STED microscopy with single light source. TeodoraŞcheul

Transcription

1 STED microscopy with single light source TeodoraŞcheul Dr. Iréne Wang, Dr. Jean-Claude Vial LIPhy, Grenoble, France

2 Summary I. Introduction to STED microscopy II. STED with one laser source 1. Two-photon single wavelength STED 2. Two colours for excitation and depletion - one source Experimental set-ups Results Conclusions and future work

3 STED microscopy - principle Stefan W. Hell et al., Opt. Lett., 1994, 19, S 1 Excitation Luminescence S 0 exc fluo Stimulated emission Energy levels and optical transitions involved in STED Excitation nm STED Luminescence 50 nm Zero intensity point Switching off the fluorescence ability of the molecules in the periphery off the focus shrinks the fluorescing area

4 STED microscopy one laser source? Ti-Sapphire femtosecond laser Single wavelength STED Microchip Nd-YAG laser Two colours for excitation and depletion

5 I. Two-photon excitation and stimulated emission depletion by a single wavelength n vib 1 n 1 S 1 exc fluo STED n vib 0 S 0 n 0 Fluorescence (arb unit) (arb unit) DCM DCM 140 Excitation and Stimulation Excitation and Stimulation Wavelength (nm) Wavelength (nm) Two photon Two photon absorption absorption cross cross section section (GM unit) (GM unit) 2hν excitation ~ I 2 (fs pulse) STED ~ I (ps pulse) Two-photon absorption and emission spectra of DCM dye

6 Experimental setup: proof of principle in solution 140 fs λ/2 plate P1 Stretcher Scheul et al. Opt. Express, 2011, 19, Ti:Sa Camera 40 ps STED Delay Line Excitation P2 λ/4 plate Sample DCM solution Monochromator PMT TCSPC board Laser source: mode-locked Ti:Sapphire laser (Chameleon CoherentTM, Ultra II): pulse duration 140 fs, repetition rate of 80 MHz. The duration of the stretched pulse: ~40 ps

7 Experimental results Excitation a STED b Exc+STED c Scheul et al. Opt. Express, CCD Images 2011, 19, d STED e f Excitation d Excitation+STED Fluorescent decays The fluorescence is quenched when the two beams overlap Normalized fluorescence signal 1,0 0,8 0,6 0,4 0,2 0.91*exp(-0.79 I STED )+0.09 λ = 680 nm λ = 700 nm 0.86*exp(-0.40 I STED ) , I STED (GW/cm 2 ) *Average powers: 25 mw for excitation beam, 80 mw for STED beam Fluorescence depletion efficiency increases with STED intensity

8 Conclusions of Part 1 We have demonstrated the possibility to quench two-photon excited fluorescence by stimulated emission with a single wavelength Standard two-photon microscope into STED microscope Outlook We are building Single wavelength STED microscope by implementing the vortex phase plate + Gaussian beam Vortex phase plate (RPC photonics) Doughnut shaped beam Test different dyes: Pyridines and Styryl dye families, for live cell imaging - large Stokes-shift red fluorescent proteins

9 II. STED with a two color microchip laser Goal: STED microscopy with a single laser source that delivers two wavelengths simultaneously. Microchip Nd-YAG 1064 nm 1064 nm 532 nm Second harmonic generation Sum frequency generation 1064 nm 355 nm 532 nm Advantages: - Two beams are intrinsically aligned and synchronized. - Low cost Q-switched Nd-YAG laser Repetition rate of 8 khz to 150 khz). Pulse duration ~1 ns Disadvantages: - Restricted dye choice (large Stokes shift and UV absorbing dyes) - The need of achromatic optics for the two wavelengths simultaneously

10 Experimental setup: confocal towards STED microscope STED Reuss et al / Vol. 18, No. 2 / OPTICS EXPRESS. Fast axis Segm.waveplate Segmented waveplate: λ/2 for 532 nm λ at 355 nm Confocal image of fluorescent beads

11 Experimental results Exc (355 nm) Exc (355 nm)+ STED (532 nm) 1,0 Abs Hoescht Em Hoescht 1,0 Abs ATTO 390 Em ATTO 390 Abs/Em (arb.units) 0,8 0,6 0,4 0,2 355 nm 532 nm Abs/Em (arb.units) 0,8 0,6 0,4 0,2 355 nm 532 nm 0, , Wavelength (nm) Wavelength (nm)

12 Conclusions of Part 2 We have build a confocal microscope with a bicolor laser source that we believe it can be turned in a STED microscope by adding a chromatic birefringent device for the beam shaping Several dyes proved to be adequate to excitation at 355 nm and stimulated emission at 532 nm Outlook Future work will be done on fluorescent beads and fixed stained cells.

13 Acknowledgements Irène Wang Jean-Claude Vial Ciro d Amico LIPhy- MOTIV

14 Thank you!