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Laboratory for Spectral Diagnosis
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Prof.Max Diem, PhD
Dept. of Chemistry and Chemical
Biology
Northeastern
University
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Updated 09/12/2008
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| 316 Hurtig Hall |
Tel: 617-373-2922
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| Boston, MA 02115 |
email:m.diem@neu.edu
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Introduction
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Since 1996, the research in Prof.
Diem's laboratory has centered around
spectral diagnosis of disease. In this work, methods to
investigate human cells, tissues or body fluids
spectroscopically are developed to arrive at a diagnosis
of disease, based on objective measurements and
mathematical-statistical procedures.
The spectroscopic techniques
utilized are methods of vibrational spectroscopy, Raman
scattering and Fourier transform infrared absorption
spectroscopy. Since disease changes the (bio)chemical
composition of cells, tissues and body fluids, molecular
fingerprint techniques such as vibrational spectroscopy
can be used to monitor these changes, and interpret them
in terms of a medical
diagnosis.
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Figure 1. Photomicrograph (left) of
a stained lymph node thin section, containing metastatic
cancer (circled), and a pseudo-color spectral map of the
same section, acquired before staining. The
spectral map was assembled by hierarchical
cluster analysis
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Since disease changes the chemical
composition on the cellular level, it is necessary to carry out
the spectroscopic measurements on a
microscopic level, matched in size to the typical size of cells.
Using modern infrared and Raman
micro-spectrometers, in which the vibrational spectrum
is measured through microscopes, it is possible to
acquire spectroscopic data from samples as small as
about 10
m
m x 10 m
m x 1 m
m, or about 10-10 g of sample, in infrared
and as low as 0.3 m
m x 0.3 m
m x1.0 m
m, or
about 10-14 g, in Raman
micro-spectro-scopy. This spatial resolution permits detection of subcellular
components (mitochondria, nucleoli, condensed chromatin) in cells, and opens
new avenues of monitoring cellular processed without the use of stains or
marker molecules, using the inherent spectral properties of molecular
constituents.
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Present Research Efforts:
1)Spectral
Histo-Pathology
.In this work on tissue diagnostics, the detection of primary
tumors in a variety of tissue types, e.g,
colon, cervix, breast, prostate, and secondary (metastatic) tumors in lymph
nodes has been pursued. Figure
1, for example, shows an infrared
pseudo-color map, based
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Figure
2. (A). Unstained tissue
section, 1.2 x 1.2 mm2,
of a colon adenocarcinoma. (B) Same tissue section,
after H&E staining, showing various
histopathological structures, and viable cancer cells
(2). (C) ANN-based tissue map, based on IR hyperspectral
data taken before staining. |
on 40,000 individual infrared
spectra acquired from a lymph node thin section, and analyzed by unsupervised
techniques of multivariate statistics (hierarchical cluster analysis), which
shows the detailed location of a colon adenocarcinoma metastasis in this lymph
node. Unsupervised methods of analysis require no reference data sets, but are
based solely on the detection of spectral similarity or dissimilarity using
pattern recognition algorithms.
The results shown in Figure 1
demonstrate that there are small, but reproducible
spectral differences between different tissue types, and
between normal and diseased tissue, which can be exploited using the statistical
methods indicated. The results of such unsupervised analyses are subsequently correlated with
tissue histo-pathology, and used to train diagnostic algorithms based, for
example, on Artificial Neural Network (ANN)
methodology. The
softwaredeveloped for the
analysis of |
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spectral imaging data sets is now commercially
available (see
www.Cytospec.com).
The research efforts on tissue
sections is at a stage where dozens of samples are being
analyzed for the presence of cancer by diagnostic
algorithms based on ANNs. Figure 2 shows a tissue
section with a colon adenocarcinoma that was analyzed in
less than a minute by an ANN to detect the cancerous
areas shown in red in Panel 2C. These efforts are aimed
at establishing diagnostic and prognostic methods that
can be used in the operating room to define the margins
of recession, and to screen excised lymph nodes for the
presence of metastatic disease. Recently, we have used
the method to detect micro-metastases (metastases
measuring less then 2 mm in size) in lymph nodes, which
are exceedingly difficult to detect in standard
histo-pathology.
