Optotune Microscopy

Transcript

Optotune Microscopy presentation Zurich, January 2017 Dr. David Leuenberger, Sales Manager Bernstrasse 388 | CH-8953 Dietikon | Switzerland Phone +41 58 856 3011 | www.optotune.com | [email protected] Optotune on a page ETH Zurich spin-off, founded 2008 Leader in tunable optics 20 sales partners in 28 countries 45 employees HQ located in Zurich, Switzerland 100% management owned 2 Agenda • Why tunable lenses for microscopy? • Integration of tunable lenses • Application examples • Conclusion 3 Starting point Today, most microscopes take 2D images, but … …Life is 3-dimensional !! 4 Starting point Modern biology wants • Imaging of 3D cell cultures • Imaging of whole embryos • In-vivo imaging in living animals Issue: • Microscopes have a limited “depth of field” (DOF) • The higher the lateral resolution, the smaller the DOF Solution: • 3D microscope 5 3D microscopy techniques Wide-field microscopy Two-photon microscopy 3 Confocal microscopy Light-sheet microscopy Need to scan along z-axis Solutions:  Motorized stages Slow bulky  Piezo-stages small travel expensive  Focus tunable lenses Fast Compact accurate 7 USP for 3D microscopy • Fast (> 100 Hz), compact and accurate 3D scanning • >100x Faster than motorized solutions • >3x cheaper than piezo stages • Larger z-range than with piezo stages (up to 600 µm with 40x objective) 8 Agenda • Why tunable lenses for microscopy? • Integration of tunable lenses • Application examples • Conclusion 9 How does it work? Membrane Lens shaper Fluid Videos available on www.optotune.com 10 Optotune’s electrically focus tunable lenses EL-10-30-TC EL-10-30-C(i) EL-10-42-OF EL-16-40-TC-5D EL-16-40-TC-20D Focal power range* 8 … 22 Dpt -1.5 … +3.5 Dpt -2 … +2 Dpt -2 … +3 Dpt -10 … +10 Dpt Clear aperture 10mm 10mm 10mm 16mm 16mm Outer diameter 30mm 30mm 42mm 40mm 40mm Wavefront quality RMS @525nm** *** <0.25 / 0.5  <0.15 / 0.25  <0.15  I: <0.15/ 0.5  II <0.25 / 0.5  I: <0.25 / 2.5  II: <0.5 / 2.5  Absolute focal power accuracy <0.15 dpt < 0.1 dpt 0.008 dpt (temp. contr.) TBD TBD Built-in sensors None Temperature Temp./Optical feedback Temp./Optical feedback Temp./Optical feedback Applications MV OCT MV Microscopy Laser marking MV/Microscopy Ophthalmology MV/Microscopy Ophthalmology * Depends on selected optical fluid 11 ** vertical / horizontal optical axis *** class I: high-mag, microscopy class II: standard grade Microscopy Digital microscopy configurations Mag. change Cam Tube Lens No Mag change Cam Relay lens ETL ETL Obj (Inf) • Large z-range 12 Relay lens Tube Lens Obj (Inf) 4f system Intermediate image plane How to integrate the EL Digital inspection microscope Scientific microscope 3. EL Pos. (camera port) Tube lens Optotune EL Zoom lens 1. EL Pos. (after objective) 13 2. EL Pos. (filter cube) Non-telecentric autofocus configuration: EL on top of objective Zeiss Axioskop http://labs.pbrc.edu/cellbiology/documents/Axioskop Manual.pdf • Zeiss Neofluar, 10x/0.3 Inf./0.17 • Zeiss LD Achroplan 20x/0.4 Korr Ph2 Inf./0-1.5 • Zeiss Plan-Neofluar 40x/0.75 Inf/0.17 Camera Teledyne Dalsa Genie TS-C1920 Optotune Lens EL-16-40-TC-VIS-20D, ANAA0380 Mounted with C-mount RMS adapters from Thorlabs - RMSA6 - Adapter with External RMS Threads and Internal C-Mount Threads - RMSA5 - Adapter with External C-Mount Threads and Internal RMS Threads 14 camera C-mount adapter Non-telecentric autofocus configuration: EL on top of objective Mag 10x 20x 40x 15 -2 dpt 0 dpt +3 dpt Non-telecentric autofocus configuration: EL on top of objective • The tunable lens was operated between -2dpt and +3 dpt (nominal tuning range) • Compact autofocus solution without the need of mechanical translation • However, in such a configuration, the field-of-view (FOV) and numerical aperture (NA) changes while focusing (non-telecentric behavior) Z-range 16 Mag change 10x 2.56mm (20D: 10.24) 7.5 % 20x 0.64mm (20D: 2.56 mm) 12.2% 40x 0.16mm (20D: 0.64mm) 23.7% Non-telecentric autofocus configuration: optical layout Objective (Olympus 40x NA 0.8)* EL Offset Lens • With the