U.S. patent application number 11/988535 was filed with the patent office on 2009-05-21 for device and method for determining the concentration of a substance.
Invention is credited to Can Ince, Egbert Gezinus Mik.
Application Number | 20090130700 11/988535 |
Document ID | / |
Family ID | 35414517 |
Filed Date | 2009-05-21 |
United States Patent
Application |
20090130700 |
Kind Code |
A1 |
Ince; Can ; et al. |
May 21, 2009 |
Device and Method for Determining the Concentration of a
Substance
Abstract
The invention provides a method for determining a concentration
of a substance in a compartment comprising exciting an endogenous
compound, or a functional part, derivative, analogue or precursor
thereof, measuring the lifetime of the luminescence and/or
transient absorption exhibited by said compound, functional part,
derivative, analogue and/or precursor, and correlating said
lifetime with the concentration of said substance.
Inventors: |
Ince; Can; (Leiden, NL)
; Mik; Egbert Gezinus; (Amsterdam, NL) |
Correspondence
Address: |
TRASKBRITT, P.C.
P.O. BOX 2550
SALT LAKE CITY
UT
84110
US
|
Family ID: |
35414517 |
Appl. No.: |
11/988535 |
Filed: |
July 6, 2006 |
PCT Filed: |
July 6, 2006 |
PCT NO: |
PCT/NL2006/000341 |
371 Date: |
November 13, 2008 |
Current U.S.
Class: |
435/29 ; 422/52;
436/172 |
Current CPC
Class: |
A61K 49/0021 20130101;
Y10T 436/207497 20150115; A61K 49/0056 20130101; A61K 49/0036
20130101; G01N 21/6408 20130101 |
Class at
Publication: |
435/29 ; 436/172;
422/52 |
International
Class: |
C12Q 1/02 20060101
C12Q001/02; G01N 21/76 20060101 G01N021/76 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 6, 2005 |
EP |
05076565.0 |
Claims
1. A method for determining a concentration of a substance in a
compartment, the method comprising: exciting an endogenous compound
or a functional part, derivative, analogue and/or precursor of said
compound, wherein said endogenous compound or functional part,
derivative, analogue and/or precursor, if excited, exhibits a
luminescence and/or transient absorption of which the lifetime is
dependent on said substance, measuring the lifetime of the
luminescence and/or transient absorption exhibited by said
compound, functional part, derivative, analogue and/or precursor,
and correlating said lifetime with said concentration.
2. The method according to claim 1, wherein said substance
comprises oxygen.
3. The method according to claim 1, wherein said luminescence
and/or transient absorption comprises delayed fluorescence and/or
triplet-triplet absorption.
4. The method according to claim 1, wherein said compound comprises
a compound capable of being excited to a triplet state.
5. The method according to claim 1, wherein said compound comprises
a porphyrin.
6. The method according to claim 1, wherein said compound comprises
a protoporphyrin.
7. The method according to claim 5, wherein said compound comprises
protoporphyrin IX.
8. The method according to claim 1, wherein said compound is
photo-excited.
9. The method according to claim 1, wherein said compartment
comprises a cell.
10. The method according to claim 1, wherein said compartment
comprises an organelle.
11. The method according to claim 10, wherein said organelle
comprises a mitochondrion.
12. The method according to claim 1, wherein said compartment
comprises at least part of a tissue.
13. The method according to claim 1, wherein said compartment
comprises an organ.
14. The method according to claim 1, wherein said compartment
comprises a tumor.
15. The method according to claim 1, wherein said compartment
comprises the microcirculation.
16. The method according to claim 12, wherein said tissue is
present in a culture medium.
17. The method according to claim 1, wherein said compartment
comprises a cell suspension.
18. The method according to claim 1, wherein said lifetime is
compared with a reference.
19. The method according to claim 1, wherein said lifetime is
measured within four hours.
20. The method according to claim 1, wherein said lifetime is
measured in the time-domain.
21. The method according to claim 1, wherein said lifetime is
measured in the frequency-domain.
22. The method according to claim 1, wherein multi-photon
excitation is applied.
23. The method according to claim 1, wherein two-photon excitation
is applied.
24. A device for determining a concentration of a substance in a
compartment, the device comprising: means for exciting an
endogenous compound or a functional part, derivative, analogue
and/or precursor thereof, wherein said endogenous compound, part,
derivative, analogue and/or precursor, if excited, exhibits a
luminescence and/or transient absorption of which the lifetime is
dependent on said substance, and means for measuring said
lifetime.
