| Cover Story | May 2006
optics helps improve ophthalmic and microscopy applications.
By Paul Bierden and
Steven Menn, Boston Micromachines Corp.
Biological imaging instruments
often have resolution limitations that restrict
the ability of researchers and clinicians to detect
critical detail, One reason is that, as light
passes through tissue to reach the object of interest
a cell, the retina or a tumor the
tissue induces wavefront aberrations in the light.
Adaptive optics can actively
correct these aberrations in the optical path
between the camera and the object being imaged.
The increased resolution allows vital information
to be extracted from biological specimens. Introduced
in the 1950s as a way to correct for air-induced
turbulence in ground-based astronomy, adaptive
optics has seen a dramatic increase in use over
the past 10 years across many fields, including
The technique is being
tested in various biological applications and
could be useful for even more. For example, retinal
imaging is currently limited in resolution and
contrast by the imperfections in the cornea and
crystalline lens, as well as by the viscous and
nonuniform nature of the vitreous humor in the
eye. Thus, clinicians are unable to view the important
In vivo cellular-level
imaging would enable early and accurate diagnosis
of diseases of the eye. And, in general biological
microscopy, aberrations induced by imaging through
thick tissue produce optical distortion that results
in reduced signal levels and degraded resolution
(Figure 1), but adaptive optics can provide a means
to create high-resolution images through a thick
All adaptive optics systems
are composed of a wavefront sensor that measures
the phase aberration in the optical wavefront,
a deformable mirror that adjusts its position
to correct for the aberration, and a control system
that takes measurements from the sensor and calculates
the required movement of the deformable mirror
( Figure 2).
The most prevalent wavefront
sensor with adaptive optics in biological imaging
systems is the Shack-Hartmann model, which breaks
the incoming wavefront into a number of small
pieces using an array of miniature lenses called
lenslets. The light from each lenslet is focused
onto a CCD camera. As the portion of the wave-
front hitting the lenslet is aberrated, the focused
spot on the CCD camera moves. The amount of aberration
in that area of the wavefront can be calculated
with simple geometry that uses the focal length
of the lenslet and the translation of the focused
spot (Figure 3).
Other techniques for wavefront
sensing, such as curvature sensors, pyramid sensors
and model free hill climbing. also
are used in bioimaging. The deformable minor is
the adaptive element of the adaptive optics system
(Figure 4). Based on information provided by the
controller, it changes its shape to coned for
the aberration in the wavefront, thus cleaning
up the image. It is essentially a thin, flexible
minor with control points behind it that adjust
shape and position.
There are several methods
for adjusting the mirrors shape. Macroscale
mirrors those with apertures between 70
and 150 mm use piezoelectric stacks to
deform the surface, while deformable mirrors based
on microelectromechanical systems (MEMS) use electrostatic
actuators to control movement In membrane-based
types, a localized electrostatic field acts directly
on the minor.
The number of control points,
or actuators, ranges from 19 for a simple membrane
mirror to more than 4000. The number of control
elements required is determined by the aperture
size of the imaging system and the complexity
of the aberration for correction. In most biological
imaging applications, 100 to 200 points are sufficient.
For many years, the only
deformable mirrors available were large-aperture
types that were designed and built for defense
and astronomy applications. Although they were
sufficient for these purposes, they had limitations
when applied to biological imaging. Their large
sizes on the order of 100 mm in diameter
complicated the optical system intended
for imaging a field of view only 5 mm in diameter.
Additionally, their cost was prohibitive for a
commercial imaging system. Despite these limitations,
most early work in adaptive biological imaging
was done with a large mirror.
In the past five to 10
years, there has been a great deal of effort to
reduce the size and cost of the deformable minor.
The ability to use semiconductor fabrication techniques,
which lend themselves to small sizes and low costs
with the economies of scale, makes MEMS the most
promising technology. Matching the performance
of their large-scale counterparts, MEMS mirrors
have met the pricing and size needs of biological
imaging systems and are being implemented around
The controller that brings
together the sensor and deformable mirror is simple.
It uses signals from the wavefront sensor to calculate
the error from the desired wavefront (usually
planar) and sends a signal to each of the actuators
in the deformable mirror to compensate for that
error. The calculation must be performed repeatedly
and faster than the changing wavefront. A standard
computer can control the adaptive optics system
because of the low actuator count (100 s) and
speed requirements (100 Hz) for biological imaging
applications (as compared with atmospheric correction
where 1000 channels and 1 kHz are required).
These components have been
integrated into a number of imaging instruments,
including scanning laser ophthalmoscopes, confocal
microscopes, two-photon microscopes, optical coherence
tomography (OCT) systems and scanning optical
According to the National Eye Institute, more
than 3 million Americans are blind or visually
impaired many suffering from one of the
three most common retinal diseases: glaucoma,
macular degeneration and diabetic retinopathy.
