Frequency (cm-1)

Imaging Chemical Changes in Disease:
From Bones to Brains
Lisa M. Miller
National Synchrotron Light Source
Brookhaven National Laboratory
Upton, NY 11973 USA
U.S. DEPARTMENT OF ENERGY
OFFICE OF BASIC ENERGY SCIENCES
Outline
•
Infrared microspectroscopy and imaging of biological tissue
•
Advantages of a synchrotron source
•
Current applications: From bones to brains
Osteoporosis &
treatments
Alzheimer’s disease
Prion diseases
Skin cancer &
therapy
Infrared Light Probes Molecular Vibrations
O
H
H
Atom Type, Size, Environment, and Bond Strength Influence Vibrational Frequencies
-C-H
=C-H
-C=C-C-Cl
-C-Br
2850-2960 cm-1
3020-3100 cm-1
1650-1670 cm-1
600-800 cm-1
500-600 cm-1
-C=O
-C-O
1640-1750 cm-1
1050-1150 cm-1
-O-H
-N-H
3400-3640 cm-1
3310-3500 cm-1
Chemical Features of Biological Components
Protein O
=
Amide I
-NH-C-CH-
Amide II
R
O
protein
CH3-(CH2)n-C-OH
Nucleic Acid
O
Absorbance
=
Lipid
C-C
stretch
CH stretch
lipid
P=O stretch
O=P-O
nucleic
acid
OH stretch
Carbohydrate
C-O stretch
carbohydrate
3000
2000
Frequency (cm-1)
1000
Protein Structure Determination with FTIR
β-turn
β-sheet
Collagen
(triple helix)
Myoglobin
(α-helix)
extended
coil
Abeta Amyloid
(α-helix + β-sheet)
α-helix
Cytochrome c
(α-helix + coil)
Secondary Structure Assignments:
1620 – 1640
1645
1652 – 1657
1660
1670 – 1695
β-sheet
extended coil (D2O)
α-helix
triple helix
β-turn
STI
(β-sheet + coil)
1700
1650
1600
Frequency (cm-1)
IR Spectroscopy vs. Micro-Spectroscopy
Bulk samples
KBr
+
solid
Spatial Resolution:
~1-3 mm
+
solution
Microscopic Heterogeneity
**
**
*
**
**
*
**
**
*
Spatial Resolution:
~ 25 - 30 µm (globar)
~ 3 - 10 µm (synchrotron)
Advantages of a Synchrotron IR Source
Higher Spatial Resolution
BRIGHTNESS (1000x)
+
BROADBAND
Better Signal-to-Noise
Faster Data Collection
Spectroscopy
(near-, mid-, far-IR)
Microspectroscopy: Globar vs. Synchrotron
Source
10 µm
Diffraction Limit
4000 cm-1 2.5 µm
2950 cm-1 3.4 µm
1650 cm-1 6.1 µm
1200 cm-1 8.3 µm
1000 cm-1 10 µm
600 cm-1 17 µm
200 cm-1 50 µm
5x5 µm
MCT-A* Signal (V)
globar
12
8
4
globar
0
0
3000
2000
Frequency (cm-1)
1000
• Conventional IRMS
is throughput-limited
synchrotron
16
synchrotron
• Synchrotron IRMS
is diffraction-limited
10 20 30 40 50 60 70
Aperture size (µm)
Imaging: Confocal vs. Array Detector
visible image
Absorbance
Absorbance
photoresist
15 µm
10 µm
6 µm
6.25 µm
array
4000 3500 3000 2500 2000 1500 1000
Frequency (cm-1)
confocal
array
3650-3000 cm-1
1750
1500
1250
Frequency (cm-1)
1000
1767-1515 cm-1
8 minutes
1 hour
Beamline U10B
Synchrotron infrared
beam pipe
Nicolet Magna 860 FTIR
Nicolet Continuµm
IR microscope
Filter cube turret
UV light source
Schwarzchild 32x
IR objective
Automated mapping stage
* 4 synchrotron infrared microscopes at the NSLS
Sample Preparation and Data Collection
Sample Preparation
+
tissue
embedding compound
(paraffin, plastic)
cut thin sections
place on substrate
Modes of Data Collection
Tissue on IRtransparent slide
(CaF2, BaF2)
Tissue on
IR-reflective
slide
Reflection
Transmission
Current Applications
Osteoporosis and Treatments:
• David Burr (Indiana University School of Medicine)
• Stefan Judex (Stony Brook University)
• Mark Chance (Albert Einstein College of Medicine)
• Cathy Carlson (University of Minnesota)
Alzheimer’s Disease:
• Judit Miklossy (Temple University)
• Laszlo Forro (Swiss FIT)
• Helene Benveniste (BNL-Medical)
Prion Diseases:
• Dieter Naumann (Robert Koch Inst)
• Michael Beekes (Robert Koch Inst)
Skin Cancer and Therapies:
• Irit Sagi (Weizmann Institute of Science)
• Abdel Mamoon (Egyptian Atomic Energy Authority)