25. The device according to claim 24, comprising a combination of a
prism and a bandpass filter.
26. The device according to claim 24, comprising a fast shutter in
front of a PMT.
27. The device of claim 24, comprising a semi-conductor device.
28. The device of claim 24, comprising an imaging device capable of
oxygen mapping.
29.-31. (canceled)
Description
[0001] The invention relates to the field of medicine. More
specifically, the invention relates to monitoring the concentration
of a substance.
[0002] Health control, diagnosis of disease and/or monitoring of
treatment of disease often involves measurement of various
parameters. One parameter is the concentration of a certain
substance, such as oxygen, within at least part of an organism.
Local tissue oxygenation is an important parameter in the diagnosis
and treatment of a wide range of diseases. Measurements of the
amount of oxygen present in a specific part of a subject are for
instance carried out during peri-operative monitoring in the
operating room and intensive care and for diagnosis of a wide range
of clinical disorders in which tissue oxygenation lies central to
the development and cure of disease. Examples include diagnosis of
cardiovascular disease, monitoring healing of decubitus and
diabetic wounds, monitoring hyperbaric correction of radiation
wounds and assessment of success of bypass surgery. Monitoring of
tissue oxygen pressure (pO.sub.2) during critical illness is
considered a major need in the adequate treatment of intensive care
patients (Siegemund et al., 1999). Assessment of tumor oxygenation
is an example wherein measuring of local tissue oxygenation is
helpful for the choice of treatment, as oxygen is an important
determinant for success of radiotherapy. Hence, the concentration
of oxygen in a tumor is preferably determined in order to determine
whether radiotherapy is recommended. Local oxygen measurements are
also applicable for the assessment of organ viability for
transplantation.
[0003] Dioxygen is a molecule of utmost biological importance
because of its role as the primary biological oxidant. Therefore,
oxygen plays a key role in the oxidation/reduction reactions that
are coupled to cellular respiration and energy supply. Adequate
measurement of oxygen concentrations in biological samples like
cells, tissues and whole organs is important to gain insight in the
determinants of oxygen supply and utilisation under normal and
pathological conditions. It is interesting to note that the
clinical interest in methods providing information about blood-flow
and oxygen delivery at the (sub-)organ level (e.g.
microcirculatory) is growing. This is amongst other things because
of increasing insight into the role of the microcirculation in
pathogenesis, and the importance of adequate tissue perfusion as
end-point of treatment (Siegemund et al., 1999).
[0004] Various techniques have been developed for direct or
indirect oxygen measurements in tissue, each having its specific
advantages and disadvantages (for a review on this subject see J.
M. Vanderkooi et al., 1991). Conventionally, measurements of tissue
oxygenation have been made by use of oxygen electrodes and
spectrophotometry of the hemoglobin or myoglobin molecule.
Reflection spectrophotometry records the difference in absorption
and scattering between a standard reference sample and a tissue
sample. The method is based on the illumination of a tissue sample
by light with a known spectral content and detection of the
diffusely reflected light from the tissue at several different
wavelengths. The spectral difference between illumination light and
detected light contains information about the wavelength-dependent
absorption and scattering within the tissue. The reference sample,
used for correction of non-ideal apparatus behavior, can be
anything with well-known absorption properties, but a white sample
(no absorption) is mostly used. The relative tissue absorbency
[E.sub.r(tissue)] can be described by the following equation:
[E.sub.r(tissue)]=log(I.sub.r(standard)/I.sub.r(tissue)) (1)
where I.sub.r(standard) and I.sub.r(tissue) are the intensity of
the diffusely reflected light from the white standard and the
tissue, respectively. Since the absorption spectra of oxygenated
and deoxygenated hemoglobin show marked differences that can easily
be detected by RS, this technique is widely used for measurement of
hemoglobin saturation in tissue. In order to derive more or less
quantitative data with RS, it is necessary to take into account the
influence of tissue optical parameters other than the hemoglobin
related ones. Different approaches for developing an appropriate
analysis algorithm are possible. One described approach is based on
the use of isobestic points (the intersection points of the curves
of oxygenated and deoxygenated hemoglobin) as reference points
within the calculation (Sato, 1979). Dummler used a somewhat
different approach for his derivation of an algorithm (Dummler,
1988) based on the two-flux theory of Kubelka and Munk (Kubelka,
1931, Kessler, 1992). The EMPHO, the Erlangen Micro-lightguide
spectrophotometer (Frank, 1989) (EMPHO II, Bodenseewerk
Geratetechnik, Uberlingen, Germany) and the O2C (Lea Medizin
Technik, Giesen, Germany) are spectrophotometers using improved
Dummler algorithms for hemoglobin saturation measurements.
