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Medical Biophysics

Robert F. Bonner, PhD, Head, Section on Medical Biophysics

To characterize or modify early stressors that drive chronic diseases, we develop new optical technologies and use them to develop effective disease-prevention strategies. One focus of this work is to adapt our prior inventions of laser capture microdissection (LCM) into a simpler system, target-expression activated microdissection (TAM), which is more easily integrated with clinical pathology and multiplex molecular analysis of targeted cells and organelles extracted from complex tissue. Integration of TAM with existing approaches to clinical molecular pathology may be important in improving patient selection for molecularly targeted therapies.

Another focus is to use integrated analysis of multispectral, multimodal clinical retinal imaging to map distributions and dynamics of retinal photochemicals and relate them to cellular dysfunction and early disease progression. We seek to use these refined noninvasive, molecular imaging methods to test our hypothesis that spectral shifts in retinal irradiance during aging induce imbalances among retinal photochemical pathways. We posit that chronic photochemical imbalances drive early age-related and Stargardt’s maculopathies, which could be mitigated or prevented by appropriate external filters (e.g., spectral sunglasses). Our noninvasive molecular mapping methods may improve characterization of early retinal disease states, including more readily reversible “preclinical” disease, and could also be used to measure the effects of benign, low-cost prevention strategies.

Laser microdissection and molecular diagnostics technology development

Integrative molecular biology requires an understanding of interactions of large numbers of pathways. Similarly, molecular medicine increasingly relies on complex macromolecular diagnostics to guide therapeutic choices. A fundamental argument for LCM of tissues is that, without separating specific cell populations from complex tissues, we would miss critical control functions of thousands of regulated transcription factors, cell regulators, and receptors that are expressed at low copy number. For example, the hallmark of cancer in any individual is a number of critical genomic alterations that drive uncontrolled growth, metastases, and tumor cell polyclonality. New cancer therapies designed for specific molecular targets require documentation of the alterations in many oncogenes within a patient’s tissues prior to treatment decisions.

In global proteomic, lipid, and other studies without robust molecular amplification methods, the quantity of isolated cells sufficient to perform accurate characterization of less abundant species in typical LCM samples is problematic. Furthermore, the low throughput, high cost, and complexity of microscopic visualization, targeting, and isolation in LCM has also limited its incorporation into routine clinical diagnostics. To address these limitations, in collaboration with NCI and CIT, we invented (US Patents #7,709,047 and #7,695,752) target-activated microdissection (TAM), in which absorptive histochemical stains determine which tissue components are bound to a flexible overlying thermoplastic film (i.e., specifically captured on the film). When using stains specific for biomarker molecules (e.g., immunohistochemistry), the TAM variant called target-expression activated microdissection (or xMD) permits rapid, automatic microtransfer of specifically labeled cells or organelles (nuclei) from formalin-fixed, paraffin-embedded (FFPE) animal and clinical tissue sections. Recently, we designed, optimized, and replicated a simple, low-cost flashlamp system for instantaneous microbonding to all stained targets within a large sample area. The new, low-cost instrument design builds on our understanding of the physics and dosimetry of LCM to achieve ultra-precise capture using a thin thermoplastic polymer coating on a flexible PET tape held in thermal contact with the surface of a stained FFPE tissue section within a vacuum slide holder. The combination of sub-millisecond flashlamp pulses and the properties of the thermoplastic layer in contact with specifically stained targets ensures high lateral precision capable of isolation of subcellular organelles, such as specifically stained nuclei from the surrounding cell cytoplasm. We also optimized system parameters in order to reliably capture unstained nuclei within those cells immunostained for specific cell membrane receptors.

