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General Properties
C43H47N2O6S2Na, Molecular weight 775.
A tricarbocyanine type of dye with infrared absorbing properties; peak absorption
at about 800nm. Has little or no absorption in the visible. It is used in
infrared photography and in the preparation of Wratten filters. It is also
used as a diagnostic aid for blood volume determination, cardiac output, or
hepatic function. --- Merck Index
Uses of ICG
The classic paper on the spectral absorption properties of ICG
is by Landsman who,
in the introduction of his paper says,
After its introduction by Fox et al. (1957) indocyanine green soon came
into general use for recording dye dilution curves, in particular for
the determination of cardiac output. The principal advantages causing
the rapid acceptance of the dye were the presence of an absorption
maximum near the isobestic point of hemoglobin and oxyhemoglobin
around lambda = 800nm, the confinement to the vascular compartment by
binding to plasma proteins, the very low toxicity, and the rapid
excretion, almost exclusively into the bile.
ICG has been used extensively in laser welding because of its absorption properties
at the laser diode wavelength of 800nm. ICG has somewhat bizarre light absorption behavior
as a function of concentration because it tends to aggegrate in water at high
concentrations. This means that the
effective absorption does not increase linearly with increasing concentration.
Furthermore, ICG tends to degrade with exposure to light. The photodegradation
is mitigated when ICG is bound to albumin, but it still proceeds slowly (days).
The photodegradation is also concentration dependent. For
details see the Landsman paper.
When ICG is added to blood plasma, it is rapidly bound to proteins in the
serum. This also changes its absorption spectrum. To summarize,
ICG absorption depends on its concentration and its solute.
Bibliography
Here are some bibliographic links.
The Molar Extinction Coefficient Defined
If the transmission through a sample is given by T, then the absorbance A is
A = -log10 T
The ideal absorbance of a dilute solute in a transparent solvent follows the
Beer's law
A = e c d
where e (liters/mole-cm) and is called the molar extinction coefficient
of the solute at the wavelength of measurement, c (moles/liter) is the
molarity of the solute, and d (cm) is the optical path length. The
linear dependence of A on c may not hold if there are
concentration-dependent effects, e.g., aggregation or acid-base
dissociation of the solute. This certainly is the case for ICG.
If the absorption coefficient µa is desired then
one uses the relations
T = exp(-µa d)
or that the absorption coefficient is directly proportional to the concentration
and the molar extinction coefficient.
µa = e c ln 10 = 2.303 e c
To predict the absorbance of a particular solution of of ICG, if
Beer's law was valid, then all that would be needed would be to multiply
the molar extinction coefficient at your particular wavelength
with the molar concentration and the
path length. Unfortunately, ICG has different molar extinction
coefficients for different concentrations. Furthermore, there are different
molar extinction coefficients if proteins (e.g., albumin) is present.
For a given concentration and wavelength, find the molar extinction coefficient from the graphs
(you might have to interpolate) and use the above equations to convert to
absorbance or absorption coefficient, depending on your application.
Landsman's ICG Data
Read Landsman's paper for the details on how these spectra were obtained.
However, Landsman plotted his results using a somewhat screwy unit. I
have digitized the data from Landsman and replotted it as wavelength
versus molar extinction coefficient.
This is plot of the digitized Landsman ICG molar extinction
coefficient of ICG in water showing how the absorption changes
as the concentration of ICG changes. If ICG strictly followed Beer's law
then each of the curves in this figure would be on top of one another.
A plot of the digitized Landsman ICG molar extinction
coefficient of ICG in plasma showing how the absorption changes
at different concentrations in plasma. Note how the primary absorption
peak is shifted to the red relative to the water absorption properties.
OMLC Measurements
If I was going to use anyone's data
I would opt for the Landsman data because they must have been much more
careful about their measurements. They also give data out to 900nm that
whereas our HP spectrophotometer only goes to 810nm. However, these do show
the absorption properties of ICG in the visible and are handy to have.
This data was taken using a cuvette consisting of two microscope slides
with a cover slip as a 155µm spacer. This allowed measurements
that did not exceed an absorbance of 2 and thus ensured that the HP Spectophotometer
remained in its linear range of operation.
A plot of the OMLC measured molar extinction
coefficient of ICG in water at 0.01mM and 1mM concentrations.
There is very little absorption in the visible and
the general trend of the absorption as a function of concentration follows
that of Landsman.
A plot of the OMLC measured molar extinction
coefficient of ICG in 22.5% albumin at 0.01mM and 1mM ICG concentrations.
The similarity to plasma is evident. The noise in the data at the low concentration
is a result of the strong absorption by albumin in the 300-350nm range.
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