High Power Diode Lasers and Multi Laser Engines, Expanding the Range of Biophotonics Applications. Konstantin Birngruber TOPTICA Photonics AG

Transcription

1 High Power Diode Lasers and Multi Laser Engines, Expanding the Range of Biophotonics Applications Konstantin Birngruber TOPTICA Photonics AG

2 TOPTICA Photonics AG Company facts Founded employees Graefelfing (Munich), Germany Victor, NY, USA Berlin, Germany Lasers for Quantum Optics Biophotonics Terahertz Test & Measurement

3 TOPTICA: Technologies Bild neu Tunable Diode Lasers OEM Diode Laser Ultrafast Fiber Laser Terahertz Tunable DL High Power DL NLO DL Photonicals Single-mode Single frequency Multi-color Picosecond Femtosecond Erbium-based Ytterbium-based Supercontinuum / discrete WL 2-color cw Femtosecond Electronic emitters Antenna modules Spectroscopy Kits Atomic Physics Laser Cooling cw Terahertz Spectroscopy Interferometry Biophotonics Microscopy CtP Printing Semicon Metrology Microscopy Spectroscopy Pulsed Terahertz Metrology Seeder for HP THz Spectroscopy (frequency-domain, time-domain) THz Imaging

4 Why Multiple Colors?

5 Why Multiple Colors?

6 Dyes & Fluorescent Proteins DAPI Alexa 488 MitoTracker Excitation UV Violet Blue Green Yellow Red Dye / FP DAPI, Hoechst, BFP, Pacific Blue. Cerulean, CFP, GFP, Alexa 488, FITC, Cy2, YFP, Alexa 514, mcitrine, Venus, DsRed, Alexa 568, Rhodamine B, TexasRed, Cy3, MitoTracker, TRITC, mcherry,... Alexa 647, Cy 5,

7 Perfect Laser Multi-Purpose Illumination for Biophotonics It doesn t exist! Reason: Strong diversification in the market asking for many specialized light sources, often contradicting each other Examples: Low power applications / high power applications Low coherence lengths / high coherence lengths Flexible / completely hands-off Low cost / high end OEM style in the hundreds / one offs highly specialized Consequence: Strong diversification in the light engine sector Goal of this presentation: give a ultra-short and incomplete overview on some Biophotonics applications and light sources for Biophotonics

8 Applications

9 Applications Spinning Disk Confocal z-stack Mouse kidney section DAPI, Alexa 488, Alexa nm, 488 nm, 561 nm Optogenetics excitation Transgenic zebrafish larvae Courtesy: Till Photonics Ion channel channelrhodopsin (488 nm) Ion pump Halorhodopsin (640 nm) Photoconvertible fluorescent protein Kaede (405 nm) Courtesy: A. Arrenberg, University of Freiburg Light Sheet Microscopy Drosophila embryo H2A-mcherry 561 nm excitation Acquision time ~ 1 hour Courtesy: L. Hufnagel, EMBL

10 Super Resolution Microscopy True super resolution methods Near-field scanning optical microscope (NSOM) Stimulated emission depletion (STED) The diffraction limited excitation beam is overlaid with a doughnut-shaped depletion beam, which depletes a specific region of the excitation beam, leaving only the center focal spot active to emit fluorescence. Excitation Beam Diameter ~ 250nm Depletion Beam Resulting Beam Diameter ~ 30nm

11 Super Resolution Microscopy Stochastic super resolution methods Stochastic Optical Reconstruction Microscopy (STORM) Photo Activated Localization Microscopy (PALM) Fluorescence Photo-activation Localization Microscopy (fpalm) During imaging, only a small subset of fluorophores is activated. The position of each fluorophore can be recorded with a normal microscope setup. The position of the fluorophore can be determined by finding the centroid position of the image of a particular fluorophore. Recorded Image (diffraction limited) Reconstructed Image

12 Super Resolution Microscopy 1 µm CDC11 CONA Localization Microscopy PTK2-Cell Actin Phalloidin-Alexa647 Precise adjustment of laser power Beam quality Simple integration in microscope control software Activation: 405 nm / Exc. 640 nm Yeast Sep7-GFP, Nanobodies-Alexa647 (red) Cell wall ConA-Alexa700 (green) Courtesy: Jonas Ries EMBL Heidelberg Custom-built single molecule microscpe

13 Spinning Disk Confocal Microscopy Example measurements performed with Spinning Disk Confocal (imic, Till Photonics, 405 nm (DAPI), 488 nm (Alexa 488) & 561 nm (Alexa 568) Multi wavelength High laser power Mouse kidney section imic with spinning disk (Till Photonics)

14 Light-sheet based microscopy Camera Emission filter Cylindrical lens Laser Sample Courtesy: L. Hufnagel, EMBL

15 Applications: Light-sheet based microscopy Drosophila embryo H2A-mcherry 561 nm excitation Acquision time ~ 1 hour High laser power High beam quality Courtesy: L. Hufnagel, EMBL

16 Controlled Light Exposure (CLEM) Microscopy Reduces light exposure to sample Minimizes phototoxicity Increases cell viability Reduces background noise

17 Controlled Light Exposure (CLEM) Microscopy Digital modulation (up to 250 MHz) Very fast rise and fall times 1 Up to 250 MHz - ns rise time Without CLEM 2 with CLEM (sub-pixel modulation) Pulse on Demand Courtesy of Erik Manders, Univ. Amsterdam