With growing rates of diabetes and an aging population,
vision scientists are actively pursuing techniques
such as scanning laser ophthalmoscopy and OCF
to improve retinal imaging and to advance the
understanding, diagnosis and curability of eye
Adaptive optics has enabled high- resolution imaging
in both retinal imaging and biological microscopy.
A long- standing goal in vision science had been
cellular-level imaging of a living human retina.
In 1999, researchers led by Austin Roorda and
David R. Williams demonstrated high-resolution
adaptive optics scanning laser ophthalmoscope
images of the retina that resolved individual
According to Williams, adaptive optics has provided
a two- to threefold improvement in resolution
for retinal imaging. This could be especially
useful because a standard perimetry test administered
by an optometrist wont detect a disease
until it affects the patients vision. However,
examination of the eyes receptors with an
adaptive optics imaging system could reveal such
a disease. If integrated into clinical instruments,
the systems could enable earlier diagnosis, improved
monitoring of therapy and a better understanding
Dr. Stephen A. Bums, a professor of optometry
at Indiana University in Bloomington, uses an
adaptive optics scanning laser ophthalmoscope
to compare photoreceptor health to changes in
retinal scattering and polarization properties
( Figure 5). The enhanced resolution has enabled high-speed
imaging to see blood cells moving in capillaries.
This sort of vital detail could aid in the early
detection of diabetic retinopathy.
Adaptive optics also can help shorten imaging
time in ophthalmic imaging. There are safety restrictions
concerning the intensity of light that can be
put in the eye, and compensating for this requires
imaging over a longer time period. Poor optical
efficiency caused by aberrations means that many
of those photons are lost, but the optics can
help more of that light make it back to the detector,
speeding the imaging time.
A similar issue exists in microscopy. Low optical
efficiencies demand an increase in illumination
power required for imaging, but this high power
gradually kills or harms the specimen. An increase
in optical efficiency permits the use of lower
illumination powers, extending the time over which
specimens can be imaged.
Martin J. Booth of the department of engineering
science at Oxford University in the UK is interested
in the application of adaptive optics to high-resolution
three- dimensional optical microscopy for biological
imaging. He is particularly interested in aberration
correction for confocal and multiphoton fluorescence
microscopy (Figure 6). Booth has shown that adaptive optics
restores fluorescence signal levels at up to a
tenfold increase, permitting the use of lower
illumination power and increasing specimen viability.
It can clearly be seen that it increases the brightness
of the fluorescence.
Adaptive correction of aberrations also can enable
imaging deep into specimens, i.e., imaging of
living tissue, while maintaining high resolution
and signal levels. Booth noted that high-resolution
optical microscopy is limited to thin specimen
sections or individual cells on microscope slides.
In many cases, it is biologically much more
interesting to look at cells and cellular processes
in their natural within living tissue,
At the Center for Automation Technology at Rensselaer
Polytechnic Institute in Troy, N.Y., researchers
led by Benjamin Potsaid have addressed the inherent
trade-off between resolution and field of view
in microscopes that use adaptive optics. The microscope
design, called the adaptive scanning optical microscope,
optically scans over a large field of view, imaging
features as small as 1.5 µm over areas as
large as 40 mm. However, aberrations introduced
by passing the light off-axis through the optical
path cause blurring toward the edge of the image.
By using adaptive optics, a deformable mirror
can correct for the position-dependent aberrations
to achieve diffraction-limited image quality over
the entire observable field.
The microscope allows researchers to observe the
effect of potential therapeutic chemicals on the
mitotic (cell division) process over a large population
of living cells in real time (Figure 7).
Although the field of adaptive optics has had
more than 50 years to mature, its application
to bioimaging is still relatively new. As with
any developing biomedical technology, challenges
exist in the transition from research to clinical
Wavefront sensors, originally designed for astronomy
and free-space communications, are not always
well-suited for microscopy and vision science,
so researchers have had to develop new methods
of wavefront sensing. Given the complexity of
these sensing systems, it may even be easier to
eliminate the sensor altogether, creating a wavefront
sensorless adaptive optics method. Based
on the principle that maximum light is measured
when the wavefront is optimized, a deformable
mirror controls the wavefront through sequential
application of various aberrations.
Although there have been many breakthroughs in
deformable mirror performance in recent years,
applications continue to push development. Bums
pointed out that aging eyes have more aberration
at high spatial frequencies. This requires more
stroke at higher-order corrections, which have
proved fundamentally difficult for membrane deformable
A number of groups are working to increase the
stroke of their mirrors to meet the needs of the
general population. For example, later this year,
Boston Micromachines Corp. will release a high-stroke
deformable mirror system that performs 12 pm of
wavefront correction. An additional configuration
implementing a novel optical design will increase
this to 24 µm, which will enable retinal
imaging in more than 95 percent of the population.
Meet the authors
Paul Bierden is president and CEO of Boston Micromachines
Corp. in Watertown, Mass.
Steven Menn is director of product marketing at
Boston Micromachines Corp.