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

1. 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?

2. 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 *

3. A High Throughput Microscope
(Molecular Devices ImageXpress Micro)
Mpx / second (4.66 Mpx sensor)
hrs / plate / 70% coverage!

4. 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:// 2

5. 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

6. 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)

7. 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

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

9. 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

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

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

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

13. 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”

14. Reflow Molding Fabrication
Pattern posts of photoresist (AZ-40XT) on silicon
Place wafer on hot 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!