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Drosophila larva heart. • Opterra design overview
• Scan Modes • Light Path • Spectral Imaging with Opterra
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).
• 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
Opterra Design Overview
50 µm slit
70 µm slit
30 µm pinholes (x32)
45 µm pinholes (x32)
60 µm pinholes (x32)
Opterra Aperture Positions
Slit scanning positions
35 µm slit 22 µm slit
Field scanning positions
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
Mag 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
Piezos move a fixed distance
Time to move depends on exposure time
It takes 2 frames for one complete “piezo sweep”
Gal
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oves
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Tim
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Galvo sweeps multiple cycles/frame, and piezos sweep only once per frame
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
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!
1. Emission light from 32 pinholes enters prism
2. Prism fans 32 points to 32 spectra
3. Galvanometer sweeps 32 points across the sample
4. Creates spectra from 32 lines on the sample
5. Shift all points by a row of pixels and repeat (16x)
PINHOLE 1
PINHOLE 32
CAMERA SENSOR
SFC with amici prism Hyperspectral 15 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
Regular Opterra image: - One exposure, one readout - 720 ms exposure time - one color
…
Spectral SFC image: - 36 Exposures, 36 readouts - 720 ms total exposure time - 15 color channels, integrated to one color “false color” image
vs.
Spectral Opterra image: - 28 Exposures, 28 readouts - 560 ms total exposure time - 15 color channels, integrated to one color “false color” image
Performance Analysis – Negligible Noise Penalty
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 Kaleidocell Root 60x Oil Objective Nuclei: EGFP Mitochondria: YFP Plastid: DSRed Plasma Membrane: GFP
Arabidopsis Root
Tail of Zebrafish Embryo 40x Oil Objective Spinal Column (sox9b): EGFP Nerve cell surface: (GFAP) A568 Cell Body: Dapi
Zebrafish Tail
Frog Egg Mitosis
Mitosis in Frog Eggs 60x Oil Objective Cell Membrane: mtag BFP Tubulin (spindles and membrane): eGFP Histone (nuclei): mCherry
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
3/28/2014 34 Bruker Confidential
• 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