This paper will review the development of wide-field and confocal microscopes with wavefront sensing and adaptive optics for correcting refractive aberrations and compensating scattering when imaging through thick tissues (Drosophila embryos and mouse brain tissue). To make wavefront measurements in biological specimens we have modified the laser guide-star techniques used in astronomy for measuring wavefront aberrations that occur as star light passes through Earth’s turbulent atmosphere. Here sodium atoms in Earth’s mesosphere, at an altitude of 95 km, are excited to fluoresce at resonance by a high-power sodium laser. The fluorescent light creates a guide-star reference beacon at the top of the atmosphere that can be used for measuring wavefront aberrations that occur as the light passes through the atmosphere. We have developed a related approach for making wavefront measurements in biological specimens using cellular structures labeled with fluorescent proteins as laser guide-stars. An example is a fluorescently labeled centrosome in a fruit fly embryo or neurons and dendrites in mouse brains. Using adaptive optical microscopy we show that the Strehl ratio, the ratio of the peak intensity of an aberrated point source relative to the diffraction limited image, can be improved by an order of magnitude when imaging deeply into live dynamic specimens, enabling near diffraction limited deep tissue imaging.
The design of an Adaptive Optics (AO) Structured Illumination (SI) microscope is presented. Two key technologies are
combined to provide effective super-resolution at significant depths in tissue. AO is used to measure and compensate for
optical aberrations in both the system and the tissue by measuring the optical path differences in the wavefront.
Uncorrected, these aberrations significantly reduce imaging resolution, particularly as we view deeper into tissue. SI
allows us to reconstruct an image with resolution beyond the Rayleigh limit of the optics by aliasing high spatial
frequencies, outside the limit of the optics, to lower frequencies within the system pass band. The aliasing is
accomplished by spatially modulating the illumination at a frequency near the cutoff frequency of the system. These
aliased frequencies are superimposed on the lower spatial frequencies of the object in our image. Using multiple images
and an inverse algorithm, we separate the aliased and normal frequencies, restore them to their original frequency
positions, and recreate the original spectrum of the object. This allows us to recreate a super-resolution image of the
object. A problem arises with thick aberrating tissue. Tissue aberrations, including sphere, increase with depth into the
tissue and reduce the high spatial frequency response of a system. This degrades the ability of SI to reconstruct at superresolution
and limits its use to relatively shallow depths. However, adding AO to the system compensates for these
aberrations allowing SI to work at maximum efficiency even deep within aberrating tissue.
KEYWORDS: Wavefronts, Wavefront sensors, Adaptive optics, Two photon excitation microscopy, Microscopes, Two photon imaging, Fluorescent proteins, Green fluorescent protein, Objectives, Control systems
A fast direct wavefront sensing method for dynamic in-vivo adaptive optical two photon microscopy has demonstrated.
By using the direct wavefront sensing and open loop control, the system provides high-speed wavefront measurement
and correction. To measure the wavefront in the middle of a Drosophila embryo at early stages, autofluorescence from
endogenous fluorophores in the yolk were used as reference guide-stars. This method does not rely on
fluorescently labeled proteins as guide-stars, which can simplify the sample preparation for wavefront measurement. The
method was tested through live imaging of a Drosophila embryo. The aberration in the middle of the embryo was
measured directly for the first time. After correction, both contrast and signal intensity of the structure in the middle of
the embryo was improved.
Optical microscopy allows noninvasive imaging of biological tissues at a subcellular level. However, the optimal performance of the microscope is hard to achieve because of aberrations induced from tissues. The shallow penetration depth and degraded resolution provide a limited degree of information for biologists. In order to compensate for aberrations, adaptive optics with direct wavefront sensing, where guide-stars are used for wavefront measurement, has been applied in microscopy. The scattering effect limits the intensity of a guide-star and hence reduces the signal to noise ratio of the wavefront measurement. In this paper, we propose to use interferometric focusing of excitation light onto a guide-star embedded deeply in tissue to increase its fluorescence intensity, thus overcoming the signal loss caused by scattering. With interferometric focusing of light, we increase the signal to noise ratio of the laser guide-star through scattering tissue by more than two times as well as potentially extending the thickness of tissue that can be corrected using AO microscopy.
Optical aberrations due to the inhomogeneous refractive index of tissue degrade the resolution and brightness of images
in deep tissue imaging. We introduce a direct wavefront sensing method using cellular structures labeled with fluorescent
proteins in tissues as guide-stars. As a non-invasive and high-speed method, it generalizes the direct wavefront sensing
method for adaptive optics microscopy. An adaptive optics confocal microscope using this method is demonstrated for
imaging of mouse brain tissue. The confocal images with and without correction are collected. The results show
increased image contrast and 3X improvement in the signal intensity for fixed mouse tissues at a depth of 70 μm. The
images of the dendrite and spines are much clearer after correction with improved contrast. The Strehl ratio is improved
from 0.29 to 0.96, a significant 3.3X improvement.
Recently, there has been a growing interest in deep tissue imaging for the study of neurons. Unfortunately, because of the
inhomogeneous refractive index of the tissue, the aberrations degrade the resolution and brightness of the final image.
In this paper, we describe an adaptive optics confocal fluorescence microscope (AOCFM) which can correct aberrations
based on direct wavefront measurements using a point source reference beacon and a Shack-Hartmann Wavefront Sensor
(SHWS). Mouse brain tissues with different thicknesses are tested. After correction, both the signal intensity and contrast
of the image are improved.
