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)

• (olympus)• (nikon)• (EG&G Wallac)• …

Zeiss 510

Zeiss 510Spectra Physics Millenia X - Tsunami

Leica TCS SP

Leica TCS SPSpectra 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

Bleach

mobile

immobile

FRAP – Fluorescence recovery after photbleaching

Variants of FP

– Blue BFP– Cyan CFP– Green GFP– Yellow YFP– Red DsRed

HcRed

• GFP timer CFP YFPCFP GFP

Fluorescence Resonance Energy Transfer FRET

•Spectral overlap•Distance <10 nm

Donor Acceptor

Interaction - FRET(Fluorescence Resonance Energy Transfer)

ProteinA CFP

ProteinB YFP

< 5-10 nm

Excitation 430-450 nm

Emission>570 nm

Donor

Acceptor

FRET: NKA – IP3R

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%

FRET based Ca2+ sensor

YFP

CFP

CaM440 nm

480 nm

YFP

CFP

CaM

535 nm440 nm

+ 4 Ca2+

Multiphoton excitation

1-photon 2-photon

Builtin confocality

1-photon 2-photon

PMT PMT

Konfokal Multifoton

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

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

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 !

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

x

y

z

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

• 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

40X/0.9NA

V/I