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BROOKHAVEN SCIENCE ASSOCIATESFunded under contract: DE-AC02-98CH10886
U.S. DEPARTMENT OF ENERGY
OFFICE OF BASIC ENERGY SCIENCES
Infrared Imaging and MicrospectroscopyInfrared Imaging and Microspectroscopy
Lisa M. MillerNational Synchrotron Light SourceBrookhaven National Laboratory
Upton, NY 11973
U.S. DEPARTMENT OF ENERGY
OFFICE OF BASIC ENERGY SCIENCES
N A T IO N AL SY N C H R O T RO N LIG H T SO U RC E
O P E R A T E D B Y B R O O K H A V E N S C IE N C E A S S O C IA T E S
Chemical Features of Biological ComponentsChemical Features of Biological Components
Protein O-NH-C-CH-
=
R
Nucleic Acid
O=P-O
O
Lipid OCH3-(CH2)n-C-OH
=
Carbohydrate
Abs
orba
nce
1000 2000 3000 Frequency (cm-1)
Amide IAmide II
CH stretch C=O esterstretch
P=O stretch
C-O stretchOH stretch
protein
lipid
nucleicacid
carbohydrate
C-Cstretch
U.S. DEPARTMENT OF ENERGY
OFFICE OF BASIC ENERGY SCIENCES
N A T IO N AL SY N C H R O T RO N LIG H T SO U RC E
O P E R A T E D B Y B R O O K H A V E N S C IE N C E A S S O C IA T E S
Protein Structure Determination with FTIRProtein Structure Determination with FTIR
α-helix
β-sheetβ-turn
extended coil
Amide I Secondary Structure Assignments:
1620 - 1640 β-sheet1645 extended coil (D2O)1648 - 1657 α-helix1660 triple helix1670 - 1695 β-sheet, β-turn
Collagen(triple helix)
Myoglobin(α-helix)
Abeta Amyloid(α-helix + β-sheet)
Cytochrome c(α-helix + coil)
STI(β-sheet + coil)
1600 1650 1700 Frequency (cm-1)
A
B
C
D
E
A
B
C
D
E
U.S. DEPARTMENT OF ENERGY
OFFICE OF BASIC ENERGY SCIENCES
N A T IO N AL SY N C H R O T RO N LIG H T SO U RC E
O P E R A T E D B Y B R O O K H A V E N S C IE N C E A S S O C IA T E S
IR Spectroscopy vs. Micro-SpectroscopyIR Spectroscopy vs. Micro-Spectroscopy
Bulk samplesKBr
solid+
Spatial Resolution:~1-3 mm
+solution
Microscopic Heterogeneity
Spatial Resolution:~ 25-30 µm (globar)~ 5-10 µm (synchrotron)
U.S. DEPARTMENT OF ENERGY
OFFICE OF BASIC ENERGY SCIENCES
N A T IO N AL SY N C H R O T RO N LIG H T SO U RC E
O P E R A T E D B Y B R O O K H A V E N S C IE N C E A S S O C IA T E S
Why SYNCHROTRON Infrared Microspectroscopy?Why SYNCHROTRON Infrared Microspectroscopy?