tunable lens on top of the objective, the FOV and NA changes while focusing (non-telecentric behavior) • The animation on the left shows this as an increasing distance between the blue (onaxis) and red (maximum FOV) foci * Japanese patent 8-292374 18 Image plane without EL Telecentric autofocus configuration: tunable lens EL with a relay system CCD with • By inserting a relay system, composed of two lenses (a 4f-system), the back focal plane (BFP) of the objective can be reimaged to an accessible location • When the EL is placed at that position, the system stays telecentric while focusing fixed image Relay lens EL at a conjugate BFP of the objective Objective (ideal lens) Tube lens Relay lens with changing WD Relay system 19 Exemplary setup of a telecentric microscope with a tunable lens autofocus solution This design principle can be found in this EL-lightsheet microscope (Fahrbach et al., Optics Express 2013) 20 Idea: Tunable camera coupler enabling fast autofocus can be retrofitted to any microscope Proposed Solution Camera USP Image sensor C-mount thread Tunable camera coupler EL-16 Different photo tube clamps for different brands Microscope Photo tube 21 Intermediate image generated by tube lens • Retrofit to existing microscope possible • Automatic user independent parfocality between eye and camera port • Fast autofocus • Focus on region of interest by clicking into image • Wide-field 3D imaging (image stacking) EL at conjugate pupil position in telecentric system features zero mag change Double telecentric lens Surface assumed to be tunable Varying object distance Magnification 1.0x 127.3mm 1.0x 122.3mm 1.0x 117.3mm  The magnification of a double telecentric system stays the same when the object distance changes if a tunable lens to perform refocusing is located at the conjugate pupil position M_016 Double Telecentric - Magnification check.zmx 22 Starting point: Typical microscope design Zebase S_006.zmx Zoom Microscope Inverted design: Object is on right hand side eye eye piece Intermediate image Tube lens Telecentric 4F system for camera path 23 stop objective object Optical design sketch with known and estimated specs General layout Tube lens IDS UI-3580CP-C-HQ • ½” image sensor • 5MP resolution Potential additional lens to shorten FL of tube lens? => Telecentricity maintained? Estimate of divergence made on real microscope 150mm 4mm 4.4mm  20mm FLTube 165mm Intermediate image 24 EL-16-TC-C 1.53° 5.63x4.22 STOP  7.036mm Design with 2 achromats + EL suffers from reduced MTF at the edge Field curvature can’t be eliminated with the two achromats => limiting MTF quality. 25 Improved design with 2 meniscus lenses to reduce field curvature Total length further reduced with two positive meniscus lenses Total length 199mm Field curvature corrected Distance to intermediate image. Stop 3.6mm Nominal: 55.89mm Distance to intermed image Nominal Distance to intermed image [mm] 55.89 Image magnification 0.35 Total thickness EL-16 incl. Cmount flange Magnification stays the same when tuning EL-16 Image quality ok. Same over entire sensor. See Telecentric Microscopy Camera Adapter (real lenses - shorter front) v09 [tune check].zmx See Telecentric Microscopy Camera Adapter (real lenses - shorter front) v09.zmx Nominal -4.0mm Nominal +4.0mm 51.89 59.89 0.35 0.35 Microscopy adapter without magnification change Zeiss Axioskop • Zeiss Neofluar, 10x/0.3 Inf./0.17 • Zeiss LD Achroplan 20x/0.4 Korr Ph2 Inf./0-1.5 • Zeiss Plan-Neofluar 40x/0.75 Inf/0.17 Camera IDS UI-3580CP-C-HQ (1/2”, 5MP) Optotune Lens EL-16-40-TC-VIS-20D Mag EL-16-40-TC-VIS-5D EL-16-40-TC-VIS-20D 10x 262um 980um 20x 64um 254um 40x 12um 56um Z-Range 28 Tuning range & wavefront Focal Power vs Current - ANAA0359 RMS Wavefront Error - ANAA0359 0.6 Focal Power (dpt) 15 10 5 0 -5 -10 -15 -20 -500 29 -250 0 (mA) Current 250 500 RMS Wavefront Error (lambda) 20 0.5 0.4 0.3 0.2 0.1 0 -15 -10 -5 0 5 Focal Power (dpt) 10 15 Almost no magnification change 10x 10 dpt 0 dpt -10 dpt 30 20x 40x Optical quality is good! 1951 USAF target Zoom-in 40x 