Spectral Imaging with the Opterra Multipoint Scanning Confocal

Spectral Imaging with the Opterra Multipoint Scanning Confocal

Outline Opterra design overview Scan Modes Light Path Spectral Imaging with Opterra Drosophila larva heart.

Opterra Design Overview Supravideo rate, high speed slit scanner. Variable pinhole, linear array field scanner. EMCCD camera-based detector. High frequency scanning minimizes phototoxicity (due to very short dwell times).

Opterra Design Overview Multiple scan modes allow for optimization of resolution, speed and light delivery Linear pinhole/slit arrays (1-D) and separate light paths for emission and excitation minimize emission crosstalk

Aperture plate Emission Apertures Excitation Apertures

Opterra Aperture Positions Slit scanning positions 22 µm slit 35 µm slit 50 µm slit 70 µm slit 30 µm pinholes (x32) Field scanning positions 45 µm pinholes (x32) 60 µm pinholes (x32)

Opterra Imaging Scanner Light Path

Excitation Emission Excitation slits and pinholes

Scan Modes: Offering Flexibility and Optimization Airy disk: best focused spot a perfect lens can make For optimal resolution, want to match objective lens ability to create a focal point with pinhole size Optimization within experiments; Flexibility between experiments. Larger pinhole: more signal, cost of resolution Slits: more signal, faster scanning, cost of resolution in one dimension Ma g NA D Pinhole Slit 100 1.49 41.07 30 35 100 1.45 42.21 30 35 100 1.30 53.49 45 50 100 0.90 77.27 60 70 60 1.49 24.64 30 22 60 1.40 26.23 30 22 60 1.25 33.38 30 22 60 0.95 38.65 30 22 60 0.85 49.09 45 35 40 1.30 21.40 30 22 40 0.95 25.77 30 22 40 0.75 37.09 30 35 40 0.60 46.36 45 35 20 0.75 18.54 30 22 20 0.75 16.32 30 22 20 0.50 27.82 30 22 20 0.45 30.91 30 22

Illumination Shaping and Scanning Laser beam is shaped into a line, then scanned

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

Field-scanning combines multiple galvo sweeps with bidirectional piezo movement

Cross talk Linear pinhole arrangement: square root of crosstalk of 2-D array scanners. Provides improved axial resolution. Detector and microscope choices. Detector: High speed or high resolution. Microscope: Inverted or upright

Zebrafish Neurons 100 um Z Range

Tumor Slice: CFP and GFP 100 um Z range

EMCCDs are monochrome devices but color is often needed for visualization or discrimination of fluorophores

Traditional Approach Narrow bandpass filters How to detect hyperspectral (i.e. >3) colors without throwing away most of our signal!

The Amici Prism >95% Transmission at 630nm

4. Creates spectra from 32 lines on the sample CAMERA SENSOR 5. Shift all points by a row of pixels and repeat (16x) 2. Prism fans 32 points to 32 spectra 3. Galvanometer sweeps 32 points across the sample PINHOLE 1 PINHOLE 32 1. Emission light from 32 pinholes enters prism

SFC Hyperspectral with amici 15 prism Color Frame Acquisition Each image captured is the full spectrum from 32 lines on the sample Between each exposure the 32 lines are shifted down by a row

Hyperspectral Stack Reconstruction

Component Analysis

Performance Analysis Negligible Noise Penalty vs. Regular Opterra image: - One exposure, one readout - 720 ms exposure time - one color Spectral Opterra SFC image: image: - 28 36 Exposures, 28 36 readouts - 560 720 ms total exposure time - 15 color channels, integrated to one color false color image

Performance Analysis Spectral Resolution Invitrogen Green-Green Beads

Performance Analysis Spectral Resolution Mathieu Ducros, et al. Spectral Unmixing: Analysis of Performance in the Olfactory Bulb In Vivo. PLoS ONE. 2009; 4(2): e4418. doi: 10.1371/journal.pone.0004418

Arabidopsis Root Arabidopsis Kaleidocell Root 60x Oil Objective Nuclei: EGFP Mitochondria: YFP Plastid: DSRed Plasma Membrane: GFP

Zebrafish Tail Tail of Zebrafish Embryo 40x Oil Objective Spinal Column (sox9b): EGFP Nerve cell surface: (GFAP) A568 Cell Body: Dapi

Frog Egg Mitosis Mitosis in Frog Eggs 60x Oil Objective Cell Membrane: mtag BFP Tubulin (spindles and membrane): egfp Histone (nuclei): mcherry

Spectral Montage: Whole Zebrafish

Zebrafish brain: Neural development

Dissected zebrafish head

Prairie View: Real Time Spectral Acquisition Selectable color bands for each image window. Real time update of spectral histogram. Multiple math modes Simple toggling between Spectral and Normal mode.

High Speed Spectral Acquisition 16 camera exposures for a full 15 channel spectral frame Leverages high speed scanning and high speed EMCCDs like the Photometrics Evolve Delta 512 x 512 sensor 15 channels spectral resolution > 4 Spectral frames per second 3/28/2014 Bruker Confidential 34

Advantages of the Opterra Spectra Negligible noise penalty 15 channel spectral resolution Facilitates multiple overlapping fluorophore separation High speed spectral capture Easily switchable between spectral and regular confocal modes Triple labeled zebrafish tail fin