Our new target-expression activated microdissection is designed to rapidly and specifically isolate specific target-cell populations based on existing immunostains for biomarkers. For example, the overexpression of an epidermal growth factor receptor (EGFR) in carcinomas is routinely detected by transmission microscopy of a tissue section labeled with an absorptive EGFR immunostain. Our new flashlamp TAM (fTAM) automatically and specifically bonds these immunostain-positive cells to a thermoplastic transfer film. Replicating the methods that we developed previously for LCM, the DNA, RNA, and proteins extracted from the specifically captured cells can be purified and submitted to comprehensive downstream molecular analyses. Advances in Next-Generation sequencing methods allow comprehensive analysis of cancer-related genomic alterations and expressed RNA from TAM–isolated cells taken from surgical specimens immunostained for any given expressed biomarker. Using multiple biomarkers differentially expressed by different discrete clonal populations within a carcinoma should enable comprehensive analysis of each subpopulation. This analysis of differences among such cancer cell subpopulations in a given patient could permit improved selection of patient-specific combination molecular-therapeutic regimens.

More generally, our TAM method allows efficient segregation of specifically stained cell populations from within complex tissues and improved molecular characterization of the purified cell populations. When integrated with evolving whole genome, exome, and proteome methods, our TAM technology could become a critical tool for integrative understanding of tissue function and pathology. We currently focus on integration of our fTAM technology with current clinically approved molecular diagnostics and with animal models of human disease to routinely prepare highly purified tissue targets suitable for subsequent comprehensive molecular analyses. The NIH Office of Technology Transfer is currently in discussions with biotechnology firms seeking to license the NIH TAM patents.

Prevention of progression of age-related macular degeneration through photoprotection

Age-related macular degeneration (AMD) is the most common cause of blindness in the aged U.S. population. Stargardt's dystrophy is a genetically linked retinal disease that causes severe visual loss at much younger ages in a manner akin to rapid geographic atrophy in AMD. Local autofluorescence changes in AMD and Stargardt’s dystrophy suggest that increased levels of retinal fluorescent bisretinoids (specifically A2E) within the retinal pigment epithelium (RPE) cause RPE dysfunction and drive early disease progression. Our biophysical model assumes that bisretinoid production in the retina principally occurs in the rod outer segments during bright daylight exposures, in which high levels of all-trans-retinal are created whenever rhodopsin bleaching rates are high. It also assumes that the RPE, without a suitable enzyme to degrade A2E, uses lipofuscin photochemistry to chemically alter and detoxify A2E segregated within lipofuscin granules. Our model predicts that the steady-state levels of A2E, the bisretinoid with greatest apparent cytotoxicity, are principally determined by the balance between green light that activates rhodopsin and shorter wavelength blue-violet light that photochemically drives bisretinoid detoxification via singlet oxygen reactions localized within the RPE lipofuscin. According to our hypothesis, normal changes in spectral transmission of the lens with age (i.e., lens yellowing) create a progressive photochemical imbalance that causes A2E levels to rise to levels that ultimately lead to progressive RPE dysfunction.

We believe that spectral photoprotection of retinal rod rhodopsin when in bright daylight (e.g., with green-light absorbing, vermilion sunglasses) should significantly reduce bisretinoid production and the accumulated RPE and photoreceptor stress. We designed spectral sunglasses that reduce rhodopsin activation 30-fold without significant reduction in photopic sensitivity or color perception.

Recently, visual cycle inhibitors that slow rhodopsin turnover up to 10-fold in both day and night have begun clinical trials. However, such inhibitors have obvious side effects (e.g., slowed dark adaptation and reduced rod sensitivity). These and other adverse effects of long-term reductions in the visual cycle are avoided with spectral sunglasses, which are used only in daylight when rhodopsin turnover rates are high, and thus reduce bisretinoid production without affecting night (scotopic) vision.

An alternative photochemical hypothesis is that violet light photo-chemistries within the retina in the presence of higher levels of retinaldehydes and accumulated bisretinoids induce chronic photo-oxidative stress from longer-lived reactive oxygen species. Such violet-blue light–driven photochemistries can be greatly reduced by yellow sunglass filters. To test these two hypotheses, we designed bicolored sunglasses in which one eye is provided with a vermilion (green-blocking) filter that specifically protects rhodopsin and the other eye with a yellow (blue-violet–blocking) filter that specifically protects against short-wavelength photochemical injury. Such sunglasses allow us to compare short-term photochemical changes between eyes in which only spectral irradiance is changed while genetics, physiology, and environmental exposures otherwise remain constant.

