Optical imaging Spectroscopic applications Microscopes conventional optical microscope

Optical imaging Spectroscopic applications

Microscopes conventional optical microscope confocal microscop measurement of fluorescence lifetime Endoscopes, telescopes Photomultipliers, microchannelplate detector gated image intesifiers Streak camera CCD and CMOS cameras

Bazsalikom levél, szekréciós mirigy az epidermisz felszínén Fluorescence from a basil leaf (cell walls fluoresce in the blue-green region, red fluorescence is due to chlorophyll) exc= 351 nm em= 385 -470 nm blue 515 -550 nm green > 650 nm red 100 m

C A 32 m B D Sorghum levél felszine: exc= 351 nm emi= 385 -470 nm kék 515 -550 nm zöld > 650 nm vörös 100 m 32 m A B C D Fluorescence layer by layer from a sorghum leaf On the surface the blue-green fluorescence of cell walls dominates, the chloroplasts lie deeper

Dane. Py infiltrated spinach leaf, at a depth of 15 m em > 650 nm 505 nm < em <550 nm exc = 351, 364 nm, Ar laser

Composite picture (green + red fluorescence) and intensity distribution along the line

Dane. Py infiltrated spinach leaf After 45 minutes photoinhibition treatment: • 2/3 of the photosynthetic activity has been lost, • only 15% protein degradation can be measured • no significant pigment bleach (total pigment) or lipid peroxidation 0’ 15’ exc = 351, 364 nm em > 650 nm 505 nm < em <550 nm 30’ 45’

Principle of multiphoton excitation

Scales in Microscopy

Conventional light microscopy is limited by diffraction

Approaches in super-resolution light microscopy SIM (Structured Illumination Microscopy) STED (Stimulated Emission Depletion Microscopy) SINGLE-MOLECULE IMAGING Schermelleh L, Heintzmann R, . et. al.

Instrumentation

N-STORM implementation by nikon

Fluorescence lifetime imaging

Measurement of temperature based on phosphorescenc lifetime

Fotoelektronsokszorozók

Fotoelektronsokszorozó működési elve

Microchannel plate detector működési elve

Kapuzható képerősítő működési elve

Példa a kapuzott képerősítő használatára

Streak camera működési elve

Streak camera használata fotokróm spirobenzopirán vizsgálatában

Egy felvétel

Quantum Efficiency (%) Typical Quantum Efficiency Curves 100 90 80 70 60 50 40 30 20 10 0 200 Back-illuminated ‘Virtual Phase’ Front-illuminated Micro. Lens front-illuminated Front-illuminat Gen III+ 300 400 500 600 700 Wavelength (nm) 800 900 1000

Interline CCD

EMCCD enhancing conventional epi-fluorescence microscopy? No Gain EM Gain BODIPY No Gain Texas Red EM Gain