A new interferometric microscopy technique processes
discarded data to produce crisp 3-D images.
Diagnosis of many of the world’s leading cancers and other diseases depends on rapid and reliable detection methods. A combination of traditional methodologies and the latest technologies have steadily increased efficiency, but old-fashioned techniques, such as invasive biopsies, retain a dominant foothold in the clinic.
Interferometric synthetic aperture microscopy (ISAM) could help phase out the need for time-consuming and invasive tissue sampling. Recently developed by a multi-disciplined team of scientists at the Univ. of Illinois at Urbana-Champaign, ISAM also represents a physics-based solution of a major conundrum in optical microscopy: the inverse scattering problem. The solution accounts for the scattering, diffraction, and dispersion of light with coherence microscopy.
ISAM is also notable for being adaptable to optical systems in most laboratories.
Making sense of scattered light
Optical microscopy has significant limitations in medicine. In addition to reduced effectiveness in denser tissues, conventional approaches suffer from shallow depth-of-focus and out-of-focus blur for those areas imaged which lay outside of the focus. The root of the problem lies in reflected light, which quickly scatters in tissue.
Available out-of-focus data in optical microscopy has usually been discarded either by confocal gating or by discarding the data digitally. ISAM was developed to make use of the out-of-focus data to increase the signal and resolution outside of the focal plane.
Pictured here are representations of a tissue phantom where the scattering points would represent cells. At left, unprocessed data of the tissue phantom are volume rendered to view distortion caused by the focusing lens. At right, the ISAM reconstruction is volume-rendered to view the spatially invariant resolution. In both images the longitudinal axis is scaled by 0.25.
Images: Beckman Institute
The problem was solved by first building an accurate physical model for the signal acquired in optical microscopy. Next, the model was reduced to a mathematical formula relating the object to the collected light. Finally, the formula was applied to the digitally acquired signal to calculate the structure of the object.
The out-of-focus data have always been available to researchers, but ISAM utilizes this data to render volumes with spatially invariant resolution. The ever-present potential for spatially invariant resolution is the result of the angular diversity of the probing beam.
Stephen Boppart, professor of electrical and computer engineering, bioengineering, and medicine at the Univ. of Illinois at Urbana-Champaign, says ISAM’s mathematical solution was inspired by data interpretation in other fields, such as computed tomography (CT) and synthetic aperture radar (SAR).
The ISAM team, including Tyler Ralston, a radar specialist at Lincoln Laboratory, Massachusetts Institute of Technology, Cambridge, looked closely at military applications of SAR which uses radar imaging to reconstruct an image from otherwise unusable data. The procedure illuminates the target from multiple different angles and collects radar returns to form the scene. The image perception is not built from point-by-point collection of data. Rather, the image is built from the computed mass as a whole.
ISAM also incorporates optical coherence tomography (OCT), which is increasingly being used for soft tissue analysis in medical imaging.
“The key innovation of ISAM is the concept that the object properties (structure) can be calculated from the field measurements,” says Ralston. “This concept has long since existed in imaging practices such as x-ray CT computed tomography and magnetic resonance imaging (MRI), but few have attempted the computations for resolving standard coherence microscopy. Also, the algorithm itself is innovative because it is analytically derived to be as efficient as possible.”
A mathematical model for ISAM, based on a scalar model for light propagation, was developed from several considerations:
1) The propagation of the focused beam from the objective into the sample;
2) Scattering within the sample;
3) The propagation of the scattered light back into the objective; and
4) The measurement of the cross-correlation with the reference pulse.
During evaluations of the Fourier transforms for one, two and three dimensions, a diagonal linear integral operator was found to be implicit in the 3-D Fourier space of the scattering potential. Based on this finding ISAM resamples and interpolates collected data in a manner similar to x-ray CT or SAR. The main difference is that the resampling grid is elliptical rather than polar. This is attributed to propagation effects stemming from the ISAM procedure itself.
Building an ISAM setup
In an ISAM system, low-coherence light is focused into a sample and the back-scattered field is interferometrically measured with a spectral detector. Image: Beckman Institute
The optimal ISAM setup is one that facilitates uniform coverage in the Fourier space with a relatively simple illumination and detection scheme. Researchers started with a fiber-optic Michelson interferometer seeded by a femtosecond pulsed laser where the axial resolution is inversely dependent on the bandwidth. A spectral interferometer was used to measure the cross-correlation between a fixed-delay reference pulse and the pulses reflected back from the sample.
