Multidimensional Imaging with the Opterra Multipoint Scanning Confocal System Atomic Force Microscopy Fluorescence Microscopy

Outline • Opterra design overview • Light Path • Scan Modes • Performance • Application data Drosophila larva heart.

Opterra allows you to pick your position in the Iron Triangle of Imaging Figure courtesy of Paul Maddox Concept originally developed by Jonas Dorn and Gaudenz Danuser 10/30/2014

Opterra Design Overview • Two modes of operation • Variable pinhole, linear array field scanner. Supravideo rate, high speed slit scanner. • EMCCD camera-based detector. • High frequency scanning minimizes phototoxicity and photobleaching • Multiple scan modes allow for optimization of resolution, speed and light delivery • Linear pinholes and separate light paths for emission and excitation minimize emission crosstalk

Overhead View of Opterra Imaging Scanner Showing Lightpath Bruker Confidential

Camera side piezo Galvanometer Scope side piezo

Aperture plate

Opterra Aperture Positions Slit scanning positions 22 mm slit 35 mm slit 50 mm slit 70 mm slit 30 mm pinholes (x32) Pinhole scanning positions

Opterra Imaging Scanner Light Path_ PINHOLE ARRAY From laser

Time to move depends on exposure time It takes 2 frames for one complete “piezo sweep” Galvo sweeps multiple cycles/frame, and piezos sweep only once per frame Time to move depends on exposure time Piezos move a fixed distance Galvo moves a fixed distance Galvanometer and Piezo Movement

Galvanometer and Piezo Movement Field-scanning combines multiple galvo sweeps with bidirectional piezo movement

Scan pattern timing Sample Piezo/Galvo/Blanking Waveforms For Single Frame piezo offset galvo offset piezo waveform galvo waveform AOTF blanking (R)est, (A)ccelerate, (F)ront porch, (S)canning, (B)ack porch, (D)ecelerate

Cross talk • Linear pinhole arrangement: < one half of the crosstalk of 2-D array scanners. • Provides improved axial resolution.

Standard Opterra camera (Evolve Delta) • 512 x 512 full frame acquisition. Up to 40 full frames per second pinhole mode. Faster acquisition from smaller region of interest. Slit scan imaging at 66 fps full frame (512x512), 500 fps with ROI, up to 1000 fps with specialized detector

Opterra benefits for Multidimensional Imaging • Less cross talk provides improved axial resolution and imaging depth • High acquisition speed provides excellent temporal resolution • Shorter exposure times results in lower phototoxicity and photobleaching • Sea urchin embryos, 50 plane Z series every 2 minutes, experiments for over 2 hours compare to 1 hour on spinning disc Generally across a number of samples exposures 5 – 10 times shorter than spinning discs

Zebrafish Neurons 100 uM Z Range Opterra confocal

Tumor Slice CFP and GFP label 100 um Z range

Z Series Collected on Timed Intervals Max projections of Z series timelapse,Rab5-GFP vesicles in axons within spinal cord of intact Zebrafish embryo. -. 12 section z series Less than 1 second per Z stack Z every 5 seconds

Green: EGFP labelled EB3, Red H2B mCherry Max projections, 5 seconds intervals, 21 plane stacks, 0.5 um spacing

Max projection of high speed z series YFP mitochondria, mCherry peroxisomes Max projection of high speed z series 11 Planes, 0.3 micron spacing 2 Color 1.2 seconds / Z stack Z every 2 seconds

Atlas Imaging for Stage Montages

Moving using the Atlas Window BRUKER

Changing Focus • Focal plane can be changed using: • • Mouse scroll wheel Prairie View’s existing Z controls Click and drag on Z slice (shown) Wheels on motor controlling hardware

Atlas Imaging BRUKEI Pruning the Grid (JXJ>

Arabidopsis 2x4 montage, 60x objective BRUKER Bruker Confidential

Tissue section, 2.4 mm by 1.2 mm area at 60 x 18x9 montage Bruker Confidential

Opterra Multipoint Scanning Confocal Summary • Flexibility in aperture selection to meet experimental needs, allows user to balance speed, resolution and sensitivity. Allows the user to determine where they want to be in the “Iron Triangle of Imaging” • Pinholes in 1-D array minimizes cross talk and improves axial resolution compared to 2-D arrays • Scanning pattern with pinholes minimizes phototoxicity • Speed provides high temporal resolution with 4-D imaging • Higher throughput on grid collection applications Bruker Confidential

© Copyright Bruker Corporation. All rights reserved.