Journal of Medical and Biological Engineering, 34(2): 172-177
172
Super Actinic 420 nm Light-emitting Diodes for Estimating
Relative Microvascular Hemoglobin Oxygen Saturation
David Townsend1,*
Francesco D’Aiuto2
John Deanfield1
1
Vascular Physiology Unit, University College London Institute of Cardiovascular Science, London WC1E 6BT, UK
2
Periodontology Unit, Department of Clinical Research, UCL Eastman Dental Institute, London WC1X 8LD, UK
Received 5 Aug 2013; Accepted 7 Feb 2014; doi: 10.5405/jmbe.1643
Abstract
This paper describes a method for comparing the absorption by hemoglobin of reflected light from a range of
high-intensity light-emitting diodes (LEDs) as part of the development of a system to obtain high-contrast video images
of microcirculation in vivo coupled with a measurement of the relative hemoglobin oxygen saturation in micro vessels.
Light from a range of high-intensity LEDs was used to image oxygenated and deoxygenated hemoglobin in vitro to
measure the difference in optical densities. The LED for which the highest difference in optical density was measured
was then incorporated in a prototype imaging device with illumination from a super actinic 420-nm LED and filtration
of the image at 410 nm and 430 nm. This device produced high contrast-video images of microcirculation at410 nm
and 430 nm. The difference in the absorption of light at the two wavelengths allowed the determination of the relative
hemoglobin oxygen saturation. The proposed method is shown to be suitable for estimating the relative oxygenated
hemoglobin from microvascular images.
Keywords: Super actinic light, Hemoglobin spectroscopy, Video microscopy, Microvasculature
1. Introduction
1.1 Microvascular video microscopy
Detailed imaging of microcirculation in vivo has been
developed with particular interest in the nail bed and sublingual
and labial capillary beds [1-6]. Orthogonal polarization spectral
(OPS) imaging and sidestream dark field (SDF) imaging have
gained in popularity, with commercially available systems such
as (Capiscope, KK Technology, UK) and (MicroScan, Micro
Vision Medical, Netherlands). These hand-held systems can be
connected to a laptop computer, making them extremely
versatile [7]. Microvascular parameters that can be easily
determined include vascular density, functional vascular
density, flow index, tortuosity, and red blood cell flow velocity
and the images can be analyzed for the determination of the
width of the glycocalyx lining the micro vessels [8].
Simultaneous imaging of the micro vessels and
spectroscopic measurement of the capillary oxygen saturation
has been described using two wavelengths of light in the red
and near-infrared regions (810 nm and 750 nm, respectively),
green light, and light with wavelengths of 420 nm and 430 nm
[9-11]. The techniques require stabilization of the tissues so
* Corresponding author: David Townsend
Tel: +44-0-2392344211; Fax: +44-0-2392492392
E-mail: [email protected]
that light can be passed through them. These methods are
unsuitable for OPS and SDF imaging where the light has to
reflect back from the underlying tissues. Some of the best
results of combining capillary imaging with oxygen saturation
measurements have been obtained for retinal vessels [12,13].
Red and near-infrared light are usually applied to the retina
where the vessels lie on the surface. A method for measuring
the oxygen saturation in individual micro vessels was described
by Tateishi [14]. The technique uses an intravital microscope to
image the micro vessel and to guide the positioning of a probe
attached to a spectrometer. Once the probe is in place over the
micro vessel, the spectrometer can record the spectral
absorption of the hemoglobin in the micro vessel. However,
this technique requires careful stabilization of the micro vessel
whilst the sampling probe is brought into position for each
point on the vessel to be measured, making measurements in
the nail bed quite practical but those in the labial and sublingual
mucosa difficult.
1.2. Hemoglobin spectroscopy
The absorption spectra for oxyhemoglobin and
deoxyhemoglobin are shown in Fig. 1. There are a number of
points along the spectrum where there is a significant
difference between the two spectra. It is at these points that it is
possible to use the measurement of the absorption of light by
the hemoglobin to determine the oxygen saturation state of the
hemoglobin. The points where the lines cross are the isobestic
points where the absorption of deoxy- and oxyhemoglobin are
J. Med. Biol. Eng., Vol. 34 No. 2 2014
identical. The minimum requirement for determining the
oxygen saturation of hemoglobin is the measurement of the
optical absorption of light at two wavelengths; the most useful
wavelengths are the isobestic points and the points of
maximum difference. The wavelengths of most interest for
oxygen saturation measurements are 410/430, 500/530, and
560/570 nm.
Figure 1. Absorption spectra of oxygenated and deoxygenated
hemoglobin between 400 nm and 630 nm (W.B. Gratzer, Med
Res Council Labs, Holly Hill London and N. Kollias,
Wellman Laboratories, Harvard Medical School, Boston).
