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Use of the Laser Speckle Flowgraphy in Posterior Fundus Circulation Research
Conflict of Interest: The authors disclose no conflicts of interest.
Grant support: This study was supported by the grants from Japan National Society for the Prevention
of Blindness (No.1316) and the Scientific Research Foundation for the Returned Overseas Chinese
Scholars of Chinese State Education Ministry( 2006 No.195).
1
Key words:
Laser speckle; Blood flowmetry; Retina; Choroid; Optic nerve head
Abstract
Objective: To review articles which aim to present an overview of the principles, progress, uses and
limitations of laser speckle flowgraphy (LSFG) in posterior fundus circulation research.
Data sources: The data used in this review was obtained mainly from the studies reported in PubMed
using the key terms “laser speckle”, “ocular blood flowmetry” and “retinal imaging”.
Study selection: Relevant literature on studies of LSFG was selected.
Results: LSFG is a unique, noninvasive imaging instrument to quantitatively visualize posterior fundus
circulation in vivo. This review delineates the LSFG principles and development, demonstrates its
extensive applicability for measurement of retina, choroid and optic nerve head circulation, compares it
with other retinal imaging technologies and discusses unresolved issues.
Conclusions: LSFG is a noninvasive, two-dimensional objective diagnostic technique that has become a
powerful method for the clinical and scientific assessment of posterior fundus circulation. Further studies
may help to develop a more comprehensive evidence-based measurement and facilitate the correlation
with other methods for chorioretinal circulation assessment.
2
[摘 要]
激光斑点血流计法是一种能在活体上动态的观察眼底血液循环并进行血流定量检测的
非侵袭性眼循环测定技术。近年来在国外尤其是日本，激光斑点血流测定技术的不断更新开发，使
其在视网膜、脉络膜及视神经乳头血液循环的临床应用及科研工作中发挥了很大的作用，日益成为
热点技术。本文就该技术的原理、发展、临床和科研上的应用及其存在的问题作一综述。
[关键词]
激光斑点, 血流计,视网膜，脉络膜，视神经乳头
3
Text
Introduction
The evaluation of tissue circulation in the human eye is essential for the investigation of physiology
and pathology of the ocular structure. Research related to ocular blood flow gives us a significant
opportunity in preventing blindness. The chorioretinal circulation is of fundamental importance in
understanding the pathological processes that occur in the ocular fundus. Nearly 30 years ago, Fercher
and Briers1 put forth the idea of estimating blood flow velocity based on the laser speckle phenomenon.
Since then, many researchers have made attempts to realize this measurement concept as a quantitative
tool for full-field blood flow assessment. Laser speckle flowgraphy (LSFG) is an apparatus for
quantitative in vivo estimation of microcirculation in the optic nerve head (ONH), choroid and retina.
Herein, we describe the basic technology, development, multiple clinical applications and limitations of
LSFG in chorioretinal circulation research.
Basic technology and progress
Laser speckle flowgraphy (Fig. 1) is an apparatus based on the laser speckle phenomenon, which targets
moving erythrocytes in the eye to estimate the circulation of ONH, choroid and retina. Laser speckle is an
interference phenomenon observed when coherent light sources (such as lasers) are scattered by a
diffusing surface. The speckle pattern is a random one with properties that can only be described
statistically. Figure 2 shows a schematic view of laser speckle flowgraphy. A fundus camera is equipped
with a diode laser (808 nm or 830 nm wavelength for tissue circulation in the ONH and choroid) and an
4
image sensor of 100×100 pixels. The subject’s fundus is illuminated by a halogen lamp for observation,
and the halogen light illumination is switched to the laser illumination at the time of measurement. The
laser beam passes through a dichroic mirror (DM1) followed by a ring mirror (Ma), which focuses the
beam on the fundus with a field of 2.1 mm in diameter in human eyes. The scattered laser light passes
through the center of Ma and is reflected by a second dichroic mirror (DM2). Thus, the light scattered
from a square field of the ocular fundus (1.06×1.06 mm in human eyes in the early model of LSFG) is
projected onto the image sensor with 100×100 pixels, where a speckle pattern appears. In accordance to
the movement of blood cells in the tissue, the structure of the pattern varies rapidly: the greater the blood
cell velocity, the greater the rate of variation. The structure of the pattern which changes rapidly according
to blood flow velocity is called “blurring”.2-4 In the previous-generation LSFG, the quantitative index of
blood flow velocity is represented as normalized blur (NB), or square blur rate (SBR), which is calculated
from the speckle pattern generated by reflected lights from the moving erythrocytes under the
illumination of a diode laser. The NB value is an approximation of the reciprocal of the speckle contrast
and is thought to serve as an index of blood flow velocity.