[0005] Drawback of these conventional techniques is that they are
either mechanically disruptive (insertion of oxygen electrodes) or
qualitative (spectrophotometry). These constrains have led to the
development of alternative methods. One of the most promising
techniques in this respect has been the use of oxygen dependent
quenching of phosphorescent dyes for measurements in the
microcirculation (Vanderkooi et al., 1987; Sinaasappel & Ince,
1996; Sinaasappel et. al., 1999).
[0006] Wilson and Vanderkooi (Vanderkooi, 1987) introduced the
oxygen-dependent quenching of phosphorescence of metallo-porphyrin
compounds for biological oxygen concentration measurements. The
technique is based upon the principle that a metallo-porphyrin
molecule that has been excited by light can either release this
absorbed energy as light (phosphorescence) or transfer the absorbed
energy to oxygen (without light emission). This results in an
oxygen dependent phosphorescence intensity and lifetime. The
relationship between the lifetime and the oxygen concentration is
given by the Stern-Volmer relationship (Vanderkooi, 1989).
Calibration constants associated with the Stern-Volmer relationship
allow oxygen concentrations to be calculated from the measured
lifetimes. The measurement of lifetimes allows quantitative
measurements without the influence of tissue optical
properties.
[0007] For in vivo measurements, Pd-porphyrin is bound to albumin
to form a large molecular complex that after injection into the
circulation remains confined, at least for a certain time, inside
the blood vessels. This allows microvascular pO.sub.2 measurements
to be made using a phosphorimeter. A phosphorimeter is a device
that measures the phosphorescence decay after a pulse of light
(time-domain device) or determines the phase-shift between a
modulated excitation source and the emitted phosphorescence
(frequency-domain device). Several of these systems have been
described in literature (Mik, 2002; Coremans, 1993; Sinaasappel,
1996 and Vinogradov, 2002). Attached to a microscope
phosphorescence lifetime measurements allow the measurement of
pO.sub.2 in single blood vessels in the microcirculation. Use of
fiber phosphorimeters allows measurement of microvascular pO.sub.2
(.mu.pO.sub.2) without having to resort to microscope techniques. A
fiber phosphorimeter has been developed for measurement of .mu.pO2
in large animal models of shock and sepsis (Sinaasappel, 1999; Van
Iterson, 1998), as well as in mice (Van Bommel, 1998) and the
analysis of the decay kinetics has been improved to provide more
reliable calculation of pO.sub.2 values from the decay kinetics
(Mik, 2002). A multi-channel implementation of this phosphorimeter
allows simultaneous detection of .mu.pO.sub.2 at different sites
and different organs. In general, the use of multi-fiber technology
is, besides imaging, a way to detect special information in optical
spectroscopy. FIG. 1 shows schematically an example of a
frequency-domain phosphorimeter of which the light source is a very
cost-effective light emitting diode (LED).
[0008] An advantage of lifetime measurements is the independence of
the concentration of the chromophore, making quantitative
measurements possible in vivo, where the precise concentration of
said chromophore cannot be predicted. An important drawback of this
technique is however that it relies on injection of
palladium-porphyrin into the circulation, making this technique
unsuitable for clinical settings because of long-term toxicity. The
use is limited to pre-clinical applications. Moreover, this
technique is only suitable for measuring oxygen levels in the
microcirculation. Since the molecules are large and
cell-impermeable, this technique cannot be applied for
intracellular oxygen measurements without disrupting the
intracellular compartment by micro-injection (Hogan, 1999).
[0009] A kind of semi-quantitative oxygen measurement using
non-specific protein phosphorescence has been used for oxygen
measurements in mitochondrial suspensions. This was based on
oxygen-dependent quenching of the phosphorescence of the amino acid
tryptophan (Vanderkooi et al., 1990). Unfortunately, this
phosphorescence cannot be used for quantitative oxygen measurements
because of the complex decay kinetics arising from the different
tryptophan containing proteins (Vanderkooi et al., 1987b). The use
of tryptophan phosphorescence for in vivo applications is
furthermore limited because of the excitation in the UV region (283
nm), resulting in extremely shallow penetration depths in tissue,
besides the well-known photo-toxicity of this high energetic
light.