SDF imaging requires light to be reflected back towards
the surface from the tissues which are deeper than the micro
vessel being imaged. The vast majority of light is lost within
the tissues; only a very small proportion is returned to the
surface to produce an image. Light is lost by a combination of
scattering and absorption. Successful imaging requires a large
difference in the absorption between the micro vessels and the
surrounding tissues while keeping the scattering of the light,
which degrades the image, to a minimum. The optimum
balance between scattering and contrast seems to be in the
green region around 520 nm. Above 600 nm, the contrast
between the tissues becomes too small to obtain a useful image,
and below 390 nm, the scattering of light becomes too great to
obtain a useful image. At 420 nm, there is good contrast and
micro vessels near the surface can be imaged; slightly deeper
micro vessels which might be visible at 520 nm may not be
seen.
Spectroscopic measurements of light intensity are made at
very specific wavelengths so the vast majority of the light
returned to the surface is not useful for such measurements.
The combination of the requirements for SDF and the
requirements for spectroscopy mean that a very intense light
source is required for illumination of the tissues. Historically,
sodium arc lamps and tungsten halogen lights have been
commonly used for illumination. These sources produce a
spectrum of wavelengths that include the wavelengths needed
for hemoglobin spectroscopy; however, they also produce a
considerable amount of ultraviolet light and heat, which need to
be filtered out of the light beam. Light-emitting diodes (LEDs)
are routinely used for spectroscopy and SDF imaging as they
have two particularly useful properties. Firstly, the electrical
energy is transformed into light very efficiently, resulting in
relatively little heat production. Secondly, the light is always
produced at a specific wavelength, which is determined by the
chemical composition of the electrodes in the LED. There has
173
been an enormous advance in the development of high-power
LEDs, particularly with aluminium gallium indium phosphide
LEDs, which produced light in the violet to amber spectrum
with a radiant power of 50 mW in 2002 and 100 mW by 2004,
and produce light with a radiant power of 400 mW today
[15,16]. Figure 2 shows the wavelengths of high-intensity
LEDs that are potentially useful for the spectroscopy of
hemoglobin.
Figure 2. Graph of absorption spectra of hemoglobin and wavelengths
of high-intensity LEDs in the range of 400 nm to 630 nm.
This study describes a method for comparing the
absorption of reflected light by oxygenated and deoxygenated
hemoglobin from a range of LEDs, and the development of a
prototype system to allow dark field illumination for the
imaging of the oral mucosa using a super actinic 420-nm LED
with filtration of the image to determine the relative oxygen
saturation of the hemoglobin in individual micro vessels. The
method makes use of high-intensity LED light combined with
recent improvements in lens technology.
2. Methods
2.1 Gradient index lens
In SDF imaging, the lens is kept in contact with the oral
mucosa and light is shone into the tissues adjacent to the lens
but not through the lens (hence sidestream dark field imaging).
This keeps reflected light from the surface of the tissue and
micro vessels to a minimum with the illumination of the tissues
produced by light reflected back to the surface from deeper
tissues. With a gradient index (GRIN) lens, light is propagated
through the lens in a sinusoidal path such that an image at one
end of the lens is reproduced at the other end if the length of
the lens is equal to the wavelength of the sinusoidal path. An
inverted image is produced at exactly one half of the sinusoidal
wavelength, as shown in Fig. 3. Only light that is incident on
Figure 3. Diagram of light propagation path through a one-pith GRIN
lens and photograph of a one-pitch GRIN lens showing image
propagation.
Oxygen Saturation from Microvascular Video Images
174
the end of the lens and within the numerical aperture of the lens
is propagated along the lens [17].
A one-pitch lens will only image the tissues that are in
contact with the end of the lens. By using a one-pitch GRIN
lens in contact with the tissues and not illuminating through the
lens, dark field illumination of the tissues at the tip of the lens
is achieved. A GRIN lens (1 Pitch, Grintech, Germany) with a
length of 10 mm and a diameter of 1 mm was used in the
present study. The lens was specially manufactured for this
study from non-toxic glass suitable for in vivo imaging. The
technique for producing non-toxic GRIN lenses using silver ion
exchange has only recently been developed.