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2) Spectral
Cyto-Pathology. The second major research area is
the detection of cellular abnormality in a sample of
cells, for example, in thin needle aspirates or
exfoliated cells. In order to succeed with this project,
the detailed spectral characteristics of cellular
components need to be investigated, as well as the
differences in spectral properties of quiescent and
proliferative cells.
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Figure 3. Principal component
analysis of cells found in normal urine. See text for
details |
The cytological efforts deal with
two major aspects. One is the development of methodology
for cancer screening in cells that are easily
accessible, such as cervical cells (from routine
gynecological exams) or bladder (urothelial) cells that
can be isolated from urine. Spectra as well as high
resolution optical images of cells are collected, and
the spectra are subject to multivariate methods of
analysis that group cellular spectra by their
similarity. Urine from a healthy donor, for example,
contains different cell types that can be distinguished by their spectral patterns.Panel 3A
shows a high resolution image of a glycogen-rich
squamous cell, and Panel 3B of a glycogen-free squamous
cell. These cells are from the distal urethra, and
cannot be distinguished by visual microscopy. Panel 3C shows an image of a urothelial
cell from the
bladder. The lower panel shows results
obtained by Principal Component Analysis (PCA) of about
100 cells. Here, every data point represents the
spectrum of one cell, and the discrimination between
urothelial (green dots) and squamous
cells (red and blue
dots) is excellent.
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Glycogen-free
(blue) and glycogen-containing (red) cells are partially
overlapping, since cells may contain a lot, very little
or no glycogen. Similar discrimination has been achieved
between normal and cancerous cells.
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Figure 4: Bright field (left) and
Rama spectral image, constructed via hierarchical
cluster analysis, of a HeLa cell in aqueous medium (60x
water immersion objective, 10 mW at 488 nm
excitation).
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3) High Resolution Raman Imaging of
Cells. The second aspect of the
cytological efforts deals with high resolution imaging,
using Raman micro-spectroscopy and hierarchical cluster
analysis, of individual cells and their constituents.
Figure 4 shows a bright field microscopic image of a
cell (left), as well as the spectral map, based on Raman
micro-spectral data. The Raman map shows the cellular
features such as cytoplasm, nucleus and nucleoli in
exquisite detail. Data analysis, as in the case of the
infrared spectral maps shown above, was carried out in a
completely unsupervised fashion, using no reference data
but only spectral similarity criteria, to construct the
image shown in Figure 4. Raman spectral imaging has been
utilized in the LSpD to study the distribution of
mitochondria in cells, the uptake of drugs into cells,
the dynamics of liposome uptake, and the condensation of
chromatin during mitosis.
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Funding (since 2000)
National Institutes of Health
CA 81675:
"Detection of Cancerous Cells via FT-IR Microscopy"
2000-2001
GM 60654:
"Detection of Cancerous Cells and Tissues by
FT-IR Microspectroscopy"
2001-2005
CA 090346:
"Infrared Microspectroscopy for
Cervical Cancer Screening"
2003-2011
CA 111330:
"Detection of Cancer in Lymph Nodes by Spectral
Imaging"
2005-
US Army
A08A-036-0118
"Automatic Microscopic Malaria Diagnosis"
(subcontract)
2008 -
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Publications
Books
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M.Diem, Introduction
to Modern Vibrational Spectroscopy, J.Wiley-Interscience, 1993(ISBN
0-471-59584-5)
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Vibrational Spectroscopy for
Medical Diagnosis, M.Diem, P.Griffiths and J.Chalmers,
Editors, J.Wiley-Interscience, 2008(ISBN:
978-0-470-01214-7)
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Recent Book Chapters (since 2005)
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M.Diem, M.Romeo, S.Boydston-White
and C.Matthäus "IR Spectroscopic Imaging:
from Cells to Tissue", in "Spectrochemical
Analysis using Infrared Multichannel Detectors",
R.Bhargava and I.W.Levin, Editors, Blackwell Publishing,
Sheffield
,
UK
, (2005), pp.189-203
(ISBN:9781405125048)
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M.J. Romeo, S.Boydston-White,
C.Matthäus, M.Miljković, B.Bird, T.Chernenko and M.Diem
"Vibrational Microspectroscopy of Cells and
Tissues", in " Biomedical
Vibrational Spectroscopy", P.Lasch and J.Kneipp,
