Optical imaging of blood flow in the microcirculation

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) • x 5/x 10 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, pp 449 -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 (33 s 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 (20 Hz – 20 KHz) Image - Moor Instruments

Scanning System Builds up image point by point, slow Image - Moor Instruments

Field Programmable Gate Array based systems 64 x 1 photodiode array FPGA implements N-point FFT and frequency weighting Parallel processing moor. LDLS 2

FPGA based systems • Sampling rate 40 KHz/pixel, 1024 point FFT • Occlusion and release test for a single pixel • 64 x 64 image (3 s/image)

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. 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 8 KHz (ideally ≥ 10 bit, 40 KHz) 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 40 KHz 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 Absolute and Average ADC Concentration Absolute Flow and Average Design modified for efficient use of silicon on-chip Only flow and concentration output (low bandwidth) 16 x 1, 4 x 4, 32 x 32 prototypes developed tailored to signals e. g. HDA amplifies ac by x 40, dc by unity

64 x 64 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: 450 Hz left, 350 Hz right (m) Amplitude : 200 m. V left, 350 m. V right • can discriminate different frequencies and amplitudes • change in amplitude along length

Rotating diffuser tests

Rotating diffuser tests Concentration Flow

Blood flow tests (64 x 64 pixels) 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

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