Pasquale Emanuele Scopelliti Silicon pixel detectors for crystallography and imaging of biological...

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

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

• 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

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

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

• 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

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

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

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

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

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

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

• 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

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

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]

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

Tritium Imaging

Spectrum in function of z

0.62 mm 0.74 mm 1 mm 1.25 mm 1.5 mm

Tritium Imaging

3H vs. 14C Distance 0.2 mm

Spatial resolution = 115 micron

Spatial resolution = 340 micron

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

• 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

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

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.

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

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

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

• 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

Best results obtained

Protein microarray - Results

Y projection X projection

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

• 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.

The End

Immobilisation chemestry

bAckUP

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Example of diagnostic application of microarray

Calibration

G = 16.6 ± 0.6 e-/ADC

CCE = 73%

Application in crystallography- Imaging

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%

Diffraction peaks

S/B increased by a factor 10

Application in crystallography- Imaging

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