High throughput microscopy with a microlens array

Antony Orth and Kenneth Crozier

8 MayCLEO 2012

2

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

-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)

N2: # of sub-fields >103 for a microscope slide > 104 for a microwell plate

With a 20x objective:

Stage translationAutofocusing~1-2 sec / FOV*

* http://www.highthroughputimaging.com/screening/imagexpress_micro.html#apps

~1-10 cm

100s of μm

A High Throughput Microscope

- 4.26 Mpx / second (4.66 Mpx sensor)

- 1.85 hrs / plate / color @ 70% coverage!

(Molecular Devices ImageXpress Micro)

http://www.highthroughputimaging.com/screening/imagexpress_micro.html#apps

5

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.htm2 http://www.highthroughputimaging.com/screening/imagexpress_micro.html#apps

6

Experimental Setup

Piezo scan

Movie of microlens apertures as sample is scanned

Microlens focal length

Bright spots in movie = fluorescence captured by individual micolenses

Each microlens = individual scanning confocal microscope

Stitch together microlens subimages to form large image

(532nm, 38 mW)

7

Reflow Molded Microlens Arrays

1.3mm

100

x 10

0 m

icro

lens

arr

ay

Molded in optical adhesive (NOA 61, n=1.56)

Pitch: 100 μmLens Diameter: 93 μm

Lens Height: 14 μm

1 mm

Pitch: 55 μmLens Diameter: 37 μm

Lens Height: 15 μm NA: 0.41 NA: 0.31

100

x 10

0 m

icro

lens

arr

ay

8

Imaging resolution

1 μm

FWHM 781 nm

37 μm diameter lenses

Focal spot size sets resolution when iris open

Bead FWHM = 787 nm +/- 39 nm ~ Focal spot FWHM

200

nm b

eads

5 μm

Microlens focal spot

9

Confocal filtering

Real images formed by microlenses.

Iris acts as confocal filter for ALL microlenses!Stopping down iris improves resolution via confocal effect.

10

Confocal filtering

Iris open Iris diameter 2 mm(0.52 Airy diameter)

5 μm 5 μm

Confocal ability adds another level of control:Can trade off signal for resolution

2 mm

50 μm

25 μm

0.85

GPx

imag

eRa

w p

ixel t

hrou

ghpu

t 4M

px/s

Uses only 0.124 Mpx sensor!Full frame sensor higher throughput

Rat Femur Slice (Cy3)

1 mm

Sam

ple

cour

tesy

of M

oone

y la

b, H

arva

rd

Rat Femur Slice (Zoom-in)

1 mm

Corti

cal B

one

Med

ulla

ry C

anal

Periosteum

80 μm

80 μm

80 μm

14

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.

20 μm

5 μm

“sph

eres

”

100

μm d

iam

. le

nses