The current state of Confocal Scanning Laser Microscopy - PowerPoint PPT Presentation

The current state of Confocal Scanning Laser Microscopy

Hjalmar Brismar

Cell Physics, KTH

• Confocal microscopy
• History
• Present
• Applications
• Areas of development
• Excitation
• Detection
• Scanning

• 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

• Microscopy (widefield, confocal, multiphoton)
• Fluorescencent probes
• Fluorescent labels, antibodies
• Genetically engineered, GFP
• Electrophysiology
• Patch clamp
• MEA, multi electrode arrays

• Marvin Minsky, 1955
• Laser (1958)1960
• Affordable computers with memory > 64kB
• CSLM 1986-87

Widefield

Confocal

• 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

• Carl Zeiss (physiology, dynamic measurements)
• Leica (spectral sensitivity)
• Biorad (multiphoton)
• (olympus)
• (nikon)
• (EG&G Wallac)
• …

Zeiss 510

Zeiss 510

Spectra Physics Millenia X - Tsunami

Leica TCS SP

Leica TCS SP

Spectra Physics 2017UV

• GFP
• FRAP
• FRET
• Multiphoton excitation

Aequoria Victoria

• 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

GFP-Tubulin in Drosophila

immobile

mobile

Bleach

FRAP – Fluorescence recovery after photbleaching

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

CFP GFP

CFP YFP

Donor

Acceptor

• Spectral overlap
• Distance <10 nm

Excitation 430-450 nm

Donor

CFP

ProteinA

< 5-10 nm

Emission

>570 nm

YFP

Acceptor

ProteinB

FRET: 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

535 nm

440 nm

YFP

YFP

CFP

CaM

CaM

+ 4 Ca2+

440 nm

480 nm

CFP

2-photon

1-photon

Builtin confocality

1-photon

2-photon

Konfokal

Multifoton

PMT

PMT

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

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

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 !

• Spectral separation
• Optical filters
• Prism or grating
• Detectors
• PMT
• Photon counting diodes

We need higher sensitivity, QE !

• Speed
• Flexibility

• Biological motivation
• Ca2+ signals
• Measurement approach
• Intracellular ion measurements
• Combined electrophysiology

Ca - wave

[Ca2+]

Models verified by experiments can provide

new information and direct the further investigations

• High resolution 3D recording of Ca2+
• High speed recording
• Combined CSLM - electrophysiology
• Big cells – hippocampal pyramidal neurons

• High time resolution (ms)
• Scan geometry  cell geometry
• 2D – cell cultures

2 s.

(Patwardhan & Åslund 1994)

z

y

x

• Z-axis precision >= optical resolution
• Bidirectional scan (to gain speed)
• Focusing distance 20-50+ um
• >100 Hz
• Nonharmonic

• Stage scan
• High mass, impossible patch clamp
• Scan objective
• Well defined mass, side effects in specimen ?
• Scan focusing lens inside objective
• Tricky optics ?

40X/0.9NA

V/I