[0010] Although both oxygen-dependent quenching of phosphorescence
and hemoglobin saturation measurements give information about the
microvascular oxygenation status, they do not provide a direct
measurement of the adequacy of tissue oxygenation. The latter is
highly dependent on factors like tissue oxygen consumption and
diffusion distances within the tissue. Additional measurements of
e.g. oxygen extraction and CO.sub.2 production are therefore often
required.
[0011] More direct spectroscopic determinations of tissue
oxygenation are also possible. One of the oldest, and most widely
used, is NADH-fluorimetry. The measurement of tissue bioenergetics
is commonly used for measurement of the adequacy of tissue
oxygenation. Oxidative phosphorylation occurring in the
mitochondria of cells is the main site for the production of ATP.
In the final step of the electron transport chain, reduced pyridine
nucleotides (NADH) is oxidized to NAD.sup.+ and H.sub.2O, utilizing
molecular oxygen. In contrast to NAD.sup.+, NADH emits blue
fluorescence (around 450 nm) when illuminated with ultraviolet
light (around 360 nm). This allows spectroscopic determination of
relative tissue NADH levels. The fluorescence intensity of NADH is
therefore an optical indicator of cellular metabolism.
[0012] Measurement of the fluorescence intensity of endogenous
mitochondrial NADH in situ can thus be used as a direct measure of
tissue bioenergetics. Since for the conversion of mitochondrial
NADH to NAD.sup.+ the availability of molecular oxygen is
mandatory, lack of oxygen results in accumulation of NADH and
subsequent increase in fluorescence intensity. The fluorescence
intensity is for instance imaged using sensitive photographic or
video techniques and can be used to study the regional
heterogeneity of tissue dysoxia on organ surfaces in vitro and in
vivo. Unwanted influence of the absorbance of hemoglobin can be
corrected by use of a two-wavelength method (Coremans, 1997).
[0013] However, even with proper calibration, exact quantification
of the NADH levels remains impossible (Masters, 1993). One of the
reasons is the contribution of cytosolic NADH and NADPH to the
total fluorescence signal.
[0014] Hence, although oxygen is one of the most important
biological molecules, concentration measurements in vivo remain
cumbersome. The same kinds of problems arise when the concentration
of another substance is measured.
[0015] It is an object of the present invention to provide an
alternative method for determining a concentration of a substance.
Preferably a method is provided wherein at least one of the above
mentioned disadvantages is overcome.
[0016] The invention provides a method for determining a
concentration of a substance in a compartment comprising: [0017]
exciting an endogenous compound of said compartment, or a
functional part, derivative, analogue and/or precursor of said
compound, wherein said compound, functional part, derivative,
analogue and/or precursor, if excited, exhibits a luminescence
and/or transient absorption, the lifetime of which is dependent on
said substance, [0018] measuring the lifetime of luminescence
and/or transient absorption exhibited by said compound, functional
part, derivative, analogue and/or precursor, and [0019] correlating
said luminescence lifetime with said concentration of said
substance.
[0020] According to the present invention an endogenous compound of
an organism, or a precursor, functional part, derivative and/or
analogue thereof, is suitable for concentration measurements of a
given substance, since it is possible to excite an endogenous
compound or a precursor, functional part, derivative and/or
analogue thereof in order to exhibit a luminescence and/or a
transient absorption, the lifetime of which is dependent on the
concentration of said substance. Hence, the lifetime of said
luminescence and/or transient absorption is correlated to the
concentration of said substance. An endogenous compound is defined
as a compound which is naturally present in said compartment,
without artificial interference by man, or which is essentially the
same kind of compound as a compound which is naturally present in
said compartment. Preferably said compound is identical to a
compound which is naturally present in said compartment. In one
embodiment said endogenous compound comprises an administered
compound which is essentially the same kind of compound as a
compound which is naturally present in said compartment. In another
embodiment said compound is present as a result of a conversion of
a precursor into at least one compound which is naturally present
in said compartment, or which is essentially the same kind of
compound as a compound which is naturally present in said
compartment. Hence, in one embodiment an endogenous compound is
derived from a precursor.