100 µm 20 µm
Biological sample are SMALL:• spatially• in concentration
BRIGHTNESS (1000x)
Higher Spatial Resolution
Better Signal-to-Noise
Faster Data Collection
Spectroscopy
+
BROADBAND
Synchrotron Advantages:
U.S. DEPARTMENT OF ENERGY
OFFICE OF BASIC ENERGY SCIENCES
N A T IO N AL SY N C H R O T RO N LIG H T SO U RC E
O P E R A T E D B Y B R O O K H A V E N S C IE N C E A S S O C IA T E S
Single Red Blood Cell: Globar vs. Synchrotron SourceSingle Red Blood Cell: Globar vs. Synchrotron Source
Diffraction Limit4000 cm-1 2.5 µm2950 cm-1 3.4 µm1650 cm-1 6.1 µm1200 cm-1 8.3 µm1000 cm-1 10 µm600 cm-1 17 µm200 cm-1 50 µm
• Synchrotron IRMS is diffraction-limited
• Conventional IRMS is throughput-limited
10 µm
1000 2000 3000 Frequency (cm-1)
5x5 µm
synchrotron
globar
P/P noise:0.0038
0.0575
0 10 20 30 40 50 60 700
4
8
12
16
MC
T-A
* Sig
nal (
V)
Aperture size (µm)
synchrotron
globar
U.S. DEPARTMENT OF ENERGY
OFFICE OF BASIC ENERGY SCIENCES
N A T IO N AL SY N C H R O T RO N LIG H T SO U RC E
O P E R A T E D B Y B R O O K H A V E N S C IE N C E A S S O C IA T E S
Beamline U10BBeamline U10B
Synchrotron infrared beam pipe
Nicolet Magna 860 FTIR
Spectra Tech ContinuµmIR microscope
Filter cube turret
UV (quartz) light source
Schwarzchild 32x IR objective
Automated mapping stage
U.S. DEPARTMENT OF ENERGY
OFFICE OF BASIC ENERGY SCIENCES
N A T IO N AL SY N C H R O T RO N LIG H T SO U RC E
O P E R A T E D B Y B R O O K H A V E N S C IE N C E A S S O C IA T E S
Chemical Content of New Bone in OsteoporosisChemical Content of New Bone in Osteoporosis
Normal illumination
Abso
rban
ce
0.5 OD
(1)
(2)
(3)
1800 1400 1000 600Frequency (cm-1)
(4)
(5)
(6)ALIZARIN(t=2 yr.)
CALCEIN (t=1 yr.)
Fluorescence illumination
• Rate of bone mineral formation is slower in osteoporosis
L.M. Miller, J. Tibrewala, C.S. Carlson. Cell. Mol. Biol., 46:1035-44 (2000).
U.S. DEPARTMENT OF ENERGY
OFFICE OF BASIC ENERGY SCIENCES
N A T IO N AL SY N C H R O T RO N LIG H T SO U RC E
O P E R A T E D B Y B R O O K H A V E N S C IE N C E A S S O C IA T E S
Prion Protein Structure and LocationPrion Protein Structure and Location
1620 1660 1700
10 µm
Frequency (cm-1)
• Hamster scrapie 263K• Dorsal root ganglia• Terminally infected
U.S. DEPARTMENT OF ENERGY
OFFICE OF BASIC ENERGY SCIENCES
N A T IO N AL SY N C H R O T RO N LIG H T SO U RC E
O P E R A T E D B Y B R O O K H A V E N S C IE N C E A S S O C IA T E S
Chemical Imaging of HairChemical Imaging of Hair
PhospholipidsPhospholipidsPhospholipidscuticle (3-5 µm)
cortex (40-100 µm)
medulla (5-10 µm)
ProteinsProteinsProteins
U.S. DEPARTMENT OF ENERGY
OFFICE OF BASIC ENERGY SCIENCES
N A T IO N AL SY N C H R O T RO N LIG H T SO U RC E
O P E R A T E D B Y B R O O K H A V E N S C IE N C E A S S O C IA T E S
Protein Aggregate Formation in Alzheimer’s PlaquesProtein Aggregate Formation in Alzheimer’s Plaques
• misfolded, aggregated protein is associated with Abeta plaques 2001000 300
200
100
0
Microns
0.0
1.0
L.M. Miller, P. Dumas, N. Jamin, J.-L. Teillaud, J. Miklossy, L. Forro (2002). Rev. Sci. Instr., 73: 1357-60.