Osteoporosis
0.5 SD
NORMAL
Osteoporotic
fractures
No
fractures
OSTEOPOROSIS
Bone Mineral Density
• Bone mineral density is lower in women with
osteoporotic fractures
1 mm
• However, there is a large overlap between the
populations
1. Is bone composition different in osteoporosis?
2. Do current osteoporosis treatments affect bone composition?
phosphate
carbonate
collagen
Absorbance
Infrared Spectroscopy of Bone
collagen fiber
formation
collagen
cross-linking
mineralization
2000
1500
1000
500
Frequency (cm-1)
Parameters:
Mineralization
(phosphate/protein)
y increases brittleness
z increases flexibility
z decreases strength
Carbonate content
(carbonate/protein)
• ~8% carbonate in bone
• content increases with bone age
• ? affect on material properties
Collagen structure
• cross-linking affects mineralization
Does the Chemical Composition of Bone Change in
Osteoporosis?
In this study:
Normal illumination
• To “induce” osteoporosis, female monkeys
have their ovaries removed (Ovx)
•
2 colonies: Ovx and Sham-Ovx
• Osteoporosis is treated with nandrolone
decanoate
•
2 colonies: Nan and Control
• Fluorescent drugs are administered and
taken up into newly remodeled bone
•
t = 0 yr. post-surgery: tetracycline
•
t = 1 yr. post surgery: calcein
•
t = 2 yr. post surgery: xylenol orange
Fluorescence illumination
CALCEIN (t= 1 yr)
XYLENOL
(t=2 yr)
T. Tague, L.M. Miller. Microscopy Today, 2: 26-32 (2000).
Bone Mineralization is Slowed in Osteoporosis
3
2
1
6
subchondral
bone
4
5
marrow
space
Absorbance
cartilage
100 µm
(1)
(2)
(3)
(4)
(5)
(6)
1800
• Rate of bone mineral formation is slower in
osteoporosis, i.e. bone quality is reduced
1400
1000
600
Frequency (cm-1)
L.M. Miller, J. Tibrewala, C.S. Carlson. Cellular and Molecular Biology, 46:1035-44 (2000).
Nandrolone Treatment Affects Carbonate Substitution
Type A / Type B Carbonate Ratio
0.8
Hydroxyapatite: Ca10(PO4)6(OH)2
Type B Carbonate
• + CO32-, - PO43• decreases Ca2+ content
0.6
0.4
Type A Carbonate
• + CO32-, - OH• increases Ca2+ content
0.2
0.0
INTACT
OVX
NAN 2yr
• Nandrolone decanoate is an anabolic steroid proposed to increase bone
mineral density (BMD)
• Carbonate composition in bone changes with nandrolone treatment
• Changes in carbonate can affect calcium content in bone
R. Huang, L.M. Miller, C.S. Carlson, M.R. Chance. Bone 30: 492-97 (2002).
Carbonate Content and Bone Mineral Density
Carbonate / Protein Ratio
Non-treated groups
Nandrolone-treated groups
R=0.67939
0.044
0.042
p=0.0151
0.040
0.040
R= -0.57061
p= 0.05269
0.038
0.036
0.036
0.034
0.032
0.032
0.030
0.028
0.52
0.56
0.60
0.64
0.68
Bone Mineral Density (g/cm2)
0.60
0.65
0.70
0.75
0.80
0.85
Bone Mineral Density (g/cm2)
R. Huang, L.M. Miller, C.S. Carlson, M.R. Chance. Bone 30: 492-97 (2002).
Bisphosphonate Treatment and Microdamage in Bone
•
Through normal activity, microdamage
occurs in bone, which is naturally repaired
by the bone remodeling process.
