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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.

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Presentation Transcript
slide2
What are we doing in Cell Physics
  • Confocal microscopy
    • History
    • Present
  • Applications
  • Areas of development
    • Excitation
    • Detection
    • Scanning
cell physics
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
Instrumentation
  • Microscopy (widefield, confocal, multiphoton)
    • Fluorescencent probes
    • Fluorescent labels, antibodies
    • Genetically engineered, GFP
  • Electrophysiology
    • Patch clamp
    • MEA, multi electrode arrays
confocal microscopy
Confocal microscopy
  • Marvin Minsky, 1955
    • Laser (1958)1960
    • Affordable computers with memory > 64kB
    • CSLM 1986-87
slide8

Widefield

Confocal

confocal evolution
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
Confocal industry
  • Carl Zeiss (physiology, dynamic measurements)
  • Leica (spectral sensitivity)
  • Biorad (multiphoton)
  • (olympus)
  • (nikon)
  • (EG&G Wallac)
slide12

Zeiss 510

Spectra Physics Millenia X - Tsunami

slide14

Leica TCS SP

Spectra Physics 2017UV

applications techniques
Applications - Techniques
  • GFP
    • FRAP
    • FRET
  • Multiphoton excitation
slide17
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
Dynamics

GFP-Tubulin in Drosophila

protein mobility bleaching experiments

immobile

mobile

Bleach

Protein mobility – bleaching experiments

FRAP – Fluorescence recovery after photbleaching

variants of fp
Variants of FP
    • Blue BFP
    • Cyan CFP
    • Green GFP
    • Yellow YFP
    • Red DsRedHcRed
  • GFP timer

CFP GFP

CFP YFP

fluorescence resonance energy transfer fret
Fluorescence Resonance Energy Transfer FRET

Donor

Acceptor

  • Spectral overlap
  • Distance <10 nm
interaction fret fluorescence resonance energy transfer
Interaction - FRET(Fluorescence Resonance Energy Transfer)

Excitation 430-450 nm

Donor

CFP

ProteinA

< 5-10 nm

Emission

>570 nm

YFP

Acceptor

ProteinB

nka ip 3 r
NKA – IP3R

After

Before

Photobleaching ofacceptor removes FRETdetected as increased donor signal

Distance < 12 nm

Ouabain binding to NKAshortens the distance – stronger interaction –increased FRET efficiency15-25%

Donor

GFP-NKA

Donor diff

Acceptor

Cy3-IP3R

fret based ca 2 sensor

535 nm

440 nm

YFP

YFP

CFP

CaM

CaM

+ 4 Ca2+

440 nm

480 nm

CFP

FRET based Ca2+ sensor
multiphoton excitation
Multiphoton excitation

2-photon

1-photon

slide27

Builtin confocality

1-photon

2-photon

slide28

Konfokal

Multifoton

PMT

PMT

slide29

0

20

80 mm

Better penetration (2-400 mm)

Enables measurements from

intact cells in a proper physiological

environment.

Electrophysiology

40

60

80

1-photon

2-photon

slide30

FRET CFP-YFP multiphoton

CFP – YFP separated by a 6 aminoacid linker

Fluorochrome distance 5 nm

2-photon @ 790 nm

2-photon @ 790 nm

790

790

YFP – Calcyon

No excitation at 790 nm

YFP excited at 880 nm

790

880

2-photon @ 790 nm

1-photon @ 514 nm

development excitation
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
Development - Detection
  • Spectral separation
    • Optical filters
    • Prism or grating
  • Detectors
    • PMT
    • Photon counting diodes

We need higher sensitivity, QE !

development scanning
Development - Scanning
  • Speed
  • Flexibility
ultrafast 3d spline scan
Ultrafast 3D spline scan
  • Biological motivation
    • Ca2+ signals
  • Measurement approach
    • Intracellular ion measurements
    • Combined electrophysiology
slide36

Ca - wave

[Ca2+]

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

approach
Approach
  • High resolution 3D recording of Ca2+
  • High speed recording
  • Combined CSLM - electrophysiology
  • Big cells – hippocampal pyramidal neurons
confocal line scan
Confocal - line scan
  • High time resolution (ms)
  • Scan geometry  cell geometry
  • 2D – cell cultures

2 s.

arbitrary scan 2d
Arbitrary scan – 2D

(Patwardhan & Åslund 1994)

design criteria
Design criteria
  • Z-axis precision >= optical resolution
  • Bidirectional scan (to gain speed)
  • Focusing distance 20-50+ um
  • >100 Hz
  • Nonharmonic
ideas for ultrafast 3d scan
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 ?