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#

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

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

Experimental Setup Piezo scan 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

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

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

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

Large-Scale Imaging With Stitching 55 μm x 55 μm 0.8 mm 2μm beads

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 μm x 55 μm 40μm 2μm beads

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

Image Reconstruction Tile red pixels for perspective view Tile sum of green pixels for full aperture view

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

Perspective Fly-Around Microlens Aperture Extracted Pixel 3.6 μm

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

Light Field Capture Microlens apertures Tile aperture images u s v t

Reflow Molding Fabrication Pattern posts of photoresist (AZ-40XT) on silicon Place wafer on hot plate @125oC for 1 min. Resist melts, surface tension provides smooth lens surface PDMS PDMS NOA 61 Microscope slide Inverse mold in PDMS Replicate melted photoresist in optical adhesive (NOA 61) with UV cure Peel off PDMS, microlens array ready for use!

Setup Revisited Image on CCD