Microlens Array 18 October FiO 2011 Antony Orth and Kenneth Crozier High Throughput Microscopy 1 httpwwwolympuscoukmicroscopy22scanRhtm High throughput fluorescence imaging by scanning sample under ID: 252812
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Slide1
Scanning Microscopy with a Microlens Array
18 October, FiO 2011Antony Orth and Kenneth CrozierSlide2
High Throughput Microscopy
1
http://www.olympus.co.uk/microscopy/22_scan_R.htm#
High throughput fluorescence imaging by scanning sample under
widefield
microscope.Slide3
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.
2
http://www.olympus.co.uk/microscopy/22_scan_R_Specifications.htmSlide4
Talk Outline
Use of microlens
arrays for fluorescence imagingExperimental setup
Array fabrication and characterization
Sample fluorescence images
Large scale imaging example
Image processing
3
Summary and outlookSlide5
Experimental Setup
4
Piezo
scan
Movie of
microlens
apertures as sample is scanned
Scan area: 20μm
x
20μm
Step size: 175nm
Frame rate: 202 Hz
Microlens
focal length
40
μmSlide6
Reflow Mold Microlens
Array1.3mm
Pitch: 55
μm
100
x
100
microlens
array
5
Lens Diameter: 40
μm
Lens Height: 15
μm
Lens array molded in optical adhesive (NOA 61,
n
=1.56)Slide7
Focal Spot Characterization
Microlens
Array
532 nm Laser
6
0.8NA Microscope Objective
FWHM = 790nmSlide8
Scanning Fluorescence Images
7
2μm, 5μm beads
Rat femur tissue section
3.6
μm
3.6
μm
FWHM = 645 nm
500nm beadsSlide9
Large-Scale Imaging With Stitching
8
2μm beads
2μm
55
μm
x
55
μm
0.8 mmSlide10
Large-Scale Imaging With Stitching
9
2μm beads
40μm
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
μmSlide11
Light Field
Parametrization
t
s
(
s,t
) position on CCD maps to initial ray angle
(
u,v
) is position in object space
10
Image on CCD
M.
Levoy
et al., J. Microscopy vol. 235 pt.2 2009 p.144Slide12
Image Reconstruction
Tile
red
pixels for perspective view
Tile sum of
green
pixels for full aperture view
11Slide13
Perspective Fly-Around
12
Microlens
Aperture
Microlens
Aperture
Extracted Pixel
3.6
μm
3.6
μmSlide14
Perspective Fly-Around
3.6
μm
Microlens
Aperture
Extracted Pixel
13Slide15
Summary & Outlook
Demonstrated parallelized point scanning fluorescence microscopy with a
microlens arrayDemonstrated pixel throughput comparable to commercial systems, but with small sensor size*
Demonstrated viewpoint selection of scene
14
*Throughput
scales with sensor size: lots of room for speed increase.
Next:
imaging through
coverslips
– more involved
microlens
designSlide16
Light Field Capture
Microlens
apertures
Tile aperture images
u
v
s
t
15Slide17
PDMS Reflow Molding Fabrication
Pattern posts of
photoresist
(AZ-40XT) on silicon
Place wafer on hot plate @125
o
C for 1 min. Resist melts, surface tension provides smooth lens surface
Inverse mold in PDMS
PDMS
Microscope slide
Replicate melted
photoresist
in optical adhesive (NOA 61) with UV cure
NOA 61
Peel off PDMS,
microlens
array ready for use!
16Slide18
Setup Revisited
Image on CCD
17