George's Seminars

Recently, spectral imaging has been successfully applied to 24-color fluorescence in situ hybridization, and it has evolved into a new powerful method for the analysis of structural and numerical chromosomal aberrations in cancer cells. The identification of chromosomal aberrations directly in interphase cells in histological or morphological preparations is possible by means of interphase cytogenetics. For cytological specimen, however, it is often impossible to use fluorescence microscopy because of prior cytological staining or auto-fluorescence. Also, re-examination of archived fluorescent specimens is hampered by fading. The need for multi-parameter cytochemical analysis, however, remains when rare or unique material is analyzed. Multi-color bright-field microscopy using enzyme precipitates has become feasible for in situ hybridization as well as immunohistochemical detection. Conventional transmission microscopy optics have allowed up to three targets to be detected simultaneously. In parallel to spectral imaging of multi-color fluorescence, we explored the potential of spectral analysis of absorbed light to increase the number of parameters in bright-field microscopy. Spectral imaging of a triple-color in situ hybridization for chromosome centromeres using tetramethylbenzidine (TMB, green), new fuchsin (NF, red), and diaminobenzidine (DAB, brown) as reporter dyes results in good spectral separation of the individual dyes. Even the colors of small spots that could not be (easily) discerned by eye were identified. The absorption peaks are wide apart and the shapes of the curves deviate clearly to allow for a spectrum-based color classification of all spots. The use of nuclear counterstains hematoxylin (blue/purple), Methyl Green or Dif-Quick (red) in various intensities did not obscure the separation of the absorption spectra of the dyes, even though counterstains co-localize with the hybridization spots and mixed spectra are measured. From these data we expect that spectral imaging will allow for reliable color separation of cytological stains and genetic markers, and will facilitate phenotype/genotype correlation in cancer cells. 

Jonathan S. Wiest¹, Kenneth Conwell II¹, Rawia Yassin², Wilbur Franklin³, Susan Proudfoot⁵, Richard Levenson⁴, and Marshall Anderson¹. University of Cincinnati, ¹Department of Environmental Health, ²Department of Pathology, Cincinnati, OH., ³University of Colorado Health Sciences Center, Denver, CO., ⁴Center for Light Microscopy and Biotechnology, Carnegie Mellon University, Pittsburgh, PA., and ⁵Lung Check, Scottsdale, AZ.

Lung cancer is the leading cause of cancer death in the United States and Western Europe. The incidence of lung cancer is expected to increase in developing countries as their smoking habits increase. The prognosis is very poor for patients whose tumors can not be completely resected with almost 90% of the patients dying from the disease within two years of diagnosis. There are four major histological types of human lung cancer: adenocarcinoma (30%), squamous cell carcinoma (25%), large cell carcinoma (15%), and small cell carcinoma (25%), with uncommon types and combined types comprising the remaining 5%. The incidence of adenocarcinoma (AC) has been increasing and AC has become the most common lung tumor type in many parts of the world. The development of lung cancer, like most cancer, is a multistep process that results from the accumulation of genetic damage as proto-oncogenes are activated and tumor suppressor genes are inactivated. The morphological changes of the preneoplastic epithelial cells in sputum that are precursors to squamous cell carcinoma have been characterized. However, the morphological changes of precursors to adenocarcinoma in sputum have not been identified. Improvements in early detection through the identification of diagnostic epithelial cells in sputum could help reduce the death rate for lung cancer. We have been analyzing sputum samples to identify epithelial cells that may be indicative of preneoplastic lesions involved in adenocarcinoma development. The ASI SpectraCube system enables the user to measure the spectrum of each image pixel within the field and allows for the differentiation of components based on subtle spectral differences in the composition of the sample. Currently, we have identified a set of cyanophilic cells that appear to show varying degrees of atypia. The diagnosis of the atypical cells was confirmed by two cytopathologists independently. Also, the cells were confirmed to be of epithelial origin by immunocytochemistry. These cells are being used with the Applied Spectra Imaging (ASI) system to create a "library" of spectra that can be used to characterize other cells with unknown degrees of dysplasia. Several different libraries have been created in an attempt to optimize the variables involved in categorizing the epithelial cells based on their spectral signature. To date we have analyzed seventy-nine sputum cytology cases from four collection sites and have observed a consistent correlation between the clinical diagnosis and the spectral characterization. No effects on classification of the atypical cells have been observed due to the various Papinacolaou staining procedures or sample preparation used at the collection sites.

Spectral imaging in preconception/preimplantation genetic diagnosis of aneuploidy: multicolor, multichromosome screening of single cells.

Published: J. Assist. Reprod. Genet. 15: 323-330.