31 Stacking of pollen images Images have been taken at 10x between -10dpt and 10dpt -10 dpt 0 dpt +10 dpt 32 Agenda • Why tunable lenses for microscopy? • Integration of tunable lenses • Application examples • Conclusion 35 Of the last 10 papers published, 6 are in microscopy • Improved quantitative phase contrast in selfinterference digital holographic microscopy • Focal Sweep Videography with Deformable Optics • Rapid 3D light-sheet microscopy with a tunable lens Microscopy • Online correction of licking-induced brain motion during two-photon imaging with a tunable lens • High-speed transport-of-intensity phase microscopy with an electrically tunable lens • Rapid quantitative phase imaging for partially coherent light microscopy Laser processing OCT R&D 36 • Simulation and realization of a focus shifting unit using a tunable lens for 3D laser material processing • Microscope-integrated intraoperative OCT with electrically tunable focus and heads-up display • Optical transport of ultracold atoms using focustunable lenses • Measurement of the M2 beam propagation factor using a focus-tunable liquid lens Wide-field microscopy A B ETL ETL Optical path of the Axiovert 35 microscope. The ETL/OL assembly can be placed at the pupil without inserting an additional relay system. TL: Tube lens. Images courtesy of F. F. Voigt, Department of Neurophysiology, Brain Research Institute, University of Zurich 37 Wide-field microscopy ETL-based focusing through a group of pollen grains. Images courtesy of F. F. Voigt, Department of Neurophysiology, Brain Research Institute, University of Zurich 38 Video Confocal microscopy Max. intensity projection of a pollen corn Images courtesy of F. F. Voigt, Department of Neurophysiology, Brain Research Institute, University of Zurich 39 Video Video Confocal endomicroscopy Focal power range -127 mm to +44.3 mm Axial scan range @ sample 700µm Ref: J.M. Jabbour et al., BIOMEDICAL OPTICS EXPRESS 2014, 5, (2), pp. 645, 2014, “Optical axial scanning in confocal microscopy using an electrically tunable lens” 40 Confocal endomicroscopy Traditional approach Optotune approach Scan through oral mucosa ex vivo Video Ref: J.M. Jabbour et al., BIOMEDICAL OPTICS EXPRESS 2014, 5, (2), pp. 645, 2014, “Optical axial scanning in confocal microscopy using an electrically tunable lens” 41 Two-photon microscopy B.F Grewe et al., Biomedical Express (2011), 2, (7), pp.2035 42 Two-photon microscopy Example: Two-photon two-layer calcium imaging in mouse neocortex Two-photon images of a stained neuronal cell population (green) Benjamin F. Grewe, BIOMEDICAL OPTICS EXPRESS (2011), 2, (7), pp. 2035 43 Video Two-photon in-vivo imaging of retinal microstructures Optical sectioning in mouse 2P fluorescence angiography. A. Two-photon images of the optic disc. The microscope objective lens and mouse were held in place, and each image was acquired at different ETL currents (10mA interval between successive images; each image is an average over 30 frames acquired at 1 fps). Arrowheads point to blood vessels visible in only a few images, but not in others. B. Images of blood vessels outside the optic disc, acquired at different scan zooms (average over 100 and 200 frames; different animal than A). The FOV of the lower image is marked by a white box. Scale bars = 50 µm. Adi Schejter, Proc. SPIE 8948, Multiphoton Microscopy in the Biomedical Sciences XIV, 894824 (February 28, 2014); doi:10.1117/12.2039375 44 Light-sheet microscopy ETL Vascular system in the brain of a zebrafish Video F. O. Fahrbach et al., Opt. EXPRESS (2013), 21, (18), pp. 21010. 45 Light-sheet microscopy Large volume scan with an ETL through the heart of a zebrafish (10x magn.) Video Courtesy of Florian Fahrbach, Michaela Mickoleit and Jan Huisken. F. O. Fahrbach et al., Opt. EXPRESS (2013), 21, (18), pp. 21010. 