1, 5-10
Fifty color-coded levels are divided
according to the NB values, and the area with fast blood velocity is displayed in red. Thus, a color map is
displayed on the color monitor to demonstrate visually the two-dimensional variation of the NB levels in
the field measured. 7- 9
In order to measure the retinal microcirculation, Tamaki et al
9
used a blue-component argon laser
(wavelength 488 nm, maximum power 3 mW) to substitute for the diode laser. However, since the
blue-component argon laser was strongly scattered in the superficial retina or strongly absorbed in the
5
retinal pigment epithelium and choroids, NB values obtained by using a blue-component argon laser
mostly reflected the retinal circulation and were only minimally affected by the choroidal circulation.
Thus, LSFG is promising for studying the physiology and pathology of retinal microcirculation.
Recently, the latest LSFG technique, known as the wide-field LSFG, has been used to measure the
hemodynamics of the posterior fundus. Use of a charge-coupled device (CCD) camera instead of an area
sensor, and the expanded laser beam with a modified prism lens composed of spherical and right angle
lenses, improved the observation angle. The new LSFG model, in which the observation field is 8.6×4.6
mm with 600×280 pixels, dramatically enlarges the observation field up to the posterior fundus, allowing
simultaneous observation of the optic disc and the macular area. The scanning speed of the image sensor
is 98 scans⁄ second in the earlier model and in the current one, 512 scans⁄ second. Every successive scan
of the image sensor results in a different profile of output signal intensity. In the current wide-field LSFG,
it uses the mean blur rate (MBR), where MBR = 2 × BR2, as an indicator of the relative velocity of the
erythrocytes. Using the rotating opal glass plate method, MBR was proved to show the best linear
correlation with the blood velocity.11
Clinical Applications
Preparation before measurement
The pupils of the subjects will be adequately dilated with 0.5% tropicamide and 0.5% phenylephrine
hydrochloride. Before starting the measurement, a rest period of 5 to 10 minutes should be suggested in
order to obtain stable systemic hemodynamic conditions, and it is also recommended to instruct subjects
6
to refrain from heavy meals or exercises the day before measurements.12, 13
Measurement of posterior fundus microcirculation
The subjects should be positioned by means of an internal fixation target so that the site of interest is in
the central area of the fundus. While taking measurements in the ONH rim tissue, it is recommended to
select a rim area free from any visible vessels (usually the temporal region). The operator will then start
the measurement. The blood velocity in the selected region is continuously calculated, displayed and
recorded. A typical color image representing the measurement of ONH and macular circulation is shown
in Figure 3.
Nowadays, the wide-field LSFG uses MBR as an indicator of the relative velocity of the erythrocytes.
Ninety-seven speckle images that are generated during four to five pulsations are averaged and used to
create a MBR map of one pulsation. By using the tracking potential of the wide-field LSFG, it is possible
to take all the superimposed images at the same location. The MBR map is animated with either
false-color or grayscale images. A composite grayscale map of a still image may be obtained by averaging
the cumulative sum of the MBR map (animation of one pulsation). During this process, any noise present
is canceled and the signals of the choroidal and retinal vessels are superimposed, which improves the
image contrast. The composite grayscale map of LSFG shows an even higher contrast image of the
choroidal vessels than fluorescein angiography (FFA) or indocyanine green angiography (ICG).
Clinical and research use
LSFG is a noninvasive technique that can measure real-time, two-dimensional relative blood flow
7
velocity and volume of ocular microcirculation using the laser speckle phenomenon.1,
5-10
The laser
speckle method has also been confirmed to correlate well linearly with the blood flow determined with
microsphere technique in the choroid7, 8, retina9, and the blood flow determined with the hydrogen gas
clearance method in the ONH 14.
With the improvement of the LSFG apparatus, various studies on ONH blood flow have been carried
out. The progress has made it possible to measure ocular blood flow, which is thought to play an
important role in the pathology of glaucoma, reliably. A number of reports have been published regarding
the effects of different kinds of medications on ONH blood flow. Tamaki et al. reported that the topical
application of timolol caused no change on ONH blood flow in the human eye, while carteolol caused an
increase.15 Ishii and Araie studied the effects of topical latanoprost and timolol in combined therapy on
retinal blood flow and tissue circulation in the ONH of the monkey. They found that the combined topical
therapy had no significant effect on the retinal blood flow, but significantly increased the ONH tissue
blood velocity in monkey eyes.