PURPOSE: Our purpose was to evaluate the utility of spectral imaging for multicolor, multichromosome enumeration in human interphase cell nuclei. METHODS: Chromosome-specific probes labeled with different fluorochromes or nonfluorescent haptens were obtained commercially or prepared in-house. Metaphase spreads, interphase lymphocytes, or blastomeres cells were hybridized with either 7 or 11 distinctly different probes. Following 46 hr of hybridization, slides were washed and detected using either a filter-based quantitative image processing system (QUIPS) developed in-house or a commercial spectral imaging system. RESULTS: The filter-based fluorescence microscope system is preferred for simultaneous detection of up to seven chromosome targets because of its high sensitivity and speed. However, this approach may not be applicable to interphase cells when 11 or more targets need to be discriminated. Interferometer-based spectral imaging with a spectral resolution of approximately 10 nm allows labeling of chromosome-specific DNA probes with fluorochromes having greatly overlapping emission spectra. This leads to increases in the number of fluorochromes or fluorochrome combinations available to score unambiguously chromosomes in interphase nuclei. CONCLUSIONS: Spectral imaging provides a significant improvement over conventional filter-based microscope systems for enumeration of multiple chromosomes in interphase nuclei, although further technical development is necessary in its application to embryonic blastomeres. When applied to preconception/preimplantation genetic diagnosis, presently available probes for spectral imaging are expected to detect abnormalities responsible for 70-80% of spontaneous abortions caused by chromosomal trisomies.

Spectral Imaging is a new way of looking at microscopic specimens. We will discuss two applications and the underlying Fourier spectroscopy/digital imaging technology. Spectral karyotyping (SKY) is a genome scanning method for identifying aberrations in all chromosomes simultaneously. SKY is a fluorescence microscope based approach that uses 5 fluorophores, singly and in combinations, to uniquely label DNA from each flow sorted chromosome. In situ hybridization with this complex DNA probe is used to identify all 24 human or all 21 mouse chromosomes in metaphase spreads. SKY is revolutionizing cytogenetics by identifying chromosome aberrations that are not apparent by G-banding, as well as to identify aberrations in solid tumor metaphases where G-banding is sub-optimal. Since SKY is a 24-chromosome approach it is much more efficient (and cost-effective) than using individual chromosome paint probes one (or two or three) at a time to identify chromosome aberrations.

George McNamara¹, Catherine Janish¹, Margaret Skokan¹, Dirk Soenksen¹, and Yuval Garini¹

1Applied Spectral Imaging, Inc., Carlsbad, CA

Spectral imaging is a new way of looking at microscopic specimens. We will discuss in back-to-back talks two scientific applications and introduce the underlying technology. Spectral karyotyping (SKY™) is a genome scanning method for identifying aberrations in all chromosomes simultaneously. In particular, SKY efficiently identifies translocations throughout the entire genome in prenatal/postnatal diagnosis, leukemia's, solid tumors and cell lines. SKY is a fluorescence In situ hybridization (FISH) technique that uniquely identifies by their emission spectra all 24 human or all 21 mouse chromosomes in metaphase spreads. SKY works by using combinations of five fluorophores to label chromosome paint probes, painting the entire genome simultaneously, and using the emission spectrum at each pixel in the spectral image to identify every chromosome. The author will explain why SKY works better than using conventional fluorescence filter sets to sequentially image the painted chromosomes (Garini et al, 1999, Cytometry, in press).

The same instrumentation has been in multicolor brightfield applications (next talk) and in Spectral FISH to identify up to 11 targets in interphase nuclei. SKY™ is revolutionizing cytogenetics by identifying chromosome aberrations that are not apparent by G-banding, as well as to identify aberrations in solid tumor and cell line metaphases where G-banding is sub-optimal. Since SKY is a genome-scanning 24-chromosome approach it is much more efficient (and cost-effective) than using individual chromosome paint probes one (or two or three) at a time to identify chromosome aberrations.

George McNamara¹, Dirk Soenksen¹, and Yuval Garini¹

1Applied Spectral Imaging, Inc., Carlsbad, CA

Spectral imaging is a new way of looking at microscopic specimens. We will discuss in back-to-back talks two scientific applications and introduce the underlying technology. Spectral Pathology (SPY) combines molecular pathology, spectral imaging and bright-field or fluorescence microscopy, to make possible objective cell identification using multiple markers. We propose that staining for multiple tumor markers followed by SPY will provide an efficient way to do immunophenotyping. For example, spectral imaging should make feasible the multiplexing of several immunohistochemical stains on a single fine needle aspirate biopsy (FNAB), to help render a definitive diagnosis from limited clinical material.

For this talk we will focus on bright-field applications. We will present examples of histological stains (H&E, Papanicolaou), multiple immunohistochemical stains, and mixed histo- and immuno-staining. We will present spectra of commonly used histological stains and immunohistochemical chromogenic products, and suggest optimal combinations.

SPY uses dye reference spectra and the absorption spectrum from each pixel in a spectral image to (1) compute the spectral optical density for every pixel and (2) generate dye component map images based on each reference spectrum. The intensity at each pixel in each map is proportional to the amount of the reference dye. The images can be saved as TIFF files and morphometry and pixel correlation maps performed. A new method for visualization of some or all component dyes, which we call Spectral UnMixing (SUN) will be explained herein and demonstrated at the accompanying workshop. SUN is a new tool that will be useful in research, clinical and education. As our instrumentation is also used by Dr. Zvi Malik for spectrally resolved morphometry (SRM; see Rothmann et al 1998 Histochem. J. 30: 539-547, for discovering nucleoli by spectral imaging of Masson’s Trichrome stain), we will compare SRM with SPY.