46 Light sheet microscopy • Goal: Optimize with help of tunable lenses the illumination light-sheet to the requirement at hand. • A telescope composed of two electrically tuneable lenses enable to define thickness and position of the light-sheet independently but accurately within milliseconds, and therefore optimize image quality of the features of interest interactively. • This technique proved compatible with confocal line scanning detection, further improving image contrast. 2x A. K. Chmielewski et al., Nature Scientific Reports 5, Article number: 9385 doi:10.1038/srep09385 (2015). 47 3D High- and super-resolution imaging using single-objective SPIM • Single-objective selective-plane illumination microscopy (soSPIM) is achieved with micromirrored cavities combined with a laser beam–steering unit installed on a standard inverted microscope. • Based on custom EL-C-10-30 focus-tunable lens (TL) from −80 mm to +1,000 mm. Remi Galland, Nature Methods, published online 11 May 2015DOI:10.1038/NMETH.3402 48 Z-stacking with inverted microscope, 100x mag Optotune EL-10-30 microtubules in HeLa cells http://scitation.aip.org/content/aip/journal/rsi/86/1/10.1063/1.4905330 49 Sanxo scope: Inspection at HD • Inspection station with 10MP camera • EL-10-30-Ci in front lens configuration with 25mm lens • Driver integrated in machine vision software “Modular X” • Features: - Click to autofocus - Focal sweep with 3D rendering 51 Microscope-integrated intraoperative OCT • Optotune’s electrically tunable lens EL-10-30-NIR-LD allowed real-time adjustment of the OCT focal plane to maintain parfocality with the microscope view. • Potential for iOCT-guided maneuvers and clinical decision-making in ophthalmic surgery Y. K. Tao et al., BIOMEDICAL OPTICS EXPRESS (2014), 5, (6), pp. 1877. 52 Autofocus for high magnification with EL-10-30-C and Optem® 70XL by Qioptiq Results: C-mount camera ½” 5MP sensor 1.5x mini tube lens P/N 29-90-28-000 Magnification 1.1x 3.5x 7.9x Z range 400mm 40mm 8mm Z resolution 100µm 10µm 2µm DOF (approx.) 1mm 0.3mm 0.1mm HFOV 4.5mm 1.4mm 0.65mm Optotune lens EL-10-30-Ci-VIS-LD-MV Optem 70XL zoom (0.75x-5.25x) P/N 399510-309 Coaxial lighting unit with lens P/N 296515-310 LED ring light (used instead) Working distance: ~90mm Optem® is a registered trademark of Qioptiq, Inc 53 • Very good image quality • C-mount threads fit perfectly • All components available off the shelf Qioptiq is integrating the EL-16-40-TC in Optem Fusion microscope • The zoom is parfocal as the EL is placed BELOW the zoom 54 Low cost AF microscope with fixed mag High mag C-mount camera Empty C-mount tube, ~50mm long • Magnification: ~5X • Z-range: ~3mm Optotune lens EL-10-30-Ci-VIS-LD M22 to C-mount adapter 25mm lens (reversed!) Edmund Optics 85358 Working distance: ~20mm 55 Phaseview provides a Z-stepping lens for microscopes • EL-10-30-VIS-LD designed into tube lens • Their own optical design, mechanics & SW • Very good image quality, also in horizontal axis! 56 Edmund optics dynamic focus VZM with the EL-10-30-Ci-VIS-LD-MV integrated • Very large focus range as EL is placed close to aperture stop • The zoom is NOT parfocal, however, as the EL is placed above the zoom 57 Optotune’s LSR boosts image quality in superresolution fluorescence microscope (STORM) Setup: LSR-3005-17S-VIS LSR off LSR on Distribution of pixel greyscale values: MRC5 cells stained with Alexa Fluor 647 Ref: P. Georgiades et al., Journal of Microscopy (2016), http://onlinelibrary.wiley.com/doi/10.1111/jmi.12453/full 58 Agenda • Why tunable lenses for microscopy? • Integration of tunable lenses • Application examples • Conclusion 59 Microscopy application overview 3D microscopy 2D microscopy • Wide-field microscopy • Digital microscopy • Two-photon microscopy • Confocal microscopy • Adapter for video port • Light-sheet microscopy ETL 60 + Conclusion • Trend towards 3D biomedical imaging • Focus tunable polymer lenses are compatible with - Wide-field microscopy - Confocal microscopy - Two-photon microscopy - Light-sheet microscopy • Tunable lenses: - Fast - Compact - Large tuning range - Broadband 61 Optotune – 1 slide Optotune Switzerland AG Bernstrasse 388 CH-8953 Dietikon Switzerland Phone: +41 58 856 3000 | Fax: +41 58 856 3001 www.optotune.com | [email protected]