ADAPTIVE OPTICS: Fluorescent microspheres enable adaptive-optics microscopy

Adaptive optics has tantalized researchers looking for ways to image through thick biological tissue–but the approach is not straightforward.

Feb 1st, 2009
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Adaptive optics has tantalized researchers looking for ways to image through thick biological tissue–but the approach is not straightforward. Now a group of researchers in California has developed a technique for directly measuring the inherent aberrations.

Barbara G. Goode

Biological microscopy of unfixed samples typically cannot capture high-quality live images that are more than 30 µm beneath the plasma membrane because of changes in the refractive index imposed by tissue composition.1-4 However, many important processes (such as stem-cell division, neurogenesis, and the key developmental events following fertilization) occur in deep tissue, so overcoming this limitation would give a huge boost to biological and biomedical investigations.

The idea of using adaptive optics (AO) to solve this problem is compelling, as AO has been applied successfully both in astronomy and in studies of the human eye.5-8 Instead of directly measuring the wavefront, though, most adaptive-optics microscopes have sought to correct the wavefront by using a hill-climbing algorithm to optimize a signal received at a photodetector.9 This approach has been popular because the alternative of adding a wavefront sensor complicates an optical system, and in biology there is no natural point-source reference such as the “guide star” used in astronomy (see What’s more, AO systems have tended to be specific to particular microscopes; no universal method for measuring the wavefront (or the results of the correction algorithm) has been available.

FIGURE 1. In the microscope with a Shack-Hartmann wavefront sensor, the 45° beamsplitter1 (Semrock, Rochester, NY) allows the laser light to be focused onto the sample, while the 90/10 beamsplitter BS2 allows the science camera and the wavefront sensor to see the fluorescent microsphere simultaneously. Lenses L1 and L2 are 65-mm-focal-length lenses that image the aperture of the objective onto the Shack-Hartmann sensor; P1 and P2 are conjugate planes. The field-stop between L1 and L2 blocks the light coming from other parts of the field of view, allowing only the light from the bead to pass. Note, the field-stop could be moved farther down the system because it is needed only by the wavefront sensor. The large distance between L1 and the aperture (P1) allows for the excitation laser (HeNe, λ = 632 nm) to be placed in this area. A source filter was added after L2 to reduce the effect of scattered light by the embryo and allow the wavefront sensor to see only the emission light. L3 demagnifies the pupil by a factor of two so that it can fit into the cooled camera (Roper Scientific, Acton, NJ).
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A team of scientists from the University of California-Santa Cruz, University of California-San Francisco, and Lawrence Livermore National Laboratory (Livermore, CA) noted that Martin Booth of the University of Oxford had described some of the difficulties associated with using a Shack-Hartmann wavefront sensor in AO microscopy. The scientists decided that most of the difficulties could be overcome with a suitable fluorescent point source, and they have proposed one. Using a Shack-Hartmann wavefront sensor and light emitted from an embedded fluorescent microsphere, they have measured wavefront aberrations induced by a drosophila embryo (see Fig. 1).

Reference source challenges

An important part of accurately measuring the wavefront is the reference source. In astronomical AO, a laser creates an artificial guide star of sufficient brightness in the mesospheric sodium layer, 90 km above sea level. While this approach requires powerful and expensive lasers, it enables the AO system to correct over a much larger portion of the sky than does the use of “natural guide stars.” The California team mimics this approach by using a crimson fluorescent microsphere 1 µm in diameter (from Invitrogen, Carlsbad, CA) as a guide/reference in its AO microscope setup (see Fig. 2).10

One of the challenges in designing a Shack-Hartmann wavefront sensor is imposed by the amount of light the reference source can provide. Fluorescent microspheres are made out of fluorescent dye and the light emitted is proportional to the radius cubed, thus smaller beads provide less light. The size of the beads should be smaller than the diffraction limit of one subaperture of the wavefront sensor. Note that this is larger than the diffraction limit of the microscope aperture by the ratio D(size of the aperture)/dLA. Because the diffraction limit of a microscope is inversely proportional to the numerical aperture (NA), smaller beads are needed for higher-numerical-aperture systems. Fortunately, the light gathered by the objective also increases with increasing NA (light gathering power of approximately NA2). Thus, increasing the wavefront sampling by a factor of four increases the size of the microsphere radius by a factor of two, and the amount of light emitted by a factor of eight. The only way to determine whether a microsphere, or any fluorescent source, will work is to image it into a Shack-Hartmann wavefront sensor using the objective. To increase the speed of the AO loop the bead size should be maximized, the researchers report.

FIGURE 2. Optical general absorption and emission curves are shown for the crimson bead (polystyrene microsphere) and excitation source at approximately 633 nm, and the edge of the source filter at 641 nm (90% pass for wavelengths greater then 641 nm).
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An emerging field in adaptive optics is tomography AO, which involves multiple light sources together with multiple wavefront sensors. The information from each sensor is processed to produce a tomographic image of the index-of-refraction changes in the optical path. One of the advantages of tomography AO is that it can provide information on the depth dependence of index-of-refraction variations in the tissue, thus allowing for the AO system to correct for the wavefront aberrations only in the optical path. This technology can also extend the isoplanatic angle by correcting wavefront aberrations that are common to a larger field of view. By depositing multiple fluorescent beads into the biological sample and using multiple wavefront sensors, the researchers discovered that it is possible to use tomographic techniques developed for astronomical AO.

FIGURE 3. The beads were imaged at different depths below the embryo surface, down to 70 µm. (Courtesy of Jian Cao, Justin Crest, Bill Sullivan).
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Measuring within an embryo

The researchers took wavefront measurements after seeding an embryo, using a hypodermic needle, with a fluorescent microsphere that served as a “guide star.” The science camera observes fluorescence in the range 641 nm and greater; the sample was illuminated by a 633 nm laser. The maximum wavefront error for a 40× objective was 1.9 µm, and 0.3 µm for the peak-to-valley and root-mean-square, respectively. The measurements also showed that the isoplanatic half-width was approximately 19 µm, resulting in a field of view of 38 µm in total.

These measurements, the researchers say, show that adaptive-optics technology is capable of improving the Strehl ratio of modern biological microscopes as much as 15 times.


This article is based on the paper “Wavefront aberration measurements through thick tissue using fluorescent microsphere reference beacons” by Oscar Azucena and Joel Kubby, Jack Baskin School of Engineering, Univ. of California, Santa Cruz; Justin Crest, Jian Cao, and William Sullivan, Molecular, Cell, and Developmental Biology, Univ. of California, Santa Cruz; Peter Kner, Department of Biochemistry and Biophysics, University of California, San Francisco; Don Gavel and Daren Dillon, Laboratory for Adaptive Optics, University of California, Santa Cruz; and Scot Olivier, Physics and Advanced Technologies, Lawrence Livermore National Laboratory. The researchers credit Steve Lane, John Sedat, and Sebastian Wachsmann-Hogiu from the NSF Center for Biophotonics Science & Technology (CBST) for support on the project. Oscar Azucena is the paper’s corresponding author; contact him at


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