pasquale emanuele scopelliti silicon pixel detectors for crystallography and imaging of biological...
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![Page 1: Pasquale Emanuele Scopelliti Silicon pixel detectors for crystallography and imaging of biological samples On behalf of: A. Bulgheroni, M. Caccia, Università](https://reader035.vdocuments.mx/reader035/viewer/2022081519/56649ef05503460f94bfff29/html5/thumbnails/1.jpg)
Pasquale Emanuele Scopelliti
Silicon pixel detectors for
crystallography
and imaging of biological samples
On behalf of: A. Bulgheroni, M. Caccia,
Università degli Studi dell’Insubria
C. Cappellini, F. Risigo, M. Jastrzab
SUCIMA collaboration
Medipix collaboration: B. Mikulec,
September 24-29 - Perugia
Lukas Tlustos SIMBASilicon Innovative Monitors for
Biomedical Applications
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Previous experiences
and knowledge in
the HEP domain
Applications
Crystallography
Bio-sample imaging
• Imaging of tritium labelled biological sample
• Protein microarray analysis and surface R&D
• Quantum imagingwith energy weighting
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• Monolithic • squared pixels, 17 m pitch• 512 x 512 pixel matrix• analog output• 30 e- noise• 50 ke- dynamic range• 10 m thickness sensitive volume• readout frequency ~ 20 MHz
Silicon pixel detectors
MIMOSA V – back-thinned
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Molecular structure reconstruction
Application in crystallography
• Monochromatic X-ray beam
• Elastic scatter produce a interference pattern
• Molecular single crystal
• Crystalline structure can be resolved from peak position
• Electron density distribution in a crystalline cell can be reconstructed from peak intensities
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The challenge is a single crystal elemental analysis
mounted on the diffractometer head
Application in crystallography
?ng~ The choice of the sample is a
stochastic procedure
X-ray Diffraction X-ray Fluorescence
80
1%
10 mm²
100
NO
point-like
Scintillator Si(Li) QE
Energy resolution %
Area
QE
Energy resolution %
Area
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• 10 minutes
• Some grams of sample
• Energy Resolution 300 eV
XRF Si(Li) commercial detector result – 4 g molecules with Cu and Br
Application in crystallography
Present in air
X –ray source
Elements in the crystal
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Setup
Monochromatic X-ray beam
17.4 keV
Tunable intensity
5 mA < I < 40 mA
20 kV < V < 40 kV
Application in crystallography
X-ray tube
collimator
cristal
MIMOSA 5
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Application in crystallography
12 h (30kevents) = 12.5 min Effective Exposure Time
1 h 5 kevent – 125 sec EET
• Cluster spectrum is good enough to determine how many elements are in the crystal
• Precise energy estimation is difficult because of charge collection inefficiency
• This analysis can be extremely fast
• This analysis can be enough for the crystallographer to confirm or refuse his hypothesis about the sample
• You can have something much more precise, but you need high statistics
Cluster spectrum
Cu
ArBr
Mo
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Application in crystallography
Ratio seed/cluster charge collected
• Select 100% charge collection efficiency events
• Less than the 10 % of total events
• In theory 1 pixel cluster events ?
Seed spectrum 12 h
This effects is under investigation
• Temperature effect
• Left over
• Physical charge sharing
Ar Cu Br Mb
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Application in crystallography
1 2
1 2
2
1
43
3
4
Peak(keV) Element(keV)
11.91 + 0.32 Br 11.92
8.09 + 0.35 Cu 8.05
2.61 + 0.37 Ar 2.9
17.01 + 0.41 Mo 17.4
• All compatible
• Energy resolution 350 eV !!!