Editors, J.Wiley (Blackwell Publishing), 2008 (ISBN
0470229454), p.121-152
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M.J. Romeo, R.K.Dukor and M.Diem,
"Introduction to Spectral Imaging:
Applications to the Diagnosis of Lymph Nodes", in
"Vibrational Spectroscopy for Medical Diagnosis",
M.Diem, P.Griffiths and J.Chalmers, Editors,
J.Wiley-Interscience, 2008, pp 1-25
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M.J.Romeo, B.Bird,
S.Boydston-White, C.Matthäus, M.Miljković, T.Chernenko
and M.Diem, "Infrared and Raman
Micro-spectroscopic Studies of Individual Human Cells",
in "Vibrational Spectroscopy for Medical Diagnosis",
M.Diem, P.Griffiths and J.Chalmers, Editors,
J.Wiley-Interscience, 2008, pp 27 - 70
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M.Romeo, C.Matthäus, M.Miljković,
B.Bird, T.Chernenko and M.Diem, "Infrared and Raman
Microscopy in Cell Biology", in "Methods in Cell
Biology, Volume 2: Biophysical Techniques", Edited by
J.J. Correia and H.W. Detrich, III, Elsevier, in press
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M.Diem, C.Matthäus, T.Chernenko,
M.J.Romeo, M.Miljković, B.Bird, J.Schubert, K.
Papamarkakis, K.Bedrossian and N.Laver "Infrared and
Raman Spectroscopy and Spectral Imaging of Individual
Cells", in "Infrared and Raman Spectroscopic
Imaging", Editors: R. Salzer, H.W. Siesler, Wiley - VCH
Publishing, in press
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C.Matthäus, T.Chernenko,
M.Miljković, L.Quintero, M.Amiji, V.Torchilin and
M.Diem, "Raman Micro-spectral Imaging of Cells,
and Applications Monitoring the Uptake of Drug Delivery
Systems", in "Confocal Raman Imaging", Hollrichter and
Dieing, Ed, Springer Verlag, Heidelberg, in
press
Papers since 2004
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M.Diem, M.Romeo, L.Miller and
P.Lasch, "Comparison of
Fourier Transform Infrared (FTIR) Spectra of Individual
Cells Acquired Using Synchrotron and Conventional
Sources", (2004) Infrared Physics & Technology, 45, 331-338
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B. R. Wood,
L. Chiriboga, H. Yee, M. A. Quinn, D. McNaughton,
and M. Diem, "FTIR mapping of the
cervical transformation zone, squamous and glandular
epithelium", (2004) Gynecologic Oncology 93(4),
59-68
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P.Lasch, W.Haensch, D.Naumann, and
M. Diem, "Imaging of Colorectal Adenocarcinoma Using
FT-IR Microspectroscopy and Cluster Analysis", (2004) Biochim Biophys Acta. 1688(2),176-86
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M.Romeo, C. Matthäus, M.Miljkovic
and M.Diem "Infrared Microspectroscopy of Individual
Human Cervical Cancer (HeLa) Cells", (2004) Biopolymer, 74, 168-171
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M.Miljkovic, M.Romeo, C. Matthäus
and M.Diem "Infrared Microspectroscopy of Individual
Human Cervical Cancer (HeLa) Cells Suspended in Growth
Medium", (2004) Biopolymers, 74, 172-175
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M.Romeo, C.Matthäus, M.Miljkovic,
S.Boydston-White and M.Diem, "Analysis of Microscopic
Infrared Spectra of Individual Dried and Live Human
Cells", (2004), Biomedical Vibrational Spectroscopy and Biohazard Detection,
Proceedings of SPIE, Volume 5321, 124-129
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P.Lasch, M. Diem and D.Naumann,
"FT-IR microspectroscopic imaging of prostate tissue
sections", (2004), Biomedical Vibrational Spectroscopy and Biohazard Detection,
Proceedings of SPIE, Volume 5321, 1-9
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M.Diem, M.Romeo, S.Boydston-White,
M.Miljovic and C.Matthäus, "A Decade of Vibrational
Micro-Spectroscopy of Human Cells and Tissue
(1994-2004)", (2004) Analyst, 129(10), 880-885
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B.Mohlenhoff, M.Romeo, B.R.Wood
and M.Diem, "Mie-type
Scattering and non-Beer-Lambert Absorption Behaviour of
Human Cells in Infrared Micro-Spectroscopy",
(2005) Biophys.J., 88(5),
3635-3640
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S.Boydston-White, T.Chernenko,
A.Regina, M.Miljković, C.Matthäus and M.Diem, "Microspectroscopy of Single
Proliferating HeLa Cells", (2005) Vibrational Spectrosc., 38, 169-177
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M.J.Romeo and M.Diem, "Infrared
Spectral Imaging of lymph nodes: Strategies for analysis
and artifact reduction", (2005) Vibrational Spectrosc., 38, 115-119
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M.J.Romeo and M.Diem, "Correction
of dispersive line shape artifacts observed in diffuse
reflection infrared spectroscopy and
absorption/reflection (transflection)
infrared micro-spectroscopy", (2005) Vibrational Spectrosc., 38, 129-132
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B. R. Wood, K. R. Bambery, L.M.
