scanning microscopy with a microlens array

Scanning Microscopy with a Microlens Array

18 October, FiO 2011Antony Orth and Kenneth Crozier

2

High Throughput Microscopy

http://www.olympus.co.uk/microscopy/22_scan_R.htm#

High throughput fluorescence imaging by scanning sample under widefield microscope.

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

Piezo scan

Movie of microlens apertures as sample is scanned

Scan area: 20μm x 20μmStep size: 175nmFrame rate: 202 Hz

Microlens focal length 40 μm

6

Reflow Mold Microlens Array1.3mm

Pitch: 55 μm

100

x 10

0 m

icro

lens

arr

ay

Lens Diameter: 40 μm Lens Height: 15 μm

Lens array molded in optical adhesive (NOA 61, n=1.56)

7

Focal Spot Characterization

Microlens Array

532 nm Laser

0.8NA Microscope Objective

FWHM = 790nm

8

Scanning Fluorescence Images2μm, 5μm beads

Rat femur tissue section

3.6 μm

3.6 μm

FWHM = 645 nm500nm beads

9

Large-Scale Imaging With Stitching

2μm beads

2μm55 μm x 55 μm

0.8 mm

10

Large-Scale Imaging With Stitching

2μm beads40μm

Highest throughput so far:

Frame rate: 202 HzSensor area: 256 x 256 pixels (0.065Mpx)Microlenses: 5000Throughput: 1Mpx/s

With optimal camera (IDT NR5-S2):

Frame rate: 1000 HzSensor area: 2560 x 1920 pixels (4.9Mpx)Microlenses: > 1,000,000Throughput: 1.2Gpx/s

55 μm x 55 μm

11

Light Field Parametrization

t

s

(s,t) position on CCD maps to initial ray angle(u,v) is position in object space

Image on CCDM. Levoy et al., J. Microscopy vol. 235 pt.2 2009 p.144

12

Image Reconstruction

Tile red pixels for perspective view

Tile sum of green pixels for full aperture view

13

Perspective Fly-AroundMicrolens Aperture Microlens Aperture

Extracted Pixel

3.6 μm3.6 μm

14

Perspective Fly-Around

3.6 μm

Microlens Aperture

Extracted Pixel

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