•
With reduced remodeling, microdamage
accumulation can occur.
•
In animals with normal bone density, high
doses of bisphosphonates increase the
occurrence of microdamage.
10 µm
Why does damage accumulation occur?
• reduced repair of microcracks
• rapid accumulation of microcracks
(changes in bone composition)
Bone Composition Differs in Microcrack Areas
Carbonate / Protein
0.00
0.025
0.050
Bone Composition Differs in Microcracks
Carbonate/Phosphate
0.006
Acid Phosphate
0.075
*
*
Collagen Structure
3
*
0.004
0.050
2
0.002
0.025
1
0.000
0.000
UD
MD
Phosphate/Protein
UD
MD
0
UD
MD
In microdamaged bone:
• Carbonate accumulation in bone is lower
1.00
• Acid phosphate content is higher
0.75
• Collagen cross-linking is disturbed,
consistent with broken/reduced cross-links
0.50
0.25
• Mineralization level is unchanged, i.e. bone
is not more brittle
0.00
UD
MD
Current Applications
Osteoporosis and Treatments:
• David Burr (Indiana University School of Medicine)
• Stefan Judex (Stony Brook University)
• Mark Chance (Albert Einstein College of Medicine)
• Cathy Carlson (University of Minnesota)
Alzheimer’s Disease:
• Judit Miklossy (Temple University)
• Laszlo Forro (Swiss FIT)
• Helene Benveniste (BNL-Medical)
Prion Diseases:
• Dieter Naumann (Robert Koch Inst)
• Michael Beekes (Robert Koch Inst)
Skin Cancer and Therapies:
• Irit Sagi (Weizmann Institute of Science)
• Abdel Mamoon (Egyptian Atomic Energy Authority)
Protein-Folding Diseases
Alzheimer’s disease
Lou Gehrig’s disease
Parkinson’s disease
~1655 cm-1
~1630 cm-1
Creutzfeldt Jakob disease
mad cow disease
scrapie
Plaque Formation in Alzheimer’s Disease
y ABeta = Amyloid beta (40,42,43 amino acids)
– Normal Abeta = 40 amino acids
– Abeta that forms plaques = 42,43 amino acids
y Fragment of larger protein APP (Amyloid Precursor Protein)
y Cut by α-secretase, β-secretase and γ-secretase
Pathology of Alzheimer’s Disease
50 µm
50 µm
• Alzheimer’s plaques are misfolded Abeta protein. They are 20 –
100 microns in size.
• Plaques are thought to disrupt communication between neurons
• Plaques may also be toxic to neurons, resulting in their death.
• Plaques have recently been identified in other organs: liver,
pancreas, ovary, testis, and thyroid.
Unanswered Questions in Alzheimer’s Disease
Abeta structural studies in AD have primarily been
studied either (1) in vitro, or (2) histologically in
tissue:
• Abeta structure in vitro is dependent upon
concentration, pH, solvent, and temperature.
50 µm
• Histological staining for amyloid plaques in other
tissues can be non-specific.
On the microscopic level in tissue, we want to understand:
1. What is the structure of Abeta in AD plaques?
2. Are the plaques and tangles identified in other tissues analogous to
those found in the brain?
3. Is Alzheimer’s disease a systemic disorder, i.e can fibrillary changes
observed in other organs aid in the early diagnosis of AD?
1626
1654
Absorbance
1666
Structure of Abeta1-42
1-42 in Solution
1740
1720
1700
1680
1660
1640
Frequency (cm-1)
1620
1600
1580
1560
Protein Aggregation in Alzheimer’s Plaques
Absorbance
MISFOLDED
NORMAL
1800
1700
1600
1500
1400
Frequency (cm-1)
1.0
• misfolded, aggregated protein is
associated with Abeta plaques
20 µm
0.0
L.M. Miller, P. Dumas, N. Jamin, J.-L. Teillaud, J. Miklossy, L. Forro. Rev. Sci. Instr., 73: 1357-60 (2002).
