Optical imaging of blood flow in the microcirculation

Steve MorganElectrical 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)

• 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

DA Beach, C Bustamante, KS Wells, and KM Foucar, Biophys. J 53, pp449-456 (1988)Dichroism signal ~3%

In vitro sickled RBCs

SSDF Imaging

Illuminate from the side to ‘back-illuminate’ RBCs

Imaging System

Illumination and Probe Design

Conventional SSDF

CC

D

Focus

Polarization sensitive

CC

D

H V

Focus

Patient Station

Polarization Images (lower lip)

Image alignment

Image alignment

xy

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

FPGA implements N-point FFT and frequency weightingParallel processing

64x1 photodiodearray

moorLDLS2

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

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

• 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

• 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

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

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

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

• can discriminate different frequencies and amplitudes• change in amplitude along length

Frequency:450Hz left, 350Hz right

Amplitude :200mV left, 350mV right

(Hz) (m)

Rotating diffuser tests

Rotating diffuser tests

Concentration Flow

Blood flow tests (64 x 64 pixels)

Unoccluded Occluded

diffuser

Blood flow sensor board

FPGA and USB board

IR and VR combined laser

Mirror

Lens

DC camera Beam splitter

Blood flow video

Actual frame rate: 1 frame/second

before 10 mins

30 mins20 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

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

Hyperspectral imaging

• Imaging oxygen saturation

• Inflammatory response

• retinal imaging

• endoscopy

Summary

Techniques for when cells are superficial and when they are obscured by overlying tissue