2.2 Experimental setup
To measure the difference in light absorption of
oxygenated and deoxygenated hemoglobin at the wavelengths
of light produced by each of the LEDs, a video microscope
(camera: UI-1546LE-M (1280×1024 pixel resolution, CMOS
rolling shutter, IDS Imaging, Germany), magnification 200x)
with two capillary tubes in contact with the tip of the GRIN
lens was used. This is the position that micro vessels would be
in when imaged in vivo. The LEDs included in this study are
listed in Table 1. Light from the LED under test was shone onto
a matte white surface placed to represent the tissues under the
tubes and reflect the light back through the capillary tubes to
the microscope, as shown in Fig. 4. The rectangular tubes have
a lumen that measures 20 µm × 200 µm (Vitrotubes 0.002 x 0.2
mm,Vitro Com , USA). When filled with blood, the 20-µm
width of the column of red cells will have a similar cross
section to that of the average vessels imaged in capillaroscopy.
Table 1. Luminous characteristics of LEDs included in tests.
Colour
Identification
Red Luxeon Rebel LMXLOrange PH01-0030
Luxeon Rebel LMXLAmber
PL01-0023
Luxeon Rebel LXMLGreen
PM01-0090
Luxeon Rebel LMXLCyan
PE01-0060
Luxeon Rebel LMXLBlue
PB01-0023
Royal Luxeon Rebel LMXLBlue PR01-0350
Super SIBDI S35L-U70
Actinic
UV Blue SIBDI S35L-U60
Spectral
Wavelength
Luminous
half width
(nm)
flux
(nm)
620
20
100 lm
597
20
140 lm
530
30
150 lm
505
30
110 lm
490
20
48 lm
460
20
840 mW
420
10
400 mW
410
10
280 mW
2.3 Samples
Citrated human blood was used for all measurements.
Blood was exposed to ambient air for ten minutes to ensure
100% oxygenation and then run into one of the capillaries. A
small amount of yeast was added to the blood, which was then
removed from the atmosphere and incubated at 35 °C for
20 min to remove all oxygen. This blood was then run into the
second capillary tube. The contrast was adjusted and a series of
ten images was recorded for each LED. For light from the
420-nm LED (SIBDI U70, SemiLEDs Inc., USA), images were
recorded with two different light filters to filter the light from
the LED (430FIB12, 410FIB12, Knight Optical UK Ltd, UK).
Figure 5 shows the empty tubes and blood-filled tubes.
Figure 5. Rectangular capillary tubes with oxygenated and
deoxygenated blood, respectively (magnification 200x), and
empty capillary tubes with light reflecting from the curved
edges of the tubes (scale bar: 100 µm).
3. Results and analysis
Image analysis was carried out using open-source ImageJ
software. The average intensity of light passing through the red
blood cell column was recorded. The light immediately
adjacent to the capillary tube represented the incident light
intensity.
The optical density of the red blood column is defined as
-Log10 of the light passing through the column divided by the
intensity of the incident intensity:
Optical Density = - Log10(transmitted light/incident light)
(1)
The hemoglobin oxygen saturation is linearly related to
the ratio of the optical densities measured at any two different
wavelengths. The optical densities of the oxygenated and
deoxygenated red blood cell columns at the wavelengths of
each LED are shown in Table 2. The greatest difference in
optical density for oxygenated and deoxygenated hemoglobin
was in the images obtained at 420 nm and filtered at 410 nm
and 430 nm.
4. Micro vessel imaging
Figure 4. Diagram and photograph of experimental
hemoglobin density measurements.
setup
for
The SIBDI 420nm Super Actinic LED gave the greatest
measurable difference in optical density so it was incorporated
into the prototype imaging capillaroscope. The prototype
capillaroscope was designed to take advantage of the optimum
wavelength for imaging, as shown in Fig. 6. When illuminated
with this LED, the oxygenated hemoglobin gives a ratio of
optical density at 410 nm/optical density at 430 nm of 1.5,
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J. Med. Biol. Eng., Vol. 34. No. 2 2014
Table 2. Average optical density of 20-µm-thick columns of red blood cells held in the micro tubes when illuminated using each light source. The
table includes the measurements for the images from illumination at 420 nm filtered at 410 nm and 430 nm.
LED colour or band-pass filter
wavelength
Red
Amber
Green
Cyan
Blue
Royal Blue
Super Actinic
UV Blue
430FIB12,
Knight Optical UK Ltd, UK
410FIB12,
Knight Optical UK Ltd, UK
Mean optical density of
oxygenated hemoglobin
(SD)
0.05 (0.03)
0.15 (0.01)
0.17 (0.03)
0.12 (0.05)
0.22 (0.03)
0.31 (0.02)
0.20 (0.03)
0.17 (0.03)
Mean optical density of
deoxygenated hemoglobin
(SD)
0.06 (0.04)
0.12 (0.01)
0.11 (0.08)
0.08 (0.03)
0.23 (0.02)
0.40 (0.03)
0.30 (0.03)
0.15 (0.02)
0.28 (0.02)
0.47 (0.03)
0.19
0.41 (0.01)
0.24 (0.02)
0.17
Figure 6. Transmission graphs for 410-nm and 430-nm filters with
420-nm LED intensity and spectrum of hemoglobin
absorption superimposed (SiBDI, Knight OpticalUK, W.B.