We demonstrated the used of an adaptive optic system in biological imaging to improve the imaging characteristics of a
wide field microscope. A crimson red fluorescent bead emitting light at 650 nm was used together with a Shack-Hartmann wavefront sensor and deformable mirror to compensate for the aberrations introduce by a Drosophila embryo.
The measurement and correction at one wavelength improves the resolving power at a different wavelength, enabling the
structure of the sample to be resolved (510 nm). The use of the crimson beads allow for less photobleaching to be done
to the science object of the embryo, in this case our GFP model (green fluorescent beads), and allows for the science
object and wavefront reference to be spectrally separated. The spectral separation allows for single points sources to be
used for wavefront measurements, which is a necessary condition for the Shack-Hartmann Wavefront sensor operation.
Adaptive optics (AO) improves the quality of astronomical imaging systems by using real time measurement of the
turbulent medium in the optical path using a guide star (natural or artificial) as a point source reference beacon [1]. AO
has also been applied to vision science to improve the view of the human eye. This paper will address our current
research focused on the improvement of fluorescent microscopy for biological imaging utilizing current AO technology.
A Shack-Hartmann wavefront sensor (SHWS) is used to measure the aberration introduced by a Drosophila
Melanogaster embryo with an implanted 1 micron fluorescent bead that serves as a point source reference beacon.
Previous measurements of the wavefront aberrations have found an average peak-to-valley and root-mean-square (RMS)
wavefront error of 0.77 micrometers and 0.15 micrometers, respectively. Measurements of the Zernike coefficients
indicated that the correction of the first 14 Zernike coefficients is sufficient to correct the aberrations we measured. Here
we show that a MEMS deformable mirror with 3.5 microns of stroke and 140 actuators is sufficient to correct these
aberrations. The design, assembly and initial results for the use of a MEMS deformable mirror, SHWS and implanted
fluorescent reference beacon for wavefront correction are discussed.
Adaptive optics (AO) improves the quality of astronomical imaging systems by using real time measurement of the
turbulent medium in the optical path using a guide star (natural or artificial) as a point source reference beacon. AO has
also been applied to vision science to improve the current view of the human eye. This paper will address our current
research focused on the improvement of fluorescent microscopy for biological imaging utilizing current AO technology.
A Shack-Hartmann wavefront sensor (SHWS) was used to measure the aberration introduced by a Drosophila Melanogaster embryo with an implanted 1 micron fluorescent bead that serves as a point source reference beacon. The measurements show an average peak-to-valley and root-mean-square (RMS) wavefront error of 0.77 micrometers and
0.15 micrometers, respectively. The Zernike coefficients have been measured for these aberrations which indicate that
the correction of the first 14 Zernike coefficients should be sufficient to correct the aberrations we have obtained. These
results support the utilization of SHWS for biological imaging applications and that a MEMS deformable mirror with 1 micron of stroke and 100 actuators will be sufficient to correct these aberrations. The design, assembly and initial results for the use of a MEMS deformable mirror, SHWS and implanted fluorescent reference beacon for wavefront correction will also be discussed.
Adaptive Optics (AO) improves the quality of astronomical imaging systems by using real time measurement of the
turbulent medium in the optical path. The measurements are then taken and applied to a deformable mirror (DM) that is
in the conjugate position of the aberrations in the optical path. The quality of the reconstructed wavefront directly affects
the images obtained. One of the limiting factors in current DM technology is the amount of stroke available to correct
the wavefront distortions which can be as high as 20 microns of optical path difference. We have developed a simulation
analysis using Galerkin's method to solve the nonlinear plate equation. The analysis uses a set of orthogonal equations
that satisfied the boundary condition to solve for the linear deformation on the mirror surface. This deformation is used
to iteratively converge to the final solution by applying the nonlinear plate equation and the nonlinear actuator forces.
This simulation was used to design a microelectromechanical DM with 10 μm of stroke.
We have designed a tip/tilt segmented mirror for the use in adaptive optics. The specific application that this device has
been designed for, tracking a laser pulse through the sodium layer of our atmosphere, is well within the capabilities of
this device. Through the use of finite element analysis simulations we have shown that the device has an operating
voltage in the 100-150 V range, full mechanical stroke of 2.8&mgr;m, and is capable of reaching its full stroke in under 60&mgr;s.
It also has shown good decoupling of the tip and tilt modes, allowing the device to track pulses that come in from any
direction. Testing of the device has shown that there is a maximum of 22 percent error between the simulation and the
testing results.
The most common detector configuration for Shack Hartmann (SH) wavefront sensors used for adaptive optics (AO)
wavefront sensing is the quad cell. Advances in detectors, such as the CCDs being developed in a project on which we
are collaborators (funded by the Adaptive Optics Development Program), make it possible to use larger pixel arrays.
The CCD designs incorporate improved read amplifiers and novel pixel geometries optimized for laser guide star (LGS)
AO wavefront sensing. While it is likely that finer sampling of the SH spot will improve the ability of the wavefront
sensor to accurately determine the spot displacement, particularly for elongated or aberrated spots such as those seen in
LGS AO systems, the optimal sampling is not dependent simply on the number of pixels but must also take into account
the effects of photon and detector noise. The performance of a SH wavefront sensor also depends on the performance
of the algorithm used to find the spot displacement. In the literature alternatives have been proposed to the common
center of mass algorithm, but these have not been simulated in detail. In this paper we will describe the results of our
study of the performance of a SH wavefront sensor with a well sampled spot. We will present results for simulations of
the wavefront sensor that enable us to optimize the design of the detector for varying conditions of signal to noise and
spot elongation. We will also discuss the application of correlation algorithms to SH wavefront sensors and present
results regarding the performance and statistics of this algorithm.
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