18 Optogenetics Light-gated ion channels in transgenic zebrafish larvae 3-laser ichrome MLE to activate: Photoconvertible fluorescent protein Kaede (405 nm) Ion channel channelrhodopsin (488 nm) Ion pump Halorhodopsin (640 nm) Optic fiber (50 µm diameter) is placed above the embedded zebrafish larva for mw light stimulations Neural circuits can be targeted and inactivated (Halorhodopsin), activated (Channelrhodopsin) or labeled (Kaede, red photoconversion) to test circuit function in a behaving animal The eye position memory circuit is inactivated (Halorhodopsin) leading to transient eye position changes Flexible & precise switching for behaviour experiments Courtesy: A. Arrenberg, University of Freiburg

19 Diode Lasers

20 ibeam smart & ibeam smart PT True one-box solution, compact size (10 x 4 x 4 cm 3 ) Highest power (up to 300 mw) Best beam properties (TEM 00 ) Unique features like FINE / SKILL Speckle Reduction

21 ibeam smart & ibeam smart PT Digital modulation (up to 250 MHz) Analog modulation (up to 1 MHz) Lowest noise (< 0.2%) Up to 250 MHz - ns rise time Spinning disk confocal image of microtubules stained with Alexa 488 Pulse on Demand

22 Multi Laser Engines

23 Typical Power Requirements Single Spot Confocal < 10 mw Spinning Disk > 50 mw Line Scanning > 50 mw Multi Focal Scanning > 50 mw TIRF up to 100 mw FRAP up to 100 mw Cytometry up to 100 mw Localization techniques (PALM) up to several hundreds of mw STED more than several hundreds of mw Important to many techniques: Exact (low ) power and low noise output Exact and fast modulation of output power

24 Other Essential Requirements Single Mode Polarization Maintaining fibers (beam quality / focus) perfect interface, interchangeability, beam quality Beam shaping capabilities Line focus, bessel beam focusing, Fast, precise power modulation capabilities Set power for different samples, each pixel, bleaching etc. Flexibility Requirements can change -> upgrades Stability End user needs to see the advantage over home build systems

25 Laser 3 Laser 2 Laser 1 Multicolor Temperature Mechanical Shocks Transportation Microscope

26 State of the Art Multi Laser Engines Most cases: breadboard approach Combination of several diode lasers DPSS, (gas lasers) (white light sources plus AOTF) Many variations: AOM, AOTF, laser cleanup, filters, polarizers, ND filters Optical interface: free-space, SM/PM fiber, MM fiber, multiple fibers Collimated, focussed beams

27 State of the Art Multi Laser Engines + very flexible + more than enough choices in the market - price point - footprint - need for periodic maintenance (alignment) - in many cases: modulation via AOTF (costs, speed, light leakage ) - in some cases: non-unified user interface Courtesy of Visitron Systems Courtesy of Picoquant

28 Next Step: Integrated solutions TOPTICA s ichrome MLE Fully automated laser combiner 10 x 10 cm 2 footprint + Footprint reduction + Automatic alignment + life time hands-off - no field upgrades

29 Multi Laser Engine: ichrome MLE Compact Multi Laser Engine 4 laser lines (diode / DPSS) Fiber delivery (SM/PM fiber) Efficient fiber coupling High passive stability Microscope Good, but not perfect Power [%] C Power after fiber delivery 15 C 405 nm 488 nm 561 nm 640 nm Time [h] Ambient Temperature [ C]

30 COOL Philosopy COOL Technology Constant Optical Output Level Extremely robust opto-mechanical setup Minimize adjustable components Maximze cemented optical components COOL DC Technology COOL AC Technology DC Dura Calibrated AC Auto Calibration Single color Multiple colors Life time hands-off On demand Feedback controlled Unique Concept in the market

31 Automatic Alignment COOL AC Technology: Constant Optical Output Level Use fiber output or external signal Plug & Play Power after fiber delivery ~ 4 µm Automatic Alignment

32 ichrome MLE High output power nw to 100 mw continuous power setting, complete on/off Direct Modulation (20 MHz) One interface for all lasers All-diode version: no AOM Power after fiber delivery DPSS *

33 Future Trends for Light Engines

34 Trend: Higher Integration Integrated solutions Footprint reduction Breadboard style with housing Stability gain Essential in applications like flow cytometry Also gaining importance in microscopy General trend: smaller is better Enabling integration into tabletop systems Omicron s LightHub

35 Trend: Hands Off Operation need for installation and manual alignment Plug & Play, lifetime hands-off Trend from research imaging systems towards turnkey systems Many end-users are not trained in opto-mechanics / lasers etc. Time saving Agilent s complex monolithic optic (CMO) technology TOPTICA s Constant Optical Output Level (COOL AC ) technology

36 Trend: Direct Modulation Availability of green laser diodes Omicron s LightHub TOPTICA s ichrome MLE + no AOTF / AOM-> cost savings / footprint reduction + faster modulation with complete off possible + less power consumption lower heat dissipation -> integration - limited power levels for SM 515 nm and 561 nm lasers Alternative: DPSS with integrated AOM available

37 Trend: Advanced Integration Easier integration into standard operating environments (unified user interface) Graphical User Interface MicroManager integration

38 Trend: Multiple fiber / exit ports Agilent s MLC 400 Andor s ALC Sourcing multiple microscope ports Enabling experiments (e.g. FRAP) Fast switching times required in some cases (< 5 ms) Various concepts Power sharing (no switching) Galvo switching Micro-switches MEMS

39 Ease of Use vs. Modularity Increasing Ease of Use Increasing Modularity

40 More Exciting Laser Engines to Come