When laser light illuminates a diffuse object, it produces a random interference effect known as a speckle pattern. If there is movement in the object, the speckles fluctuate in intensity. These fluctuations can provide information about the movement. A simple way of accessing this information is to image the speckle pattern with an exposure time longer than the shortest speckle fluctuation time scale—the fluctuations cause a blurring of the speckle, leading to a reduction in the local speckle contrast. Thus, velocity distributions are coded as speckle contrast variations. The same information can be obtained by using the Doppler effect, but producing a two-dimensional Doppler map requires either scanning of the laser beam or imaging with a high-speed camera: laser speckle contrast imaging (LSCI) avoids the need to scan and can be performed with a normal CCD- or CMOS-camera. LSCI is used primarily to map flow systems, especially blood flow. The development of LSCI is reviewed and its limitations and problems are investigated.
The retina/choroid structure is an example of a complex biological target featuring highly perfused tissues and vessel flows both near the surface and at some depth. Laser speckle imaging can be used to image blood flows but static scattering paths present a problem for extracting quantifiable data. The speckle contrast is artificially increased by any residual specular reflection and light paths where no moving scatterers are encountered. Here we present results from phantom experiments demonstrating that the static and dynamic contributions to laser speckle contrast can be separated when camera exposures of varying duration are used. The stationary contrast parameter follows the thickness and strength of the overlying scatterer while the dynamic proportion of the scatter resulting from vessel flows and Brownian motion is unchanged. The importance of separating the two scatter components is illustrated by <i>in vivo</i> measurements from a scarred human retina, where the effect of the un-perfused scar tissue can be decoupled from the dynamic speckle from the intact tissue beneath it.
How does speckle contrast K, measured at camera exposures T around 10 ms, give us information about temporal autocorrelation of the speckle pattern with time constants τ < 1 ms, corresponding to Doppler shifts in the KHz range? We explore the implications of this question and show that for any particular assumed temporal speckle autocorrelation function, K measured at T >> τ accurately measures τ, but that K measurements at T < τ are required in order to determine the actual shape of the autocorrelation function. Determining the shape of the autocorrelation function is important if we wish to distinguish between different types of flow or movement in tissue, for example distinguishing Brownian motion or the randomly-oriented flows in capillary networks from more ordered flow in resolvable vessels.
Measurements of flow in retinal vessels is presented and compared with in vitro measurements on whole blood in
capillaries ranging from 75 to 200μm diameter. The viewing angle of the capillaries and their range of size allows size-dependent
effects to be investigated when estimating flow within actual vessels.
Retinal measurements show a pulse effect. When this is removed by synchronisation, multi-exposure measurements
show different spectral signature from single speed scatterers. Multi-exposure measurements of the retina demonstrate a
varying contribution of stationary scatter across the field. Unlike scattering in dermal tissue, photons in retinal vessels
must return by multiple scatter from moving Red Blood Cells, whose motion is directed. Speckle estimates of flow in
retinal vessels are therefore possible.
Laser speckle and laser Doppler perfusion measurements apply different analyses to the same physical phenomenon and
so should produce the same results. However, there is some evidence that laser Doppler can measure perfusion at greater
depths than laser speckle. Using phantom measurements and comparison to spatially modulated imaging, we show why
this might be the case.
Various implementations of imaging laser Doppler and speckle systems have different optical setups, producing different
effective distances between the illumination and detector points on the surface of the tissue. Separating the effective
source and detector regions in tissue measurements biases the measurements towards deeper tissues, and when the
effective source and detector regions coincide, the measurement is biased towards surface tissues. Probe-based or
scanning laser Doppler systems with point illumination can separate the source and detector regions to interrogate deeper
tissues, while whole-field imaging laser Doppler systems and laser speckle contrast systems have broad illumination
covering the measurement areas. The volume of tissue informing a measurement at any point in a whole-field system,
and hence the depth sensitivity, is determined by the optical properties of the tissue at the working wavelength.
Recent success in reconciling laser Doppler and speckle measurements of dermal perfusion by the use of multi-exposure
speckle has prompted an investigation of speckle effects arising from directed blood flow which might be expected in the
small blood vessels of the eye.
Unlike dermal scatter, the blood in retinal vessels is surrounded by few small and stationary scatterers able to assist the
return of light energy by large-angle scatter. Returning light is expected to come from multiple small angle scatter from
the large red blood cells which dominate the fluid.
This work compares speckle measurements on highly scattering skin, with measurements on flow in a retinal phantom
consisting of a glass capillary which is itself immersed in an index matching fluid to provide a flat air-phantom interface.
Brownian motion dominated measurements when small easily levitated scatters were used, and flow was undetectable.
With whole-blood, Brownian motion was small and directed flows in the expected region of tens of mm/s were
detectable. The nominal flow speed relates to the known pump rate; within the capillary the flow will have a profile
reducing toward the walls.
The pulsatile effects on laser speckle contrast in the retina are discussed with preliminary multi-exposure measurements
on retinal vessels using a fundus camera. Differences between the multiple exposure curves and power spectra of
perfused tissue and ordered flow are discussed.