[0021] It is of course possible to provide a compartment with a
compound which is the same kind of compound as an endogenous
compound. This is for instance done to increase the concentration
of said endogenous compound. Hence, a method of the invention is
not limited to exciting compounds which are already naturally
present in a compartment. Exciting an administered compound which
is essentially the same kind of compound as an endogenous compound,
or which is a functional part, derivative and/or analogue of an
endogenous compound, is also within the scope of the present
invention. Hence, one embodiment of the invention comprises
exciting an endogenous compound, or a functional part, derivative
and/or analogue of an endogenous compound, which has been
administered to a compartment. Additionally, or alternatively, a
method of the invention comprises exciting an endogenous compound
which is already naturally present within said compartment. Yet
another embodiment of the invention comprises administering a
precursor of an endogenous compound, which is capable of being
converted into at least one endogenous compound, and exciting a
compound derived from said precursor. In one embodiment said
precursor is excited.
[0022] A functional part of a compound is defined as a part which
has the same kind of properties in kind, not necessarily in amount.
Preferably said functional part exhibits a luminescence and/or
transient absorption property which is the same--in kind, not
necessarily in amount--as said compound. Most preferably said
functional part comprises the same delayed fluorescence and/or
triplet-triplet absorption properties as said compound in kind, not
necessarily in amount. A functional derivative of a compound is
defined as a compound which has been altered such that the
luminescence and/or transient absorption properties of said
compound are essentially the same in kind, not necessarily in
amount. A derivative can be provided in many ways, for instance by
addition, deletion and/or substitution of at least one atom or
group, by an esterification, et cetera.
[0023] A person skilled in the art is well able to generate
analogous compounds. An analogue has essentially the same
luminescence and/or transient absorption properties of said
compound in kind, not necessarily in amount.
[0024] As used herein, the phrase "endogenous compound" also
encompasses a functional part, derivative and/or analogue of an
endogenous compound.
[0025] A compartment is defined as an area with properties that
make it distinguishable from other areas. Said compartment for
instance comprises an organism as a whole, or a part of an organism
such as for instance an organ, a tissue, a cell, an organelle, a
tumor and/or the microcirculation of an organism, or a part of said
organ, tissue, cell, organelle, tumor and/or microcirculation. In a
preferred embodiment said compartment comprises a mitochondrion. In
one embodiment said compartment comprises a part of an organ,
tissue or cell. With a method of the present invention it is
possible to measure a concentration of a substance in several parts
of an organ, tissue, or cell, such that concentration of a
substance at several sites is determined. In one preferred
embodiment a concentration gradient is determined.
[0026] In yet another embodiment said compartment comprises an in
vitro compartment, such as for instance a culture medium, a cell
suspension, a bioreactor or a tissue or organ cultured in vitro. In
one embodiment said compartment comprises an enclosed area, such as
an organism, cell, organelle (preferably a mitochondrion) or
bioreactor. In an alternative embodiment said compartment is not
enclosed. Examples of such compartments are parts of a tissue,
organ and/or tumor. Although no exact borders of such compartment
are present, usually tissue present within 20 cm, preferably within
15 cm of a given site of interest is considered. In one embodiment
said compartment comprises a tumor, because information about the
concentration of a substance such as oxygen in a tumor is desired
in order to determine whether a certain treatment such as
irradiation and/or photodynamic therapy is suitable. In one
preferred embodiment a concentration gradient through at least part
of an organ, wound and/or tumor is determined.
[0027] With a method of the invention it is possible to measure the
concentration of any substance capable of influencing a
luminescence lifetime and/or transient absorption lifetime of an
endogenous compound, or a functional part, derivative, analogue
and/or precursor thereof, that has been excited. In a preferred
embodiment said substance comprises oxygen. The invention is
further exemplified by the preferred embodiments relating to
determination of oxygen concentration. It is to be understood
however that a method of the invention is also applicable to
determining a concentration of another substance capable of
influencing a luminescence lifetime of an excited endogenous
compound.
[0028] In order to determine oxygen concentration within an
organism, phosphorescent dyes such as metallo-porphyrins are
currently often injected into the circulation. However, as already
mentioned, such methods have the disadvantage of long-term
toxicity. With a method of the invention, wherein an endogenous
compound and/or a precursor thereof is used, this problem is
circumvented.
