George McNamara

Spectra links

mpmicro (zipfile)

Temporal Area Map & Histogram For Analysis of Cell Motility and Chemotaxis (TAM webpage)

Crusade for better micrographs

prism

Boswell-McNamara Fluorescence Spectra Web Site (introduction page)

Tiki God

Tiki Goddess

See also the tiki_goddess website

Geo's favorite places

Geo's EXE's (zip collection)

NIH Biosketch

 

 

 

George's Seminars

I have not given many presentations since moving to CHLA, but have plenty of PowerPoint presentations and welcome opportunities to speak.

2001-2003 Talks

I have presented several talks at CHLA, and taught several Adobe Photoshop classes as well. 

 

2000 Talks

Duke University, October 2000, lectures on SKYomics and on Spectral Imaging in Pathology. The main goal was to help Professor Garnett Kelsoe design a set of novel experiments using SKY and SKY&FISH.

1999 talks

1999 Southern California Optical Biology Users Group Meeting
May 13-14, 1999 at U.C. Irvine.
"GFP2" The Second International Symposium on Green Fluorescent Protein
May 22-27, 1999 at The Town and Country Resort & Conference Center San Diego, CA
Biological Stain Commission 1999 Annual Meeting
June 4-5, 1999 aboard the Hotel Queen Mary, Long Beach, CA.
All three were titled "Spectral Karyotyping and Spectral Microscopy".

1998 Meetings

On July 26, 1998 I hosted a session on Spectral Imaging at the Fifth U.S.-Japan Joint Meeting of Histochemistry and Cytochemistry, on the UCSD (La Jolla) campus. Please see http://www.hcs.microscopy.com for details on the meeting. The abstracts for our session's talks are below (and were published in the Journal of Histochemistry and Cytochemistry, http://www.jhc.org/http://www.jhc.org, or subscribe to the journal for $98 per year). I also presented back-to-back talks at the January 1999 International Society for Analytical and Molecular Morphology conference (ISAMM'99).
Here's a customer and I at one of the receptions at the HCS meeting (the SKY metaphase was added to the image. It was not floating above Ulli Weier's head at the UCSD Price Center auditorium).

 

Histochemical Society 1998 Abstracts

 

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Spectral Imaging: Novel Methods For Quantitative Pathology. Sunday July 26, 1998 
Multicolor spectral imaging of chromogenic dyes in cytological specimen
Merryn V.E. Macville1,2, Thomas Ried1, 1National Human Genome Research Institute, National Institutes of Health, Bethesda, 2Institute of Pathology, University of Nijmegen, The Netherlands
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. 
 
Spectral imaging of sputum cytological samples using the Applied Spectral Imaging system.
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.
J. Fung1,2, W. Hyun3, P. Dandekar1, R.A. Pedersen1, H.U. Weier1. 1Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, Life Sciences Division, E.O. Lawrence Berkeley National Laboratory, University of California, Berkeley, 3Laboratory for Cell Analysis, Cancer Center, University of San Francisco.
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 Pathology (SPY) and Spectral Karyotyping (SKY)
Dirk Soenksen and George McNamara, Applied Spectral Imaging, Inc., Carlsbad, CA
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.
Spectral pathology (SPY) is a new application for both bright-field and fluorescence pathology/histology samples as well as interphase cytogenetics. The key is to use the spectral information from each image pixel to generate chromogen component maps. Each map is an image whose intensity is proportional to dye concentration. In absorption measurements this is achieved by the novel ability to compute the spectral optical density for every pixel. For the Feulgen reaction, for example, such a map would then be proportional to DNA concentration at each pixel. The map data can be saved as tagged image file format (TIFF) images and further analyzed using conventional image analysis. From an H&E stain image we generate maps of hematoxylin and eosin. Preliminary results suggest that the hematoxylin component map can be used to identify normal, cancer, and apoptotic nuclei. The number of chromogens is not limited to two. It is expected that SPY will routinely be suitable for quantitative analysis of many chromogens simultaneously, i.e. ER, PR, DNA ploidy, p53 status and a counterstain. We will present SPY results from classical pathology samples and discuss our spectral methods for separating multiple immunostains from each other.
 For more information:
SpectraCube, SKY™: http://www.spectral-imaging.com
George McNamara, Ph.D. (SPY), Randy Knudtson (SKYTM)), Applied Spectral Imaging, Inc., Carlsbad, CA 92009 Tel: 760-929-2840, geomcnamara@earthlink.net
Jacob Kaufmann, Applied Spectral Imaging, Ltd., Migdal Haemek, Israel, Tel: +972 6 654 7567, asi-ltd@spectral-imaging.com
SD-200 Information Group, R&D Section, Life Science Dept., Shimadzu Corp., Kyoto, Japan, Tel: + 075 823 1351, sd-info@shimadzu.co.jp
Dr. Michael Koehler, ASI, Edingen-Neckarhausen, Germany, Tel: +49 (0) 6203-923800, michael.koehler@spectral-imaging.com
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I presented back to back SKY and SPY talks at the 1999 International Society for Analytical and Molecular Morphology conference (www.ismm.org). The abstracts are listed below. As noted in the abstracts I hosted two workshop sessions where I did do spectral imaging on cytogenetics (SKY™), standard interphase FISH (including the Oncor/Ventana INFORM Her-2/neu FISH test), spectral FISH, and spectral pathology (SPY) samples. At one of the SPY sessions we did spectral imaging of Progesterone Receptor (DAB stained) tissue sections counter stained with hematoxylin. We succeeded in imaging these samples, though I was disappointed that no one gave me many color immunostained slides to SPY on.

 

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Spectral Karyotyping (SKY™): Identification of Chromosome Translocations and Numerical Aberrations by 24-Color

Fluorescence In Situ Hybridization

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.
Spectral imaging system workshops will be held Days 3 and 4 (Jan. 13 and 14) (see http://www.ismm.org/finalprogram.htm). We will also be available during breaks and evenings. Attendees are welcome to bring their own prepared microscope slides (contact GM at 760-929-2840 x 17 for instructions). We will bring SKY™, FISH, GFP/BFP, H&E, and SPY slides.
For further information about SKY™, SPY and spectral imaging contact the author at GeoMcNamara@earthlink.net or check our web site, www.spectral-imaging.com

 

Spectral Pathology (SPY): Multicolor Immunophenotyping of

Cancer in a Single Tissue Section

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.
Spectral imaging system workshops will be held Days 3 and 4 (Jan. 13 and 14) (see http://www.ismm.org/finalprogram.htm). We will also be available during breaks and evenings. Attendees are welcome to bring their own prepared microscope slides (contact GM at 760-929-2840 x 17 for instructions). We will bring SKY™, FISH, GFP/BFP, H&E, and SPY slides.
For further information about TAM, EFA, imaging, microscopy, SKY™, SPY and spectral imaging contact the author at GeoMcNamara@earthlink.net.

Here is the binary version of my temporal area map Rorschach test for you to stare at. Email me with how many face profiles you see (to make it easy for me, email me a picture with each profile sector painted a different color. Whoever sends me the largest set of believable profiles will get his or her 'portrait' and name added to this site. Only faces, please!

 

Thanks for reading this far.

 
George
 
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Copyright ©2000-2003 George McNamara

This page was last updated on 09/02/03 .