Plaque Structure in the Pancreas
1630/1655 cm-1
0.0
10 µm
1.3
Evidence of amyloid formation in pancreas:
• Thioflavin-S fluorescence
• misfolded protein aggregates by IRMS
Other Directions in AD Research
• Can bacteria initiate amyloid plaque formation?
Absorbance
1.0
0.8
α-helix
1.3
β-sheet
0.6
0.4
0.0
0.2
50 µm
50 µm
0.0
1800
Neuronal cells infected with
spirochetes for 6 weeks
1700
1600
Frequency
1500
(cm-1)
1400
Infrared image: 1630 / 1655 cm-1
J. Miklossy, A. Kis, A. Radenovic, L.M. Miller, L. Forro, et al. Neurobiology of Aging, in press (2005).
Current Applications
Osteoporosis and Treatments:
• David Burr (Indiana University School of Medicine)
• Stefan Judex (Stony Brook University)
• Mark Chance (Albert Einstein College of Medicine)
• Cathy Carlson (University of Minnesota)
Alzheimer’s Disease:
• Judit Miklossy (Temple University)
• Laszlo Forro (Swiss FIT)
• Helene Benveniste (BNL-Medical)
Prion Diseases:
• Dieter Naumann (Robert Koch Inst)
• Michael Beekes (Robert Koch Inst)
Skin Cancer and Therapies:
• Irit Sagi (Weizmann Institute of Science)
• Abdel Mamoon (Egyptian Atomic Energy Authority)
Prion Protein Diseases
Scrapie (1732)
Mad Cow Disease (1987)
Creutzfeldt-Jakob Disease (1920/21)
Kuru (1957)
New-variant Creutzfeldt-Jakob Disease (vCJD) (1996)
Chronic Wasting Disease, Feline Spongiform
Encephalopathy, Transmissible Mink Encephalopathy
263K Scrapie-hamster
“Infectivity” of Misfolded Prion Proteins
•
Prion proteins naturally occur in the nervous
tissue.
•
Their functions is unclear, but it is thought that
they may have antioxidant function.
•
Prion proteins can misfold and become
“infectious”, causing more normal protein to
misfold.
•
Misfolded prion proteins form aggregates.
They are small and diffuse, about 5 – 30
microns in size.
•
Plaques are thought to disrupt communication
between neurons
•
Plaques may also be toxic to neurons,
resulting in their death.
NORMAL
MISFOLDED
Unanswered Questions in Prion Diseases
Prion protein structural studies have primarily
been studied either (1) in vitro, or (2) with
immunohistochemistry:
• PrPSc-folding in vitro is dependent upon
concentration, pH, solvent, and
temperature.
• Antibodies to the prion protein are not
structure-specific, and only show where the
protein is located in high concentration.
Variant Creutzfeldt Jakob disease
On the microscopic level in tissue, we want to understand:
1.
How and why do prion proteins misfold?
2.
How is the misfolded prion protein infectious, i.e. how does the misfolded
protein “catalyze” further misfolding?
3.
What is the structure of the misfolded prion protein within the tissue?
Prion Protein Structure and Location
1700
1660
1620
Frequency (cm-1)
• Hamster scrapie 263K
• Dorsal root ganglia
• Terminally infected
10 µm
J. Kneipp, L.M. Miller, M. Kittel, M. Beekes, D. Naumann. BBA, 1639: 152-58 (2003).
Not All Cells are Affected
D
1658
1659
1700
1655
1600
1500
-1
Frequency (cm )
Q. Wang, A. Kretlow, M. Beekes, D. Naumann, L.M. Miller. Vibr. Spectr., in press (2005).
Control Animals Do Not Exhibit Misfolded Prions
K
20 µm
C
1660
D
1638
1638
1660
1700
1600
1500
Frequency (cm-1)
1700
1600 1500
Frequency (cm-1)
Importance of a Synchrotron IR Source
Absorbance
10 µm
10 µm
30 µm
unstained cell
stained cell
1645
1700
1650
1600
1550
1500
Frequency (cm-1)
