Closed loop adaptive optics in
fluorescence microscopy
Michael Shaw1, Carl Paterson2
1National
2Photonics
Physical Laboratory, Teddington, Middlesex, TW11 0LW, UK
Group, The Blackett Laboratory, Imperial College, London, SW7 2BW, UK
Active & Adaptive Optics Meeting, PHOTONEX, 18 October 2011
Contents
The problem: aberrations in optical microscopy.
AO microscopy: state-of-the-art.
Direct wavefront sensing in microscopy.
Fluorescence microscope incorporating closed loop AO.
System testing and preliminary results.
Future work and summary.
Introduction
Fluorescence microscopy is a very widespread tool in
the life sciences due in part to the specificity of
fluorescent labels and its ability to image live
specimens.
However, the images formed by such systems are
degraded by wavefront aberrations due to:
Imperfections in the optical system
A difference between the immersion medium of the
objective lens and the specimen
The non-uniform refractive index of the specimen itself.
Adaptive optic techniques offer a way to correct
aberrations and restore image quality.
Simulation showing effect increasing
aberration amplitude with imaging depth
AO microscopy limitations
In many biological specimens imaging depth is limited by light scattering.
However correction of low order refractive aberrations has significant potential to
improve imaging of many commonly studied organisms such as C. Elegans and
Drosophila.
Beam scanning microscopes such as confocal laser scanning microscopes, which are
widely used for high resolution 3D fluorescence imaging, operate at scan speeds of up
to 100s of KHz (pixel dwell time of few µs).
Isoplanatic patch spans many pixels so lower frequency measurement and
correction of wavefront is acceptable.
AO microscopy: state-of-the-art
There are specific challenges in implementing an adaptive optic system in a
microscope, principally direct wavefront sensing is often difficult.
A number of different schemes have been developed for different imaging modalities.
Class
Description
Examples
Image optimisation
Optimise an image quality metric using
iterative changes to the DM
Hill climbing, GA and random search to
maximise signal (Wright, 2005)
Modal wavefront sensing (Neil, 2000)
Image-based wavefront sensing
Infer the wavefront aberration from the
effect that changes to the illumination
have on the image
Pupil segmentation (Ji, 2010)
Coherence gated WFS (Rueckel, 2006)
Direct wavefront sensing
Measurement of the wavefront using a
wavefront sensor or interferometer
Artificial or natural guidestars (Azucena,
2011 & Tao, 2011)
Confocal WFS (Cha, 2010)
Direct wavefront sensing approaches offer the potential of high speed correction for
imaging dynamic systems (live specimens) and minimising light exposure (reduced
photobleaching, adverse phototoxic reactions etc.).
Direct wavefront sensing using artificial guide stars
Direct wavefront sensing can be achieved using
point objects which provide a source of reference
wavefronts.
Seeding a biological specimen with small fluorescent
particles enables measurement and correction of the
local wavefront aberration.
For simple WF measurement, particles need to be
smaller than the PSF of the Shack-Hartmann
wavefront sensor (d ~ 1.22λ/NA) and we want
approximately one microsphere per isoplanatic
patch.
For our SHS, NA = 0.04, dAiry = 21 µm @ 635
nm.
Fluorescent emission from a point object
in an aberrating medium collected by a
microscope objective
Adaptive optic fluorescence microscope system
Stage scanning epifluorescence /
confocal (w / wo pinhole)
microscope system with closed
loop AO.
Object / confocal pinhole imaged
onto cooled CCD camera.
AO system comprised of:
37 channel Piezoelectric DM, 50
mm Ø (OKOTech)
Shack-Hartmann wavefront
sensor
4f optical relays to conjugate
defining aperture, DM, objective
exit pupil, SHS.
Separate illumination for widefield
imaging.
