Spectral Imaging with the Opterra Multipoint Scanning Confocal

Outline

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

Aperture plate

Emission Apertures

Excitation Apertures

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

Opterra Imaging Scanner Light Path

Excitation slits and pinholes

Excitation Emission

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

Laser beam is shaped into a line, then scanned

Illumination Shaping and Scanning

Piezos move a fixed distance

Time to move depends on exposure time

It takes 2 frames for one complete “piezo sweep”

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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

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

…

Hyperspectral Stack Reconstruction

Component Analysis

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

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 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

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

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

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