High throughput microscopy with a microlens array Antony Orth and Kenneth Crozier 8 May CLEO 2012

Microscopy with lens arrays What is high thoughput microscopy? Experimental setup – confocal system Lens array characteristics, resolution Effect of confocal filtering Large scale imaging What’s next?

High Throughput Microscopy ~1-10 cm Microscope field of view (FOV) << sample size. Sub-fields of large sample imaged sequentially. Sub-fields stitched together to form large continuous image. Histological slide scanning High content screening (HCS) Stage translation Autofocusing ~1-2 sec / FOV* 100s of μm With a 20x objective: N2: # of sub-fields >103 for a microscope slide > 104 for a microwell plate * http://www.highthroughputimaging.com/screening/imagexpress_micro.html#apps

A High Throughput Microscope (Molecular Devices ImageXpress Micro) - 4.26 Mpx / second (4.66 Mpx sensor) - 1.85 hrs / plate / color @ 70% coverage! http://www.highthroughputimaging.com/screening/imagexpress_micro.html#apps

What limits high throughput microscopy? Specs sheet for typical systems advertise ~1s per image. Camera sensors are ~1-5Mpx, so throughput is ~1-5Mpx/s, far below the throughput available with digital cameras.1,2 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. 1http://www.olympus.co.uk/microscopy/22_scan_R_Specifications.htm 2 http://www.highthroughputimaging.com/screening/imagexpress_micro.html#apps

Experimental Setup Piezo scan (532nm, 38 mW) Microlens focal length Piezo scan Bright spots in movie = fluorescence captured by individual micolenses Each microlens = individual scanning confocal microscope Stitch together microlens subimages to form large image Movie of microlens apertures as sample is scanned

Reflow Molded Microlens Arrays 1 mm 1.3mm 100 x 100 microlens array 100 x 100 microlens array Pitch: 55 μm Pitch: 100 μm Lens Diameter: 93 μm Lens Height: 14 μm Lens Diameter: 37 μm Lens Height: 15 μm NA: 0.41 NA: 0.31 Molded in optical adhesive (NOA 61, n=1.56)

Imaging resolution 1 μm Microlens focal spot 5 μm FWHM 781 nm 37 μm diameter lenses Microlens focal spot 5 μm Focal spot size sets resolution when iris open Bead FWHM = 787 nm +/- 39 nm ~ Focal spot FWHM 200 nm beads

Confocal filtering Real images formed by microlenses. Iris acts as confocal filter for ALL microlenses! Stopping down iris improves resolution via confocal effect.

Confocal filtering Confocal ability adds another level of control: 5 μm 5 μm Iris open Iris diameter 2 mm (0.52 Airy diameter) Confocal ability adds another level of control: Can trade off signal for resolution

Raw pixel throughput 4Mpx/s 2 mm 50 μm Raw pixel throughput 4Mpx/s 0.85 GPx image 25 μm Uses only 0.124 Mpx sensor! Full frame sensor higher throughput

Rat Femur Slice (Cy3) Sample courtesy of Mooney lab, Harvard 1 mm

Rat Femur Slice (Zoom-in) Cortical Bone 80 μm Medullary Canal Periosteum 80 μm 80 μm 1 mm

Summary & Outlook Built a parallelized scanning microscope using refractive μlenses Fabricated 10,000 element μlens arrays. NA: 0.41 (37μm diameter), NA: 0.31 (93 μm diameter). Constructed a 0.85 Gpx image with <790nm resolution. Resolution of <700nm can be achieved using confocal filtering. Demonstrated imaging of microspheres, rat femur section. Throughputs up to 4Mpx/s using 352 x 352 px sensor. Lots of room for scaling. Have recently achieved imaging through a coverslip. Next step: image microwell plate, multiple wells at once. 100 μm diam. lenses 20 μm 5 μm “spheres”

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!