Advances in lasers, optics, and imaging for the life sciences

May/June 2010

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O P H T H A L M O L O G Y / M E D I C A L I M A G I N G

The organ we most depend on for pro-viding input about our environment—

the eye— is helpful for the diag-nosis of diseases such as diabe-tes. Also, because the retina is part of the brain, its assess-ment enables identification of neurological effects—and thus the eye warrants significant research attention.

A number of technology advances are fueling progress in retinal imaging—both for research and clinical application.

In vivo researchMice have played a key role for research into biological functions such as vision, and are especially attractive for genetic research because they have a particularly “plastic” genome. They can be genetically engineered to have various diseases that mimic human dystrophies such as diabe-tes and Alzheimer’s disease.

“A barrier to effective animal research has been the difficulty of imaging the retina in these tiny mammals,” says Bert Massie of Phoenix Research Laboratories (Pleasanton, CA; www.phoenixreslabs.com), explaining that the mouse eye, at 3 mm in diameter, is a fraction of the size of the 25-mm human eye. “Attempts to use cameras designed for the human

eye have produced limited results and at great expense,” he notes.

Addressing the great need for reti-nal imaging specifically in small animal research, Phoenix Research Labs provides instrumentation that incorporates multi-ple imaging modalities important for eye research. These include white light (also called brightfield) imaging; angiography, in which injected fluorescein enables reti-nal blood flow study; and imaging of flu-orescent probes such as green fluores-cent protein (GFP) where the challenge is to observe a low level of fluorescent return. When Phoenix Research Lab-oratories developed its Micron III reti-nal imaging microscope specifically for laboratory rodents, a key design objec-tive was to deliver these imaging modal-

ities—along with a wide field of view; resolutions below 5 μm; and easy, rapid imaging (a mouse or rat can be imaged by a solo technician in less than a minute). (See Fig. 1.)

Key to achieving these design objec-tives is the system’s CCD camera. Massie explains that a stan-dard, single-chip CCD camera could

not deliver the performance needed because it uses a mask over the pixels with an array of filters to capture a color image. Since most of the spatial infor-mation is in the green, the pattern of fil-ters provides twice the number of green samples as red or blue, and the software interpolates the colors where measure-ments are not made. By contrast, a three-chip CCD uses a prism to separate the colors, each of which is then sent to one of three CCDs. This allows equal spatial resolutions for each color, for good color images and for fluorescent studies at arbi-trary wavelengths. The Micron III uses Toshiba Imaging’s (Irvine, CA; cameras.toshiba.com) 56 × 44 × 44 mm IK-TF7 three-chip CCD camera to provide high resolution (XGA) and a full pixel-inde-

Retinal imaging advances research, disease diagnosisRecent instrumentation progress is enabling superior imaging of the retina in both laboratory animals and humans. The effect is better research for neurological disorders, for instance, as well as disease diagnosis.

FIGURE 1. Phoenix Research Laboratories designed its Micron III retinal imaging microscope for easy operation by a single technician.

B y Barbara G . Goode

O P H T H A L M O L O G Y / M E D I C A L I M A G I N G

pendent readout of 30 frames per second and 1000 to 1 dynamic range. Important to live animal retinal imaging tasks, three 1/3-in. progressive scan CCDs eliminate image jitter.

Another key to improved research is the ability to perform in vivo imaging of the mice. Traditionally, researchers must breed large colonies of animals, sacri-fice a segment of the colony at each stage of disease progress, and then remove the retina and flat-mount it to a micro-scope slide for examination. The process is complicated, and precludes longitudi-nal studies. The Micron III addresses this need as well.

The results are providing new insights into the eye. A typical set of images dem-onstrates the system use in a variety of observational modalities, from bright field to fluorescein angiography to GFP (see Fig. 2). New attachments are in devel-opment for imaging the anterior segment and for performing visual function tests.

Research to clinical Imagine Eyes (Orsay, France, www.imag-ine-eyes.com), developer of the com-pact adaptive optics retinal camera pro-totype described in the article, “Adap-tive optics approaches clinical ophthal-mology,” http://bit.ly/cC4gkp), says it expects to launch its second-generation

system this Fall. The system’s first

generation had a prototype interface and a 4 × 4° field of view at ±2–3 μm.  The goals of the sec-ond generation are a more ergonomic interface, a wider imaging field and better image con-trast. It is geared to enable clinical and academic users with minimal training to capture and stitch together compo-nent images to form large, wide-f ield views. “This second generation will be

a crucial step in launching a commercial camera as quickly as possible,” says Mark Zacharria, director of marketing commu-nications at Imagine Eyes.

Meanwhile, the company will pro-vide component technologies to basic research as well as systems to academic and industrial users.

