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

1. High Throughput Microscopy
High throughput fluorescence imaging by scanning sample under widefield microscope.

2. 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.

3. 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

4. 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

5. 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

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

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

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

9. 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

10. 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 p.144

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

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

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

14. 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

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

16. 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!

17. Setup Revisited
Image on CCD