In clinical studies, we are seeking to test predictions of our biophysical model and disease hypothesis, i.e., that spectral imbalances in retinal illuminance raise levels of toxic photochemicals in the RPE, which lead to progressive dysfunction including rod rhodopsin loss in early stages of AMD and Stargardt’s macular dystrophy. We have been exploring several retinal pathologies in which bisretinoid photochemistries appear to drive progression and for which our photoprotection sunglasses might demonstrate a clear clinical effect on RPE autofluorescence and subsequently the rate of early lesion progression.

We have been developing new noninvasive optical imaging and analysis techniques to measure and correlate local rod rhodopsin levels and bisretinoid autofluorescence in patients with these retinal diseases. One such development is a new, noninvasive, autofluorescence imaging method to map, with about 30 micron resolution, rhodopsin densities in patients. The confocal scanning laser ophthalmoscope (cSLO) at 488nm or 515nm permits continuous autofluorescence imaging at low-light doses that only bleach about 1% of the rod rhodopsin per image. After 25–40 sec of imaging, the rhodopsin bleaching achieves steady state in which more than 95% of the rhodopsin is “bleached” (activated rhodopsin forms in which its absorption peak at 507nm is lost). The normal optical density of the photoreceptor layer is about 0.3 at 488–515nm in a dim light–adapted subject (i.e., rhodopsin more than 99% unbleached) and consequently reduces the laser light reaching the fluorescent bisretinoids in the RPE by about 50%. By registering and determining the fractional increase in detected autofluorescence for each pixel during cSLO image sequences from the initial unbleached to the steady-state highly bleached rhodopsin states, we can obtain a high-resolution map of rhodopsin density (pixel size of about 10 micron).

In preliminary tests of this method on NEI patients, we observed uniform local loss of rhodopsin within a central hypofluorescent lesion associated with photoreceptor outer segment disruption (detected by optical coherence tomography, OCT) in a young Stargardt’s patient. The measured reduction in bleachable rhodopsin extended into the lesion’s hyperfluorescent rim, even though the rim exhibited a normal photoreceptor organization in the OCT images. We applied these methods to several patients with reticular pseudo-drusen, a retinal pathology associated with a high risk of progression to the geographic atrophy form of age-related macular degeneration. In this pathology, accumulated RPE waste products appear to be ejected through their apical membranes into the extracellular space around photoreceptor outer segments, resulting in progressive rod outer segment loss (supraRPE drusen). In these patients, large reticular arrays of such lesions appear to expand circumferentially and radially in the macula outside the fovea within a couple of years and are associated with a reticular or grid-like pattern, whose centers exhibit increased blue-green light reflection (backscatter) and decreased RPE autofluorescence. Each of the 100–micron drusen with local extracellular debris is surrounded by more normal-appearing retina with intact outer segments and normal RPE autofluorescence. Our “dark-adapted” rhodopsin density maps within these lesions show greatly reduced rhodopsin within the central hyper-reflective, hypofluorescent reticular druse, with a radial gradient of rising rhodopsin and bisretinoid fluorescence levels approaching normal midway between drusen. Thus, the relationship of RPE autofluorescence and overlying dark-adapted rod rhodopsin levels can be resolved within retinal lesions. The identified NEI reticular drusen patients appear to be a particularly suitable population to test whether, by largely blocking bisretinoid formation, our rod-sparing vermilion sunglasses could alter the autofluorescence patterns, raise dark-adapted rhodopsin levels and rod sensitivity locally within the defined lesions, and slow the expansion of the fields of reticular drusen.

In collaboration with applied mathematicians at the University of Vienna and University of Maryland, we are developing new quantitative analysis tools for multimodal retinal image sets in order to identify and monitor earlier changes in macular pathology in AMD and a wider variety of retinal diseases. Such methods could improve statistical characterization of early disease states and may thus facilitate studies of early intervention for disease prevention, such as the effects of spectral photoprotection.