16
To evaluate the effect of combined topical application of latanoprost
and beta blockers on ONH blood flow in normal tension glaucoma (NTG) patients, Sugiyama and
associates measured ONH blood flow by LSFG and concluded that topical latanoprost-carteolol
combined therapy increased ONH blood flow in NTG patients, but latanoprost-timolol therapy didn’t.17
Yaoeda et al (2000) 18 measured the ONH microcirculation using LSFG and scanning laser Doppler
flowmetry (SLDF). Only a weak correlation between the blood flow indexes of the ONH was detected by
the two non-invasive imaging techniques because of basic differences in the principles of measurement.
Using LSFG, Yaoeda and associates(2000)19 measured the microcirculation in ONH of normal volunteers
8
and found that there were significant differences of ONH blood flow between the right and left eyes and
between the superior and inferior temporal neuroretinal rims. Their study using the LSFG (2003) showed
a significant correlation between a decrease in ONH blood flow velocity and visual field loss in NTG
subjects.20 However, whether diversified antiglaucoma medications and intra ocular pressure (IOP) affect
ocular blood flow in normal subjects or patients with NTG and primary open-angle glaucoma (POAG)
remains controversial. The effects of different kinds of antiglaucoma medications on ONH blood flow
require additional research. Future work should comprehensively assess ocular blood flow in response to
standardized stimuli in order to better understand the pathophysiology of glaucomatous optic
neuropathy.21-25
Maeda et al (2009) used LSFG to examine the optic disc blood flow in a nonischemic type central
retinal vein occlusion case. Their clinical study helped verify the implications of LSFG when evaluating
the hemodynamics of the ONH circulation, that LSFG may be a promising clinical tool for monitoring the
progression of retinal vascular diseases.26
Using LSFG and ICG to observe the choroidal circulation, Isono et al
27
(2003) produced a panoramic
composite 3-mm-square NB map by combining the adjacent 1.5-mm-square ones. It showed that the
vascular pattern of the panoramic map was similar with that of the ICG angiography. Branch retinal artery
occlusion was then induced in monkey eyes, the panoramic NB maps of LSFG taken before and after the
experimental BRAO were compared and the vascular patterns showed no change. This suggested that the
vascular pattern seen in the panoramic composite NB map was mainly choroidal vessels in origin. LSFG
may noninvasively visualize the hemodynamics of choroidal circulation in various choroidal diseases,
9
which is comparable with that of ICG angiography. Recently, Hirose et al
28
(2008) studied the ability of
LSFG to quantitatively evaluate blood flow velocity at the macula in patients with Vogt-Koyanagi-Harada
disease before and after systemic corticosteroid therapy. The results showed that systemic corticosteroid
therapy improved inflammation-related impairment in choroidal blood flow velocity at the macula and
also confirmed that LSFG was an effective method for clinical use, capable of providing results within 60
seconds without contrast agents. Watanabe et al
29
(2008) compared the images of choroidal vasculature
obtained by wide-field LSFG, the latest LSFG technique, and ICG in eyes with polypoidal choroidal
vasculopathy (PCV). They further proved that the choroidal vasculature on the grayscale composite MBR
map of wide-field LSFG indicated the fine resolution similar to the ICG results and the MBR map
showed the watershed zone and highest signal intensity in the macula. Furthermore, wide-field LSFG is
the first instrument that can noninvasively generate images of the hemodynamics of the entire posterior
fundus.
Enaida and associates investigated the changes of chorioretinal blood flow using LSFG in efficacy of
intravitreal bevacizumab injection in a patient with proliferative diabetic retinopathy (PDR). LSFG
measured MBR in a previously confirmed area, with neovascularization elsewhere (NVE),
neovascularization of the disc (NVD), and without neovascularization. MBR was then compared before
and after bevacizumab treatment in each area. Regression of blood flow was observed at the area of NVE
and NVD after bevacizumab treatment. However, in the area of without neovascularization, reduction of
MBR was not observed. Their study verified the practical usefulness of LSFG in measuring chorioretinal
blood flow changes by drug intervention.30
10
Comparison with other measurement systems
The evaluation of chorioretinal microvascular blood flow is of fundamental importance for
understanding a number of physiological and pathological processes that occur in the posterior segment
of the eye. Conventionally, we use the dye dilution methods31, 32 (FFA or ICG with or without scanning
laser ophthalmoscope) to observe the circulation of the fundus. These techniques require the
administration of exogenous substances, making them difficult to perform repeated measurements with
short time intervals and the risk of severe complications exists. Furthermore, analysis of the obtained data
is time consuming, making quantitative analysis difficult. Based on the studies we cited above, 27-29 LSFG
may noninvasively visualize the hemodynamics of chorioretinal circulation in various posterior segmental
diseases, which is comparable with the dye dilution techniques.
