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Optics

Focus

Confocal theory

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Dr. Marvin MinskyConfocal microscope: 1957

Confocal theory

LASER

LightAmplification by theStimulatedEmission ofRadiation

ScanningConfocalMicroscope

Laser

DichroicMirror

Conventional / Confocal

Fluorescence

400 nm

Wavelength and Light Properties

575 nm

E = h c

HighEnergy

LowEnergy0,5*

*2

Multiphoton exitation

800 nm

800 nm

1200 nm

1200 nm

1200 nm

400 nm

Dr. Maria Göppert-Mayer : theory of two-photon quantum transitions (two-photon absorption and emission) 1931,

LASER500 nm

Pulsed LASER1000 nm1200 nm800 nm

nm500 nm800 1200

Temporal photon concentration

Prof. Watt W. Webb et al.Two-photon laser scanning fluorescence microscopy:

1990

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Spatial photon concentration

Confocal / Multiphoton

Luo at al. Cell Structure and Function 2006, 31: 63Comparison of photoactivation of PA-GFP in vivo with single-photon (405 nm) and multiphoton (790 nm) laser light.

PhotoconversionExcitation area

Thorsten at al. Nature 2004; 427: 159T-cell priming by dendriticcells in lymph nodes occurs in three distinct phases

In VIVO Imaging