3
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Conclusions
• Direction where to go
Cooling
Application in crystallography
• Two analysis possibilities
Fast and dirty
Longer and accurate350 eV energy resolution
Duty cycle
• Note Companies extremely interested
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3H 14C 32P 33P
3H is better and also the most challenging
Biological samples Imaging
Sensitive volume
Image blurring
MPV 3.8 keV
End point 18.6 keV
Mean 5.7 keV
θ
Autoradiography
Sample
Requirements for the application
• High sensitivity to low energy electrons
• Fast read-out
• High imaging capability
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• Phosphor imaging plate
Sensitivity two orders of magnitude higher respect with films
Reusable
5 orders of magnitudes dynamic range
Low image resolution in case of low energy source
Only one label detectable
Existing devices
Biological samples Imaging
• Films
Very poor detection efficiency. Weeks of exposition are needed
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Minimal sensitivity
Tritium Imaging
Tritium standards Slide with 14 dots
4X5 mm2
9.8 kBq 7.4 10-2kBq
4.86 kBq 4.0 10-2 kBq
2.76 kBq 2.1 10-2kBq
1.26 kBq 1.1 10-2kBq
7.3 10-1kBq 5.8 10-3kBq
3.3 10-1kBq 2.8 10-3kBq
1.6 10-1kBq 0 kBq
2.8 10-3kBq
12 h 100,000 frames – 9200 hits
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Tritium ImagingImaging in function of z
0.60 mm 0.75 mm
1 mm 1.25 mm 1.5 mm
9.8 kBq Tritium dot activity
n° o
f hits
per
fram
e
distance [mm]
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Tritium Imaging
Image quality
• Take the projection of the dot
• Calculate the slope of the projection
• The width of the slope function in corrispondence of the dot edges is a figure of merite of the image quality
Distance [mm]Distance [mm]
pixe
l
pixe
l
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Tritium Imaging
Spectrum in function of z
0.62 mm 0.74 mm 1 mm 1.25 mm 1.5 mm
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Tritium Imaging
3H vs. 14C Distance 0.2 mm
Spatial resolution = 115 micron
Spatial resolution = 340 micron
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Tritium Imaging
Conclusions for tritium imaging
• Better image quality with 3H respect with 14C Importance of low-energy source Importance of thin sensitive volume
• The distance from the source is critical for: Image quality Spectrum quality Efficiency
• Sensitivity of the sensor is very high up to 2.8 10-3 kBq It is able to cover the range intensity needed in almost all the applications
• Next step Imaging of a real sample
•High spatial resolution with tritium 115 micronAt least comparable with other devices
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• Spot dimensions are 50-200 microns diameter
Protein microarray
• There are 106 different proteins produced in human cells
• You need to study proteins properties and interactions
• You need high troughput low-cost analysis instrument
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Fluorescence analysis
PMT
• No imaging capability
• Low-sensitivity
CCD
Protein microarray
• Single photon sensitivity
• Low QE = 20 %
• Imaging capability
• High QE
Requirements
• High QE
• High spatial resolution
• Imaging capability
• Fast readout
• Low-cost
• Scanning tecnique
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Protein microarray
Two different isotopes with different decay energies replace the fluorescent markers
32P
33P
1710 keV 695 keV
249 keV 76 keV
End point Mean energy
Separablespectra
MAPS
• Single dacay sensitivity
• Real time
• High spatial resolution
• High image quality
Nano patterned surfaces
• High density
• High functionality
• Stability of the process
• High QE =100%
• Riproducibility
• Specific immobilisation
• Technique exploits CMOS sensors and nanostructured surfaces to detect radiolabelled proteins.
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PEG (Poly Ethylene Glycol)
PAA (Poly Acrylic Acid)
Protein microarray – Surface
Traditional surface
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
NH 2
NH2
NH2
NH 2
NH2
NH2
NH2
NH2
NH 2
NH2
NH2
NH 2
NH2
NH2
NH2
NH2
NH 2
NH2
NH2
NH 2
NH2
NH2
NH2
NH2
NH 2
NH2
NH2
NH 2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH 2
-COOH + -NH2 = CON
H
+ H2O
Amminic Binding
Activation Incubation Washing
• pH solution
• Temperature
• Time of reaction
• pH solution
• Temperature
• Time
• EDC and NHS quantity
• Temperature
• Method
• Solution
Thanks to Dr. Mila Silvia
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PEG (Poly Ethylene Glycol)
PAA (Poly Acrylic Acid)
Surfaces by Joint Research Center – Ispra (Italy)
Protein microarray – Surface
Nanocraters
Valsesia et al. Adv. Funct. Mater. 2006,16, 1242
Atomic Force Miscroscope Imaging
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Flu
ores
cenc
e (a
.u.)
Scan direction (nm)
Fluorescent confocal microscope BSA (Bovine Serum Albumin)
Protein microarray – Surface
Valsesia et al. Adv. Funct. Mater. 2006,16, 1242
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• Hybrid• squared pixels, 55 m pitch• 256 x 256 pixel matrix• leakage current compensation• energy windowing with lower and upper thresholds, tunable on each pixel by a 3 bit DAC • 13 bit counter • max counting frequency ~ 1MHz• max readout frequency ~ 100 MHz• 250 m thickness sensitive volume
Medipix2
Best results obtained
Protein microarray - Results
• Medipix
Because very fast and real time
• Millimetric spot
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Best results obtained
Protein microarray - Results
Y projection X projection
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Protein microarray
Conclusions for protein microarray application
• High sensitive low-cost detection method is needed in this field in order to push surface R&D
• Results with the Medipix are very encouraging from the point of view of the sensor
• Surface properties investigation is going on
• Measure with MIMOSA 5 are coming
• Mesure with two separable spectra markers: energy weighted imaging
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• non HEP applications may really be a great fun!
• HEP sensors most often are NOT what you really need but they are the “workhorse” for a demonstrator program and define the guidelines for application specific developments
Final Conclusion
• Thanks to the collegues of Department of Structural & Functional Biology, University of Insubria and to Ispra JRC researchers.
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The End
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Immobilisation chemestry
bAckUP
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bAckUP
Example of diagnostic application of microarray
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Calibration
G = 16.6 ± 0.6 e-/ADC
CCE = 73%
Application in crystallography- Imaging
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Application in crystallography- Imaging
X-ray tube
collimator
attenuator
cristal
nail
MIMOSA 5I = 3.9 keV
Pd = 2.8 keV
EQ FY
70% 0.1
Lα
0.0585%
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Diffraction peaks
S/B increased by a factor 10
Application in crystallography- Imaging
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bAckUP
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bAckUP