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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
thro
ughp
ut 4
Mpx
/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
“sp
here
s”
100
μm d
iam
. le
nses