A measurement system using the blue field entoptoscope and a microcomputer was devised by Riva &
Petrig in 1980. Blue-field entoptic stimulation (BFS)
33-36
is a noninvasive experimental method of
inspecting average leucocyte velocity around the fovea in the inner nuclear and outer plexiform layers.
One fundamental difference is that LSFG traces moving erythrocytes as opposed to leucocytes. As
leucocytes are larger than erythrocytes and may move more slowly in capillaries, BFS may underestimate
blood velocity compared with blood flow measured by LSFG. Secondly, BFS can take measurements only
in the macular area compared with LSFG, which can observe all parts of the fundus. Moreover, its
psychophysical and subjective nature limits its usefulness.
11
The principles of measurements of LSFG and SLDF are similar in the use of the scattered reflection of
the laser light from moving red blood cells and the obtained data are both in arbitrary units. However the
difference in laser wavelength (LSFG 830 nm, SLDF 780nm) may influence blood flow indexes due to
tissue permeability. The depth of measurement in LSFG exceeds the postlaminar region of the ONH,
however, in SLDF the depth is limited to the prelaminar.18, 36-41 Moreover, a unique advantage of laser
speckle imaging is that blood flow can be obtained in a snap shot without line scanning as is the case for
laser Doppler flowmetry.42
Laser Safety
The American National Standards Institute has determined that the maximum permissible retinal
exposure for viewing the diffuse reflections of a diode laser is 460mW/cm2 for 10 seconds. The maximum
retinal exposure with the current LSFG is about 90 mW/cm2, and the exposure time was 10 seconds,
considerably within the permissible limits.7-9 , 43 Thus LSFG causes no potential hazards to human eyes.
Limitations and progress
In spite of efforts by many researches, there are still some limitations of the instrument. Firstly, during
the measurement, it is difficult to acquire a clear image in subjects with corneal opacities or cataracts. The
quality of the measurement mainly relies on the clarity of the ocular media. Moreover, the subject’s
fixation ability is of key importance to maintain optimal outcome, as any vibration of the eyeball, called
the fixation nystagmus, gives rise to unwanted noise in the LSFG system.
12
44
Although, use of the tracking
potential of the wide-field LSFG allows adjustment for small eye movements, measurement in subjects
with low fixation ability will result in greater variability. Secondly, it should be noted that the map of the
NB or MBR value may not display the actual blood flow variation if the flow becomes extremely fast.7, 8,
45
Agreement between the value and tissue blood flow is thought to rely on the fact that the tissue blood
velocity is relatively low. No previous studies have established the upper limit of the blood flow velocity
that can be measured by LSFG．The present LSFG system indicates the blood flow volume, rather than
blood flow velocity in the strict sense, but can display, in a relative scale.7-9, 14, 21 In addition, the values
obtained by LSFG are in arbitrary units, which makes it difficult to make direct comparisons among data
obtained from different sites of measurement or in the eyes of different subjects and to correlate them
well using a different apparatus such as SLDF. 14, 18, 19, 33-38
LSFG was initially developed to measure blood flow in the retina. Nowadays, its primary application
has been extended to image blood flow in the brain and skin. These studies indicate the usefulness of
LSFG measurements for assessing tissue circulation in different fields. 46, 47 More recently, Srienc et al
used LSFG in conjunction with confocal microscopy to monitor light-evoked changes in blood flow of rat
retinal vessels. By this dual imaging technique, it was possible to stimulate retinal photoreceptors and
measure vessel diameter with confocal microscopy while simultaneously monitoring blood flow with
LSFG. The combined use of LSFG and confocal microscopy ensures the feasibility for studying the
regulation of blood flow in the normal or pathological retina.48
In summary, the multiple approaches of LSFG to functional imaging have opened up research
13
development respecting a wide range of ocular diseases. Progress of the technology will improve our
ability to provide earlier diagnosis, better understanding and novel treatment in fighting blindness.
14
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Figure Legends:
Figure 1. The laser speckle flowgraphy system.
Figure 2. Schematic diagram of laser speckle flowgraphy. DM1 and DM2 indicate the diachronic mirrors;
Ma is the ring mirror.
Figure 3. False color composite map shows simultaneous observation of the optic disc and the macular
area using wide-field LSFG.
21
Fig. 1. The laser speckle flowgraphy system.
22
Computer
system
Area sensor
Display
Subject’s eye
Observer
Ma
DM1
DM2
Halogen lights
Laser head
Fig 2. Schematic diagram of laser speckle flowgraphy. DM1 and DM2 indicate the diachronic mirrors; Ma
is the ring mirror.
23
Fig 3. False color composite map shows simultaneous observation of the optic disc and the macular area
using wide-field LSFG.
24