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Optical imaging of blood flow in the microcirculation
Steve MorganElectrical Systems and Optics Research Division,
University of Nottingham, UK
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Imaging the microcirculation
Imaging when superficial tissue is relatively thin• eye, mouth, nail fold• cells can be visualized• capillaroscopy for sickle cell anaemia
Imaging when superficial tissue is relatively thick• skin • indication of flow in the microcirculation• full field laser doppler blood flowmetry
• Other techniques
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Capillaroscopy
• Find a site where there is very little scattering
• ‘Windows’ (eye, nailfold, under tongue, lower lip)
• x5/x10 microscope objective
• Polarized light capillaroscope
• Aim to detect dichroic (sickled) red blood cells in sickle cell anaemia.
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Capillaroscopy (Sub-lingual)
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• Genetic disorder affecting RBCs
• Haemoglobin polymerizes on de-oxygenation– Polymerisation on a cellular and sub-cellular level
• Effects–Painful Crises–Organ Damage
• Currently no in-vivo assessment
Sickle Cell Anaemia
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DA Beach, C Bustamante, KS Wells, and KM Foucar, Biophys. J 53, pp449-456 (1988)Dichroism signal ~3%
In vitro sickled RBCs
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SSDF Imaging
Illuminate from the side to ‘back-illuminate’ RBCs
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Imaging System
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Illumination and Probe Design
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Conventional SSDF
CC
D
Focus
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Polarization sensitive
CC
D
H V
Focus
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Patient Station
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Polarization Images (lower lip)
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Image alignment
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Image alignment
xy
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Image segmentation
Segmentation
#%
LD Determination
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Capillaroscopy summary
• Camera sensitive to changes in polarization ~0.5% but dichroism not observed in vivo.
• instrumentation; resolution, dynamic range
• Clinical reason? Just isn’t present under the tongue or to the extent observed in vitro
• future – increase magnification, CMOS cameras, single cell oxygenation
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Full field laser Doppler blood flow imaging
Imaging when superficial tissue is relatively thick• skin • indication of flow in the microcirculation• full field laser doppler blood flowmetry• Inflammatory responses, wounds, vein viewing
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Full field laser Doppler blood flow imaging
vascular response to an intradermal injection of 20 µl of 1 µM histamine into the volar surface of the forearm of a healthy volunteer (33s intervals).
Image – GF Clough, MK Church, University of Southampton
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Single point blood flow imaging
Originally single point measurement system, measuring doppler shift from moving RBCs (20Hz – 20KHz)
Image - Moor Instruments
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Scanning System
Builds up image point by point, slow
Image - Moor Instruments
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Field Programmable Gate Array based systems
FPGA implements N-point FFT and frequency weightingParallel processing
64x1 photodiodearray
moorLDLS2
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FPGA based systems
• Sampling rate 40KHz/pixel, 1024 point FFT
• Occlusion and release test for a single pixel
• 64 x 64 image (3s/image)
0 5 10 15 20 250
2
4
6
8
10x 10
13 Flux trace
Time [s]
Flu
x [a
.u.]
Occlusion & Release of a Finger
Black ground noise
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FPGA based systems(forearm)
In collaboration with Moor Instruments
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FPGA based systems(back of hand)
In collaboration with Moor Instruments
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Commercial CMOS camera systems, (Serov et al)
• High readout rate CMOS camera • Requires high data rate between sensor and processor
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Commercial CMOS camera, Serov et al
• Full field imaging• Uses commercial CMOS camera and processing on a PC• Requires high data rate between sensor and processor• Data restricted to 8 bit at 8KHz (ideally ≥ 10bit, 40KHz)• No anti-aliasing filter
Proc. SPIE Vol. 6080 608004-1
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• Arrays of photodetectors with on- chip processing• Fabricated using a standard CMOS process • Can be tailored to signals of interest
• Compact, portable design
Smart CMOS sensors
Processing electronics
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Off-Chip processing of Doppler signals(single channel)
Low passfilter
Band passfilter
Divider
Frequency weighted filter 0.5
Square andAverage
ConcentrationSquare and
Average
Flow
Optical detection & linear amplification
Beclaro (1994), Laser Doppler, Med-Orion.
• For full field requires each pixel to be sampled at 40KHzand transferred to a processor• High data rate required
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On-Chip Processing of Doppler signals
• Design modified for efficient use of silicon on-chip• Only flow and concentration output (low bandwidth)• 16x1, 4x4, 32x32 prototypes developed• tailored to signals e.g. HDA amplifies ac by x40, dc by unity
HDAOptical detection
(normalized)
Frequency weighted filter
Absolute and
Average
ConcentrationAbsolute
andAverage
Flow
ADCBand passfilter
ADC
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64x64 array
• pixel size = 55μm x 55μm, 2~3 speckles per pixel• 4 ADCs and on-chip processing
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Test configuration, vibrometer
• Provides a reproducible, predictable source of Doppler signals
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Test configuration, vibrometer
• can discriminate different frequencies and amplitudes• change in amplitude along length
Frequency:450Hz left, 350Hz right
Amplitude :200mV left, 350mV right
(Hz) (m)
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Rotating diffuser tests
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Rotating diffuser tests
Concentration Flow
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Blood flow tests (64 x 64 pixels)
Unoccluded Occluded
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diffuser
Blood flow sensor board
FPGA and USB board
IR and VR combined laser
Mirror
Lens
DC camera Beam splitter
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Blood flow video
Actual frame rate: 1 frame/second
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before 10 mins
30 mins20 mins
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Other techniques
• in vivo flow cytometry• photoacoustic imaging• Doppler OCT• Laser speckle contrast analysis• hyperspectral imaging
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In vivo flow cytometry
Georgakoudi et al Cancer Researh 64, 5044–5047, 2004
Line illumination count fluorescent fluctuations of labelled cells
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Photoacoustic imaging(wang JBO 15:011101-9 (2010)
• Use light to excite u/s in tissue• Used to image vessels but also blood cells• Also Doppler version
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Doppler OCT(Makita et al opt express 14:7821 (2006)
• Short coherence length interferometry overcomes scattering• Imaging of retinal vessels
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Chick embyro heart(Moor Instruments)
Laser Speckle Contrast Imaging(alternative to laser doppler)
• Full field imaging• Indirect measure of fluctuations• Reduction in spatial resolution, spatial averaging
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Hyperspectral imaging
• Imaging oxygen saturation
• Inflammatory response
• retinal imaging
• endoscopy
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Summary
Techniques for when cells are superficial and when they are obscured by overlying tissue