Gratzer, Med Res Council Labs, Holly Hill London and N.
Kollias, Wellman Laboratories, Harvard Medical School,
Boston).
with the ratio for deoxygenated hemoglobin of 0.5 (410-nmand 430-nm-filtered images 0.41/0.28 = 1.5 for oxygenated
hemoglobin and 0.24/0.47 = 0.5 for deoxygenated hemoglobin).
To confirm the ability of the capillarosope to identify changes
in the hemoglobin oxygen saturation, a calibration procedure
was carried out. Fully oxygenated blood mixed with yeast was
run into a single capillary tube and incubated at 35 °C. This
was imaged by the capillaroscope as the oxygen was consumed.
The results are shown in Fig. 7.
Difference between oxygenated and
deoxygenated optical densities
0.01
0.03
0.08
0.04
0.01
0.09
0.10
0.02
A series of micro vessel images were then obtained in the
labial sulcus of a member of the research team. These images
were analyzed to determine if the measured optical densities
matched the expected ratios from the in vitro studies. Imaging
was carried out in the labial mucosa using the 420-nm LED to
illuminate the tissues (SIBDI U70 420nm Super Actinic;
operating current: 350 mA; radiometric power: 400 mW). The
reflected light was collimated and passed through a beam
splitter. One half of the split beam was filtered at 410 nm and
the image was captured on an IDS LE camera. The other half of
the split beam was filtered at 430 nm and the image was
captured on a second IDS LE camera (LE, IDS Imaging,
Germany) (430FIB12, 410FIB12 Knight Optical UK Ltd, UK).
The image of a capillary was selected and the optical density of
the capillary was measured at five points: two points on the
afferent vessel, two points on the efferent vessels, and one point
on the tip. The measurements were repeated for the image
filtered at 410 nm and 430 nm. Figure 8 shows the imaging
setup and Fig. 9 shows the density graphs of the afferent and
efferent vessels.
Figure 8. Experimental setup for in vivo imaging.
Figure 7. Graph showing the ratio of optical density at 410 nm/optical
density at 430 nm of blood hemoglobin in a capillary tube as
the oxygen saturation drops from fully oxygenated to
deoxygenated, as measured by the capillaroscope (vertical
lines indicate standard deviation).
Figure 9. Capillary density measurements with illumination at 420 nm
and filtration at 410 nm and 430 nm. The graph represents the
optical density measured along the line marked on the
capillary image (capillary scale is shown in Fig. 10).
176
Oxygen Saturation from Microvascular Video Images
The incident light was measured adjacent to the capillary
and the transmitted light was measured as the minimum
intensity within the capillary. The optical density was
determined by calculating the logarithmic value of the
transmitted light divided by the incident light (see Eq. (1)).
The hemoglobin oxygen saturation is proportional to the
ratio of the optical density at 410 nm divided by the optical
density at 430 nm. Figure 10 shows the ratio determined at
each of the five points along the capillary loop. The value for
blood flowing into the capillary loop (1.1) and that for the
blood flowing out of the loop are close to the values from the in
vitro capillary tube laboratory tests.
eliminating or compensating for the irregular reflected light
intensity, the measurements are only an estimate of the
hemoglobin oxygen saturation. This study described a method
that produces coherent results that are in line with results
obtained from in vitro measurements.
6. Conclusion
This study presented a system that combines the recent
developments in video capillaroscopy for determining the
hemoglobin oxygen saturation in micro vessels using
illumination at 420 nm with a super actinic LED coupled with
filtration of the reflected light at 410 nm and 430 nm.
Acknowledgments
Figure 10. Capillaroscopic image with the ratio of optical density at
410 nm/optical density at 430 nm showing a reduction of the
ratio along the length of the capillary loop (scale bar:
100 µm).
FD holds a Clinical Senior Lectureship Award supported
by the UK Clinical Research Collaboration. FD and JD work at
University College London which received a proportion of
funding from the Department of Health’s National Institute of
Health Research (NIHR) Biomedical Research Centres funding
scheme. This research forms part of a PhD thesis supported by
Solent HNS Trust and Health Education England.
5. Discussion
References
The use of a beam splitter combined with illumination at
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hemoglobin oxygen saturation was described by Ellis, who
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density of the hemoglobin but the tissues deep to the area being
imaged also contain oxygenated and deoxygenated hemoglobin,
which will affect the intensity of the reflected light. Without
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