The spectrum measured on the line camera corresponded to the Fourier transform of the cross-correlation signal, from which the amplitude and phase of the reflected field from the sample are inferred. A microscope objective was used to fix the focus on a single plane in the sample, and galvanometer-driven mirrors were used to direct the scanning beam.
Early on, the team analyzed how OCT scans the beam by looking at a cross-sectional image with a single strip focus. Examining the overlap above and below the focus, they noted the beam waist became larger with signals coming back from numerous positions. In interferometric microscopy, a light beam focused onto a sample assumes an hourglass shape. The 3-D wavefront resembles a parachute as it approaches the focus, and looks more like a bowl away from the focus. By breaking the parachute into multiple rays, each ray points to the focus at different angles. As the beam sweeps across the plane, the interferometer captures the phase information about the light from all depths. The image is reconstructed from that data.
However, to collect the necessary data, the microscopy setup must track the phase of the wavefront propagated through the sample. Because ISAM is a multiplexed measurement method, the signal-to-noise ratio will decrease if the measurement is not phase stable. To achieve this, a coverslip was placed over the sample. The coverslip and the top reflection from the air-coverslip interface acted as a fixed reference delay relative to the object. The delay fluctuations of the interferometer were removed from each cross-correlation interferogram by locating the air-coverslip reflection in each interferogram, estimating the phase and group delay of the reflection, and applying the opposite phase and group delay to the entire interferogram.
The reference surface provides a way to calibrate the phase and can be adapted for other geometries, including catheter-based rotational setups.
As a test image, the team used ISAM with a 0.05-numerical aperture objective on a tissue phantom made of titanium dioxide scatterers with a mean diameter of 1 µm uniformly suspended in silicone. The system reconstructed a data set of a volume 360 µm x 360 µm (transverse) x 2,000 µm (axial). The data set contained three pairs of en face sections for both the unprocessed data and the ISAM reconstructions. The sections were relatively evenly spaced, by more than 500 µm, from the focus.
As expected, the interference in the unprocessed data between signals scattered from adjacent scatterers was easily seen. ISAM accounted for the diffraction of the beam and separated the superimposed signals from the scatterers to produce resolved point images on all planes. The system was also successfully tested on resected human breast tumor tissue.
The ISAM advantage
An interferometric microscope can be adapted to an ISAM system by introducing devices that can ensure phase stability and compute the collected data using the new algorithm. A high-numerical aperture objective and a way to perform 2-D scanning are also required.
However, Boppart and Ralston are confident the system will be attractive to a wide variety of applications because such a system can be built on existing interferometric systems in nearly any setting. Plus, the flexibility of the technique means it can be adapted to several geometries, including catheter-based rotational scopes.
Boppart says ISAM could profoundly affect analysis in the fields of cell and tumor biology. Biopsies are still the “gold standard,” he says, but in vivo imaging would be far preferable in many circumstances.
“In medicine we rely on histological sections to make our diagnosis. Any time we’re doing that we are significantly undersampling tissues,” he says. Even if 10 to 20 tissue sections are taken, he adds, the technique is hardly comprehensive.
Interferometric microscopy, on the other hand, is extremely accurate, and ISAM could deliver more than a thousand en face planes in 2 mm that could be computationally processed in real-time to form high-resolution 3-D images. Potential applications include developmental biology, neuronal imaging, tissue engineering, cellular scaffolds, in vivo organ examinations, biopsies, and ophthalmic imaging.
In some ways, says Ralston, ISAM is similar to ultrasound, except that it can achieve higher resolutions within tissues to see cellular structures.
“This particular project has been especially exciting because there is a great potential to impact a wide range of application-specific studies. Furthermore, it offers so much to the field of microscopy which will be useful for years to come,” says Ralston.
—Paul Livingstone
ISAM’s product line potential
Optical coherence tomography is developing rapidly, particularly
in the ophthalmology market, but also in cardiovascular and gastrointestinal imaging and oncology. Interferometric synthetic aperture miscroscopy (ISAM) could improve functionality in these applications. A few of the companies that could benefit include:
• Bioptigen Inc., Research Triangle Park, N.C., www.bioptigen.com
• Carl Zeiss MicroImaging, Thornwood, N.Y., www.zeiss.com/micro
• Imalux, Cleveland, Ohio, www.imalux.com
• LightLab Imaging, Westford, Mass., www.lightlabimaging.com
• Optovue Inc., Fremont, Calif., www.optovue.com
• Thorlabs, Newton, N.J., www.thorlabs.com
