Optical imaging of blood flow in the
microcirculation
Steve Morgan
Electrical Systems and Optics Research Division,
University of Nottingham, UK
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
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.
Capillaroscopy (Sub-lingual)
Sickle Cell Anaemia
• 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
In vitro sickled RBCs
DA Beach, C Bustamante, KS Wells, and KM Foucar,
Biophys. J 53, pp449-456 (1988)
Dichroism signal ~3%
SSDF Imaging
Illuminate from the side to ‘back-illuminate’ RBCs
Imaging System
Illumination and Probe Design
Conventional SSDF
Focus
CCD
Polarization sensitive
Focus
CCD
H
V
Patient Station
Polarization Images (lower lip)
Image alignment
Image alignment
Dy Dx
Image segmentation
Segmentation
LD
Determination
#%
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
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
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
Single point blood flow imaging
Originally single point measurement system, measuring
doppler shift from moving RBCs (20Hz – 20KHz)
Image - Moor Instruments
Scanning System
Builds up image point by point, slow
Image - Moor Instruments
Field Programmable Gate Array based systems
64x1 photodiode
array
FPGA implements N-point FFT and
frequency weighting
Parallel processing
moorLDLS2
FPGA based systems
• Sampling rate
40KHz/pixel, 1024
point FFT
13
10
Occlusion & Release of a Finger
Black ground noise
8
Flux [a.u.]
• Occlusion and release
test for a single pixel
Flux trace
x 10
6
4
2
0
0
5
10
15
Time [s]
• 64 x 64 image
(3s/image)
20
25
FPGA based systems
(forearm)
In collaboration with Moor Instruments
FPGA based systems
(back of hand)
In collaboration with Moor Instruments
Commercial CMOS camera systems, (Serov et al)
• High readout rate CMOS camera
• Requires high data rate between sensor and processor
Commercial CMOS camera, Serov et al
Proc. SPIE Vol. 6080 608004-1
•
•
•
•
•
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
Smart CMOS sensors
Processing
electronics
• Arrays of photodetectors with on- chip processing
• Fabricated using a standard CMOS process
• Can be tailored to signals of interest
• Compact, portable design
Off-Chip processing of Doppler signals
(single channel)
Optical detection
& linear
amplification
Band pass
filter
Low pass
filter
Square and
Average
Divider
Frequency
weighted
filter 0.5
Square and
Average
Concentration
Flow
• For full field requires each pixel to be sampled at 40KHz
and transferred to a processor
• High data rate required
Beclaro (1994), Laser Doppler, Med-Orion.
On-Chip Processing of Doppler signals
Optical
detection
(normalized)
HDA
Band pass
filter
Frequency
weighted filter
•
•
•
•
ADC
Concentration
Absolute
and
Average
ADC
Absolute Flow
and
Average
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
64x64 array
• pixel size = 55μm x 55μm, 2~3 speckles per pixel
• 4 ADCs and on-chip processing
Test configuration, vibrometer
• Provides a reproducible, predictable source of Doppler signals
Test configuration, vibrometer
(Hz)
Frequency:
450Hz left, 350Hz right
(m)
Amplitude :
200mV left, 350mV right
• can discriminate different frequencies and
amplitudes
• change in amplitude along length
Rotating diffuser tests
PC + ADC
20mm
32x32 array
100mm

Lens B
Rotating
Laser
Lens A
Static, 20mm diameter
Rotating diffuser tests
Concentration
Flow
Blood flow tests (64 x 64 pixels)
PC
64x64 array
Lens B
5mW laser
diffuser
Unoccluded
Occluded
IR and VR combined laser
FPGA and USB board
Mirror
Blood flow sensor board
DC camera
Lens
Beam splitter
diffuser
Blood flow video
Actual frame rate: 1 frame/second
before
20 mins
10 mins
30 mins
Other techniques
•
•
•
•
•
in vivo flow cytometry
photoacoustic imaging
Doppler OCT
Laser speckle contrast analysis
hyperspectral imaging
In vivo flow cytometry
Georgakoudi et al
Cancer Researh 64, 5044–
5047, 2004
Line illumination count
fluorescent fluctuations of
labelled cells
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
Doppler OCT
(Makita et al opt express 14:7821 (2006)
• Short coherence length interferometry overcomes scattering
• Imaging of retinal vessels
Laser Speckle Contrast Imaging
(alternative to laser doppler)
Chick embyro heart
(Moor Instruments)
• Full field imaging
• Indirect measure of fluctuations
• Reduction in spatial resolution, spatial averaging
Hyperspectral imaging
• Imaging oxygen saturation
• Inflammatory response
• retinal imaging
• endoscopy
100%
50%
Summary
Techniques for when cells are superficial and when they
are obscured by overlying tissue