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Scanning Microscopy with a Microlens Array

Scanning Microscopy with a Microlens Array. 18 October, FiO 2011 Antony Orth and Kenneth Crozier. High Throughput Microscopy. High throughput fluorescence imaging by scanning sample under widefield microscope. http://www.olympus.co.uk/microscopy/22_scan_R.htm#.

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Scanning Microscopy with a Microlens Array

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  1. Scanning Microscopy with a Microlens Array 18 October, FiO 2011 Antony Orth and Kenneth Crozier

  2. High Throughput Microscopy High throughput fluorescence imaging by scanning sample under widefield microscope. http://www.olympus.co.uk/microscopy/22_scan_R.htm#

  3. 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

  4. 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

  5. Experimental Setup Microlens focal length 40 μm Scan area: 20μm x 20μm Step size: 175nm Frame rate: 202 Hz Piezo scan Movie of microlens apertures as sample is scanned

  6. Reflow Mold Microlens Array 1.3mm Lens array molded in optical adhesive (NOA 61, n=1.56) 100 x 100 microlens array Pitch: 55 μm Lens Diameter: 40 μm Lens Height: 15 μm

  7. Focal Spot Characterization 0.8NA Microscope Objective 532 nm Laser FWHM = 790nm Microlens Array

  8. Scanning Fluorescence Images 2μm, 5μm beads 3.6 μm FWHM = 645 nm 500nm beads 3.6 μm Rat femur tissue section

  9. Large-Scale Imaging With Stitching 2μm 55 μmx 55 μm 0.8 mm 2μm beads

  10. Large-Scale Imaging With Stitching Highest throughput so far: Frame rate: 202 Hz Sensor area: 256 x 256 pixels (0.065Mpx) Microlenses: 5000 Throughput: 1Mpx/s With optimal camera (IDT NR5-S2): Frame rate: 1000 Hz Sensor area: 2560 x 1920 pixels (4.9Mpx) Microlenses: > 1,000,000 Throughput: 1.2Gpx/s 55 μmx 55 μm 40μm 2μm beads

  11. Light Field Parametrization (s,t) position on CCD maps to initial ray angle (u,v) is position in object space t s Image on CCD M. Levoy et al., J. Microscopy vol. 235 pt.2 2009 p.144

  12. Image Reconstruction Tile red pixels for perspective view Tile sum of greenpixels for full aperture view

  13. Perspective Fly-Around Microlens Aperture Microlens Aperture Extracted Pixel 3.6 μm 3.6 μm

  14. Perspective Fly-Around Microlens Aperture Extracted Pixel 3.6 μm

  15. 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

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