• Misfolded prion protein within tissue
is β-sheet structure
1656
10 µm
aperture
30 µm
aperture
• Proteins are generally located near
(or in) the cell membrane
• Not all neurons are affected equally
J. Kneipp, L.M. Miller, M. Kittel, M. Beekes, D. Naumann. BBA, 1639: 152-58 (2003).
Current Applications
Osteoporosis and Treatments:
• David Burr (Indiana University School of Medicine)
• Stefan Judex (Stony Brook University)
• Mark Chance (Albert Einstein College of Medicine)
• Cathy Carlson (University of Minnesota)
Alzheimer’s Disease:
• Judit Miklossy (Temple University)
• Laszlo Forro (Swiss FIT)
• Helene Benveniste (BNL-Medical)
Prion Diseases:
• Dieter Naumann (Robert Koch Inst)
• Michael Beekes (Robert Koch Inst)
Skin Cancer and Therapies:
• Irit Sagi (Weizmann Institute of Science)
• Abdel Mamoon (Egyptian Atomic Energy Authority)
Gelatinase B is Implicated in Tumor Growth
• Tissue is made of cells and extracellular matrix (ECM)
• For tumor to grow, these must be degraded
Normal tissue
Tumor
• Gelatinase enzyme is implicated in degradation of ECM
Green Gelatin Degradation by Gelatinase B (MMP-9)
Absorbance
0.80
undegraded
degraded
0.60
0.40
0.20
0.00
1800
1700
1600
1500
Frequency (cm-1)
•
•
Green gelatin can be used to visibly
identify degradation of gelatin films by
gelatinase B
The structure of degraded gelatin can be
determined with IR micro-spectroscopy
20 µm
0.68
0.74 0.80
1630 / 1660 cm-1
S. Federman, L.M. Miller, I. Sagi. Matrix Biology, 21: 567-77 (2002).
Gelatin Degradation by HT-1080 Tumor Cells
20 µm
• IR micro-spectroscopy can be used to
identify gelatin degradation by HT-1080
tumor cells
0.60
0.65
0.70
0.75
0.80
0.85
1625 / 1650 cm-1
S. Federman, L.M. Miller, I. Sagi. Matrix Biology, 21 567-77 (2002).
Human Skin Melanoma and Photodynamic Therapy
Absorbance
Indocyanine Green
• Human melanoma cells
• 100 µM indocyanine green
• Ti-Sapphire laser (780 nm)
• 50 – 150 mW / cm2
• 10 – 30 min irradiation
• Re-incubate 48 hours
400
500
600
700
800
900
Wavelength (nm)
• Assess cell death with trypan blue staining
• Probe chemical composition with FTIR
microscope (~50 cells per time point)
Affect of PDT on Melanoma Cell Composition
% Survival
(of control)
Absorbance
Control
0J
50 J
4000
3500
3000
2500
2000
Frequency (cm-1)
1500
100 %
100 %
95 %
100 J
85 %
150 J
80 %
1000
Cluster Analysis of PDT Therapy
Increasing Heterogeneity
50 J
CNT
• Indocyanine dye has little
affect on cellular
composition
• Increasing light dose has
increasing affect on
composition
• Largest affect in protein
and nucleic acid structure
0J
100 J
150 J
Other Applications in the Biosciences
Osteoarthritis:
• Cathy Carlson (Univ. of Minnesota)
• David Hamerman (AECOM)
Biopolymerization:
• Richard Gross (Polytechnic Univ)
• Bo Chen (Polytechnic Univ)
Dilated Cardiomyopathy:
• Wasiem Sanad (Charite Hospital,
Berlin)
Agricultural Science:
• Peiqiang Yu (Univ. Saskatchewan)
• David Wetzel (Kansas State Univ.)
Chronic Lymphocytic Leukemia:
• Nicholas Chiorazzi (North ShoreLIJ, NY)
Bioremediation:
• U. Ghosh (Stanford Univ.)
• M. Fuhrmann (BNL Environ. Sci.)
Apoptosis:
• Jean-Luc Teillaud (Inst Curie)
• Paul Dumas (LURE)
• Nadege Jamin (LURE)
Cosmetics:
• Paul Dumas (LURE)
• Stefania Nuzzo (L’oreal)
Acknowledgements
The Group
Meghan Ruppel
Adele Qi Wang
Randy Smith
Ariane Kretlow
Tejas Telivala
Michael Appel
William Little
Ted Feldman
$$$
• US Department of Energy
• National Institutes of Health
• DOE Cooperative Research Grant
• BNL-LDRD Grant
• BNL-SBU Seed Grant