AO fluorescence
microscope schematic
Adaptive optic fluorescence microscope system
Excitation path
Objective lens
& sample
Wavefront sensing
& imaging arm
DM
laser
Retroreflector for
WFS calibration
AO fluorescence microscope photograph
Adaptive optic fluorescence microscope system
Emission path
Objective lens
& sample
Wavefront sensing
& imaging arm
DM
laser
Retroreflector for
WFS calibration
AO fluorescence microscope photograph
Adaptive optic fluorescence microscope system
Position of the confocal pinhole:
Confocal pinhole before WFS allows rejection of
wavefronts originating away from focal plane, but…
Pinhole acts as spatial filter > affects accuracy of
wavefront measurement.
May be preferable to move pinhole to back focal
plane of L8.
Simulate using double fft with a mask at the
common focus.
Optical layout (left), intensity and phase at input and output (centre) and
rms wavefront error vs pinhole radius (right) for propagation of first order
spherical aberration through a 4f relay with a confocal pinhole.
AO fluorescence
microscope camera &
SHS schematic
Deformable mirror
The aberration modes which the DM can generate / correct
depends on the illuminated aperture and how this is mapped onto
the exit pupil of the microscope objective.
The DM influence functions were measured using a phase
stepping Fizeau interferometer.
The influence matrix was inverted (svd) to explore how the
aperture ratio affects the ability of the mirror to correct for the
spherical aberration introduced by a planar refractive index
mismatch – a very common aberration in practical confocal
microscopy.
Influence functions (top) and orthogonal
modes (bottom) of the piezoelectric DM
Actuator layout (left), residual WFE (centre)
and optimum mirror fit (right) to aberration
from NA = 1.2 objective focussing 100 µm
into water. Optimum aperture ratio is 0.59.
System calibration
Calibrate WFS using reference beam (assumes no
aberration in detection arm).
System aberrations measured by exciting a single
fluorescent microsphere mounted on the surface of a
glass slide:
DM replaced with reference flat: 0.11 λ rms (@ 635
nm)
DM with all actuators at 0V: 0.22 λ rms (@ 635 nm)
Calibrate DM influence matrix using fluorescent
emission from a single microsphere on the surface of a
glass slide:
After DM feedback: 0.06 λ rms (@ 635 nm).
WFS calibration arm
schematic
Fluorescent microspheres on glass surface
1 µm Ø Crimson Fluospheres dried onto a glass slide and imaged in widefield
mode before and after AO correction.
AO off
AO on
Image & intensity
projections of two 1 µm
diameter Crimson
Fluospheres on the surface
of a glass slide
Artificial guide star phantoms
Phantoms prepared by seeding polyacrylamide gel with different concentrations
of 1µm Ø, Crimson Fluospheres.
CLSM image of
polyacylamide gel seeded
with 1 µm diameter
Fluospheres
Approximate particle densities of: 1 particle per (60 µm)3 and 1 particle per (120
µm)3.
Wavefront sensing with guide star phantoms
Measure wavefront aberration vs. penetration depth by exciting individual
microspheres throughout the gel matrix.
Expect linear increase in
spherical aberration with
imaging depth.
Spherical aberration coefficient and rms wavefront error vs. depth measured using
crimson microspheres in polyacrylamide gel
Wavefront aberration (top) and calculated far-field intensity (bottom) measured from Crimson fluorescent microspheres with
penetration depth increasing left to right.
Summary and Future Work
Direct wavefront sensing and closed loop AO has significant potential to improve
the performance of fluorescence microscopes, particularly when imaging deep
inside biological specimens.
We have developed a stage scanning fluorescence microscope (capable of
confocal, epifluorescence and widefield imaging modes) equipped with a closed
loop adaptive optic system.
Initial results and testing indicate that we are able to perform accurate wavefront
measurements and use the AO to correct some imaging aberrations.
Future work:
Further optimsation of AO feedback setup and system calibration
Add a 488 nm laser for fluorescence imaging (convenient for many fluorescent labels
e.g. GFP)
Imaging of model biological systems seeded with fluorescent microspheres
Investigation of the use of natural guidestars.
Title of Presentation
Name of Speaker
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measurement science & technology through these organisations
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