Scanning Microscopy with a Microlens Array

18 October, FiO 2011Antony Orth and Kenneth Crozier

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High Throughput Microscopy

http://www.olympus.co.uk/microscopy/22_scan_R.htm#

High throughput fluorescence imaging by scanning sample under widefield microscope.

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What limits high throughput microscopy?

• Specs sheet for typical systems advertise ~1s per image.• Camera sensor typically ~1Mpx, so throughput is ~1Mpx/s,

far below the throughput available with digital cameras.

• Limiting factors: – Motorized stages have small bandwidth.– Scanning procedures (focusing, moving FOV) become temporally

expensive.– Motion blur/lighting.

• Can we alter optics to alleviate these problems?– Break up imaging into small, parallelized fields of view.

http://www.olympus.co.uk/microscopy/22_scan_R_Specifications.htm

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Talk Outline

• Use of microlens arrays for fluorescence imaging– Experimental setup– Array fabrication and characterization– Sample fluorescence images– Large scale imaging example– Image processing

• Summary and outlook

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Experimental Setup

Piezo scan

Movie of microlens apertures as sample is scanned

Scan area: 20μm x 20μmStep size: 175nmFrame rate: 202 Hz

Microlens focal length 40 μm

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Reflow Mold Microlens Array1.3mm

Pitch: 55 μm

100

x 10

0 m

icro

lens

arr

ay

Lens Diameter: 40 μm Lens Height: 15 μm

Lens array molded in optical adhesive (NOA 61, n=1.56)

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Focal Spot Characterization

Microlens Array

532 nm Laser

0.8NA Microscope Objective

FWHM = 790nm

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Scanning Fluorescence Images

2μm, 5μm beads

Rat femur tissue section

3.6 μm

3.6 μm

FWHM = 645 nm500nm beads

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Large-Scale Imaging With Stitching

2μm beads

2μm55 μm x 55 μm

0.8 mm

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Large-Scale Imaging With Stitching

2μm beads40μm

Highest throughput so far:

Frame rate: 202 HzSensor area: 256 x 256 pixels (0.065Mpx)Microlenses: 5000Throughput: 1Mpx/s

With optimal camera (IDT NR5-S2):

Frame rate: 1000 HzSensor area: 2560 x 1920 pixels (4.9Mpx)Microlenses: > 1,000,000Throughput: 1.2Gpx/s

55 μm x 55 μm

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Light Field Parametrization

t

s

(s,t) position on CCD maps to initial ray angle(u,v) is position in object space

Image on CCD

M. Levoy et al., J. Microscopy vol. 235 pt.2 2009 p.144

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Image Reconstruction

Tile red pixels for perspective view

Tile sum of green pixels for full aperture view

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Perspective Fly-Around

Microlens Aperture Microlens Aperture

Extracted Pixel

3.6 μm3.6 μm

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Perspective Fly-Around

3.6 μm

Microlens Aperture

Extracted Pixel

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Summary & Outlook

• Demonstrated parallelized point scanning fluorescence microscopy with a microlens array

• Demonstrated pixel throughput comparable to commercial systems, but with small sensor size*

• Demonstrated viewpoint selection of scene

• *Throughput scales with sensor size: lots of room for speed increase.

• Next: imaging through coverslips – more involved microlens design