Clinical functional imagingSpeaking at the BiOS Hot Topics ses-sion during Photonics West 2010, Profes-sor Amiram Grinvald, founder and CEO of Optical Imaging Ltd. (www.opt-imag-

ing.com) and a faculty member of the Weizmann Institute of Science (Rehovot, Israel), described the application of his company’s Retinal Function Imager (RFI), an FDA-approved hardware-and-software system providing noninvasive, ophthalmic functional imaging—in < 1 s to 10 min—to the resolution of single red blood cells moving through capillaries. 1 The system is partially based on a technique described in Grinvald’s 1986 paper in Nature for functional imaging of the brain based on intrinsic signals. 2 Alternative technolo-gies, such as PET and f-MRI, still provide only 1–10% of the resolving power of Grin-vald’s approach in both the temporal and the spatial domains, he says.

RFI enables direct visualization of ret-inal blood dynamics without the injec-tion of contrast agents, and clearly reveals the motion of individual red blood cells and blood cell clusters, thus enabling quantitative detection of abnormal blood flow velocity in capillar-ies, arterioles, and venules. This opens the door to many new diagnostic pos-sibilities—for instance, a significant velocity decrease in arteries and veins may indicate non-proliferative diabetic retinopathy. And increased blood flow velocity in the retinal arteries and veins can be an early indicator of diabetes mellitus in patients without any sign of diabetic retinopathy.

The basic RFI 3000 offers both blood flow velocity mapping and capillary per-fusion mapping (CPM), which enables analysis of a series of images to reveal motion and microvasculature detail—often in greater detail than the gold stan-dard for clinical retinal imaging: fluores-cein angiography (FA).

The higher-end RFI 3006 adds a mul-tispectral imaging and analysis module, for instance, to provide insight into oxy-gen use and other functions. Based on a fast-switching filter wheel, it overcomes issues such as poor signal-to-noise ratio that have typically hampered such anal-yses. The module enables assessment of the oximetric state of the retina and visualization of choroidal vessels. This latter option provides ICG-like images (using the contrast agent IndoCyanine-Green) without the use of ICG.

FIGURE 3. Detected using qualitative oximetry with Optical Imaging’s Retinal Functioning Imager (RFI), perfusion deficits and abnormalities in a patient with sickle cell retinopathy appear as regions of color distinct from their surroundings. (Image courtesy Richard B. Rosen and Teerapat Jittpoonkuson, New York Eye and Ear Infirmary)

FIGURE 2. Multi-modal imaging enables generation of various depictions from a single instrument: a retinoblastoma, a juvenile eye tumor as it appears in a mouse (top left); an anterior defect in an albino rat (top right); a retinal defect in a mouse depicted by an angiogram (bottom left); and GFP expression in a rat (bottom right). (Images courtesy Phoenix Research Laboratories)

O P H T H A L M O L O G Y / M E D I C A L I M A G I N G

Retinal ref lectance changes in response to photic stimulation carry information about metabolic pro-cesses—which is the basis for the 3006’s metabolic functional imaging capabil-ity. RFI can image under near-IR light, outside the absorption range of the

photoreceptors, and thus can be used to optically monitor retinal activity in response to a well-defined visual stimu-lus. The metabolic state of retinal com-ponents is determined by comparing post and pre-stimulated images, reflect-ing changes in absorption outside the

absorption range of the photoreceptors or scattering. It has been found in ani-mal model experiments that the func-tional signal is very sensitive for the detection of induced glaucoma.

The system features a 12.3-mm square sensor with resolution of 1024 × 1024 pix-els that capture images at 60 Hz. Its light source is a stroboscopic xenon with eight flashes at 100-Hz maximum frequency and a 10-s inter-series recharging inter-val. Imaging optics include a Topcon TRC-50DX fundus camera with three-position variable field angle. Software modules enable capture, sophisticated analysis, storage and retrieval, image processing, and display of related imag-ery as well as relevant historical and clini-cal information. «References1. D. Izhacky et al., Jpn. J. Ophthalmol.,

53(4):345–51, July 2009.2. A. Grinvald et al., Nature, 1986, 324:

361–364.

FIGURE 4. Capillary perfusion mapping (CPM; left) enables better visibility of capillaries compared with the gold standard imaging method, fluorescein angiogram (FA; right). In addition, CPM is completely noninvasive, and as such it allows patient follow-up not feasible with FA after treatment with drugs or surgery. (Image courtesy Optical Imaging Ltd.)

PhoenixRESEARCH LABORATORIES, INC.231 Market Place, Suite 296San Ramon, CA 94583Ph. 925 485 1100Fax 925 485 1155www.phoenixreslabs.com

Bert Massie, Ph.D.bertmassie@phoenixreslabs.com