectral overlap
• Distance lt10 nm

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Interaction - FRET(Fluorescence Resonance Energy
Transfer)
Excitation 430-450 nm
Donor
CFP
ProteinA
lt 5-10 nm
Emission gt570 nm
YFP
Acceptor
ProteinB
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FRET NKA IP3R
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NKA IP3R
After
Before
Photobleaching ofacceptor removes FRETdetected
as increased donor signal Distance lt 12
nm Ouabain binding to NKAshortens the distance
stronger interaction increased FRET
efficiency15-25
Donor GFP-NKA
Donor diff
Acceptor Cy3-IP3R
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FRET based Ca2 sensor
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Multiphoton excitation
2-photon
1-photon
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Builtin confocality
1-photon
2-photon
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Konfokal
Multifoton
PMT
PMT
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0
20
80 mm
Better penetration (2-400 mm) Enables
measurements from intact cells in a proper
physiological environment. Electrophysiology
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60
80
1-photon
2-photon
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FRET CFP-YFP multiphoton
CFP YFP separated by a 6 aminoacid
linker Fluorochrome distance 5 nm
2-photon _at_ 790 nm
2-photon _at_ 790 nm
790
790
YFP Calcyon No excitation at 790 nm YFP excited
at 880 nm
790
880
2-photon _at_ 790 nm
1-photon _at_ 514 nm
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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 !

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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
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
z
y
x
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Design criteria

• Z-axis precision gt optical resolution
• Bidirectional scan (to gain speed)
• Focusing distance 20-50 um
• gt100 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
V/I
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