0.00
0.10
0.20
0.30
0.40
0.50
Log
(1/R
)
1400 1500 1600 1700 1800 Frequency (cm-1)
NORMAL
MISFOLDED
U.S. DEPARTMENT OF ENERGY
OFFICE OF BASIC ENERGY SCIENCES
N A T IO N AL SY N C H R O T RO N LIG H T SO U RC E
O P E R A T E D B Y B R O O K H A V E N S C IE N C E A S S O C IA T E S
Metal Accumulation in Alzheimer’s PlaquesMetal Accumulation in Alzheimer’s Plaques
Cu Kα
Zn Kα
• Zn and Cu are associated with Abeta plaques
U.S. DEPARTMENT OF ENERGY
OFFICE OF BASIC ENERGY SCIENCES
N A T IO N AL SY N C H R O T RO N LIG H T SO U RC E
O P E R A T E D B Y B R O O K H A V E N S C IE N C E A S S O C IA T E S
Correlation Between Metal Accumulation and Protein AggregationCorrelation Between Metal Accumulation and Protein Aggregation
Zn
Abeta
misfolded protein
Zn + Abeta
Abeta + misfolded protein
Zn + misfolded protein
Metal
Thioflavin SFluorescence
MisfoldedProtein
composite
U.S. DEPARTMENT OF ENERGY
OFFICE OF BASIC ENERGY SCIENCES
N A T IO N AL SY N C H R O T RO N LIG H T SO U RC E
O P E R A T E D B Y B R O O K H A V E N S C IE N C E A S S O C IA T E S
Chemical Imaging of Mitotic CellsChemical Imaging of Mitotic Cells
Max
Min-10 0 10 20 300 5 10 15 20 25 30 350
5
10
15
20PROTEIN (1650 cm-1)
-10 0 10 20 300 5 10 15 20 25 30 350
5
10
15
20LIPIDS (2925 cm-1) Max
Min
OPTICAL IMAGE
10 µm 10 µm
N. Jamin, J.L. Teillaud, P. Dumas, G.L. Carr, G.P. Williams (1998). PNAS 95: 4837-40.
U.S. DEPARTMENT OF ENERGY
OFFICE OF BASIC ENERGY SCIENCES
N A T IO N AL SY N C H R O T RO N LIG H T SO U RC E
O P E R A T E D B Y B R O O K H A V E N S C IE N C E A S S O C IA T E S
Chemical Changes in Apoptotic CellsChemical Changes in Apoptotic Cells
ell
ptosis l)
ptosis ell)
108512
40
1541
1651
1043
1175
1223
1455
1539
1655
1043
1175
1223
1455
154316
55
1000 3000 Frequency (cm-1)
2000
Abs
orba
nce
20 µm
20 µm
1175-1180 cm-1
5 µm
How does apoptosis proceed in real time?
L.M. Miller, P. Dumas, N. Jamin, J.-L. Teillaud, J. Miklossy, L. Forro (2002). Rev. Sci. Instr., 73: 1357-60.
U.S. DEPARTMENT OF ENERGY
OFFICE OF BASIC ENERGY SCIENCES
N A T IO N AL SY N C H R O T RO N LIG H T SO U RC E
O P E R A T E D B Y B R O O K H A V E N S C IE N C E A S S O C IA T E S
Impact of NSLS-II: Schematic Brightness ComparisonImpact of NSLS-II: Schematic Brightness Comparison
Frequency/Photon Energy (log scale)
Brig
htne
ss (
log
scal
e)
NSLS-II (500ma, 20mr)
VUV (700ma)
40m
r
90m
r
Mid-IRFar-IRTHz
400
cm-1, 2
5 µm
, 50
meV
4000
cm
-1, 2
.5 µ
m, 0
.5 e
V• Mid IR: NSLS-II will have equal brightness to NSLS • Far-IR and multi-point imaging: disadvantaged by NSLS-II
X-ray
Infrared
Opening Angle
IR flux is primarily dependent upon ring current in mid-IR
U.S. DEPARTMENT OF ENERGY
OFFICE OF BASIC ENERGY SCIENCES
N A T IO N AL SY N C H R O T RO N LIG H T SO U RC E
O P E R A T E D B Y B R O O K H A V E N S C IE N C E A S S O C IA T E S
SummarySummaryOverview
• Infrared microspectroscopy and imaging can be used to image the chemical makeup of biological tissues and cells
• The spatial resolution of synchrotron IRMS is diffraction-limited at 2-20 µm
Importance of Research• Chemical imaging of mineralized tissues, in situ protein, lipid, nucleic acid
content, protein structure
Scientific Challenges and Opportunities• Multi-technique imaging: combining x-ray, visible, IR imaging techniques• Improve spatial resolution• Improve data collection rates
Impact of NSLS-II• NSLS-II brightness will slightly improve the mid-IR imaging capabilities• NSLS-II will prevent the creation of large opening-angle beamlines for (1) far-IR
applications, and (2) line-source (i.e. multi-point) imaging
Synergy and Demand• Combination of IRMS with x-ray imaging techniques• Currently 4 IRMS bending magnet beamlines; need at least this many for NSLS-II
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