United States Patents

Emmert-Buck MR, Tangrea MA, Bonner RF, Chuaqui R, Pohida TJ. Target activated microtransfer. #7,709,047; May 4, 2010.
Bonner RF, Pohida TJ, Emmert-Buck MR, Tangrea MA, Chuaqui R. Target activated microtransfer. #7,695,752; April 13, 2010.
Bonner RF, Goldstein SR, Smith PD, Pohida TJ. Method of laser capture microdissection from a sample utilizing short pulse. #6,897,038; May 24, 2005.
Liotta LA, Emmert-Buck M, Krizman DB, Chuaqui R, Linehan WM, Trent JM, Bonner RF, Goldstein SR, Smith PD, Peterson JI. Isolation of cellular material under microscopic visualization. #6,867,038; March 15, 2005.
Bonner RF, Goldstein SR, Smith PD, Pohida TJ. Non-contact laser capture microdissection. #6,743,601; June 1, 2004.
Goldstein SR, Bonner RF, Smith PD, Peterson J, Pohida TJ. Mechanical handling systems for laser capture microdissection. #6,720,191; April 13, 2004
Liotta LA, Buck MF, Weiss RA, Zhuang Z, Bonner RF. Isolation of cellular material under microscopic visualization. #6,569,639; May 27, 2003.
Bonner RF, Goldstein SR, Smith PD, Pohida TJ. Precision laser capture microdissection utilizing short pulse length. #6,420,132; July 16, 2002.
Bonner RF, Liotta L, Buck M, Krizman DB, Chuaqui R, Linehan WM, Trent JM, Goldstein SR, Smith PD, Peterson JI. Isolation of cellular material under microscopic visualization. #6,251,516; June 26, 2001.

Publications

Ehler M, Rajapakse V, Zeeberg B, Brooks B, Brown J, Czaja W, Bonner RF. Analysis of temporal-spatial covariation within gene expression microarray data in an organogenesis model. Bioinformatics Res Applications. LNICS 2010;6051:38-49.
Ehler M, Rajapakse VN, Zeeberg BR, Brooks BP, Brown J, Czaja W, Bonner RF. Nonlinear gene cluster analysis with labeling for microarray gene expression data in organ development. BMC Proc 2011;5,Suppl 2:S3.
Hanson JC, Tangrea MA, Kim S, Armani MD, Pohida TJ, Bonner RF, Rodriguez-Canales J, Emmert-Buck MR. Expression microdissection adapted to commercial laser dissection instruments. Nat Protoc 2011;6:457-467.
Tangrea MA, Hanson JC, Bonner RF, Pohida TJ, Rodriguez-Canales J, Emmert-Buck MR. Immunoguided microdissection techniques. Methods Mol Biol 2011;755:57-66.
Zeeberg BR, Liu H, Kahn AB, Ehler M, Rajapakse VN, Bonner RF, Brown JD, Brooks BP, Larionov VL, Reinhold W, Weinstein JN, Pommier YG. RedundancyMiner: de-replication of redundant GO categories in microarray and proteomics analysis. BMC Bioinformatics 2011;12:52.

Collaborators

Brian Brooks, MD, PhD, Ophthalmic Genetics and Visual Function Branch, NEI, Bethesda, MD
Stephen Byers, PhD, Lombardi Cancer Center, Georgetown University, Washington, DC
Emily Chew, MD, Clinical Branch, NEI, Bethesda, MD
Catherine A. Cukras, MD, Clinical Branch, NEI, Bethesda, MD
Denise Cuningham, CRA, RBP, MEd, Clinical Branch, NEI, Bethesda, MD
Wojciech Czaja, PhD, Norbert Weiner Center, University of Maryland, College Park, MD
Martin Ehler, PhD, Universität Wien, Vienna, Austria
Philip G. McQueen, PhD, Mathematical and Statistical Computing Laboratory, CIT, Bethesda, MD
Sanford Meyers, MD, Retina Consultants, Des Plaines, IL
Nicole Y. Morgan, PhD, Laboratory of Bioengineering and Physical Science, NIBIB, Bethesda, MD
Erik Olson, PhD, MBA, Ventana Medical Systems, Tucson, AZ
Tom Pohida, MSE, Computational Biology and Electronics Laboratory, CIT, Bethesda, MD

Contact

For more information, email bonnerr@mail.nih.gov.

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