Miller, M. Quinn, L. Chiriboga, M. Diem, and D.
McNaughton, "Comparison of FTIR multi-channel detector
images and synchrotron maps recorded from thin -
sectioned cervical biopsies", (2005) Proc.SPIE, Vol 5651, 78-84
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C.Matthäus, M.Miljković, M.Romeo,
S.Boydston-White and M.Diem, "Raman and Infrared
Micro-spectral Imaging of Mitotic Cells", (2006) Appl.Spectrosc., 60(1), 1-8
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S.Boydston-White, M.Romeo,
T.Chernenko, A.Regina, M.Miljković and M.Diem,
"Cell-cycle-dependent variations in FTIR micro-spectra
of single proliferating HeLa cells: Principal component
and artificial neural network analysis", (2006) Biochimica et Biophysica Acta,
1758,
908-914
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M.Romeo, B.Mohlenhoff, M.Jennings
and M.Diem, "Infrared micro-spectroscopic studies of
epithelial cells", (2006) Biochimica et Biophysica Acta,
1758,
915-922
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P.Lasch, M.Diem, W.Hänsch and
D.Naumann, "Artificial Neural Networks as Supervised
Techniques for FT-IR Microspectroscopic Imaging", J.Chemometrics, 20(5),
209-220
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M.Romeo,
B.Mohlenhoff and M.Diem "Infrared
Micro-Spectroscopy of Human Cells: Causes for the
Spectral Variance of Oral Mucosa (Buccal) Cells", (2006)
Vibrational Spectrosc., 42, 9-14
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C.Matthäus, T.Chernenko, J.Newmark,
C.Warner and M. Diem "Label-free detection of
mitochondrial distribution in cells by nonresonant Raman
Micro-Spectroscopy" (2007), Biophys.J., 93, 668-673
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C.Matthäus, T.Chernenko, A.Kale,
V.Torchilin and M.Diem "Raman-microspectroscopic Imaging
of Liposomal Drug Delivery Systems Inside Individual
Cells", (2008) Molecular Pharmaceutics, 5(2), 287-293.
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B.Bird, M.Romeo and M.Diem,
"Cytology by Infrared Micro-Spectroscopy: Automatic
Distinction of Cell Types in Urinary Cytology", (2008),
Vibrational Spectrosc., in
press,
doi:10.1016/j.vibspec.2008.03.006
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C.Matthäus, T.Chernenko,
L.Quintero, L.Milan, A.Kale, M.Amiji, V.Torchilin and
M.Diem "Raman Microscopic Imaging of Cells and
Applications Monitoring the Uptake of Drug Delivery
Systems", (2008), Proc.SPIE, Vol.
6991 06/1 -
06/8
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B.Bird, M.Miljković,
M.J.Romeo, J.Smith, N.Stone, M.W.George and
M.Diem, " Infrared Micro-Spectral Imaging: Automatic
Distinction of TissueTypes in Axillary Lymph Node
Histology", J.Clin.Pathology, in press
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B.R.
Wood, T.Chernenko, C.Matthäus, M.Diem, U.Bernhard,
C.Jene, A. Brandli, D.McNaughton, M.J.Tobin, A.Trounson,
and O.Lacham-Kaplan, "Spectroscopic method for detecting
changes in the molecular architecture of cells ", Anal.Chem. 2008,
accepted
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