[0029] Preferably, a method of the invention is provided wherein
said endogenous compound comprises a compound capable of being
excited to a triplet state since molecular oxygen is a molecule of
which the ground state is a triplet state. Oxygen is therefore
capable of quenching an excited triplet state. Hence, a compound
capable of being excited to a triplet state is particularly
suitable for determining an oxygen concentration with a method of
the present invention. As used herein, quenching an excited triplet
state means causing relaxation of an excited triplet state to occur
at a rate that is higher than the rate of spontaneous relaxation.
Spontaneous relaxation means relaxation without the presence of a
substance capable of accelerating relaxation. For instance, in the
presence of oxygen the lifetime of an excited triplet state is
shortened as compared to the lifetime of an excited triplet state
in the absence of oxygen.
[0030] Luminescence for instance comprises phosphorescence and/or
fluorescence. Fluorescence and phosphorescence lifetime
measurements are based on the fact that after pulsed excitation the
emitted signal does not vanish instantaneously, but decays with a
certain lifetime. Energy transfer between the excited molecules and
quencher molecules in its environment causes shortening of the
luminescence lifetime. Preferably said luminescence comprises
delayed fluorescence. Delayed fluorescence is a phenomenon which
occurs in the case of a bi-directional intersystem-crossing. For
instance, repopulation of a S1 state from a T1 state results in
delayed fluorescence. Delayed fluorescence presents itself as
another component of fluorescence besides prompt fluorescence,
having a decay time equal to the lifetime of a triplet state if the
time needed for intersystem-crossing is much shorter than the
lifetime of the T1 state. Compared to prompt fluorescence, delayed
fluorescence is measured much longer after a molecule has been
photo-excited, thus avoiding interference of the emitted light
pulse and the measured fluorescence.
[0031] Transient absorption is defined as a temporary absorption
change after photoexcitation. Such temporary absorption change is
measured using any method known in the art. In one preferred
embodiment said transient absorption comprises triplet-triplet
absorption. A preferred method of the invention therefore comprises
measuring a triplet-triplet absorption. This is for instance
performed with a MicroScan. Triplet-triplet absorption from the
first excited Triplet state (T1) to the second excited triplet
state (T2) is a process that can only occur after previous
population of the first excited Triplet state and during the
existence of this T1 state. If for example the T1 to T2 transition
occurs with the absorption of light of a certain wavelength
.lamda., than a transient absorption of light of wavelength .lamda.
is observed after photo excitation of the compound. This transient
absorption has a lifetime equal to the T1 lifetime and is therefore
also a means to measure the T1 lifetime. Triplet-triplet absorption
measurements require a second light source (with another wavelength
as the main excitation source).
[0032] In one aspect of the invention said endogenous compound
comprises a porphyrin. A porphyrin chelated to an iron atom
constitutes the haem molecule. Haem is one of the central molecules
involved in oxygen transport (haemoglobin and myoglobin) and oxygen
utilisation (cytochromes in the mitochondrial respiratory chain).
Porphyrins are derivatives of porphine. Porphine possesses a
ringsystem (FIG. 2) with four pyrolrings and is a chemically very
stable molecule that can be found as "chemical fossil" in oil.
Porphine and its derivatives are of biological importance because
of their central role in most vital processes were oxygen turnover
takes place. For example in plants derivatives of porphine are key
substances in the photosynthesis process. This is the process were
oxygen is produced out of carbon dioxide and light. In mammals on
the contrary, porphine derivatives like heme and cytochrome C play
central roles in oxygen transport and oxygen consumption.
[0033] Preferably, said endogenous compound comprises a
protoporphyrin. An even more preferred embodiment provides a method
of the invention wherein said compound comprises protoporphyrin IX
or a functional part, derivative and/or analogue thereof.
Protoporphyrin IX (PpIX) is the final precursor in the synthesis of
haem and present in many cells and tissues. Protoporphyrin IX
(PpIX, structure formula in FIG. 2) is synthesized inside the
mitochondria were it becomes heme after inclusion of an iron atom
by the enzyme ferrochelatase. Since the ferrochelatase activity is
rather slow (speed limiting step), adding the precursor
5-aminolevulinic acid (ALA) results in a temporary rise in
intramitochondrial PpIX levels. Hence, if desired, the level of
PpIX in a compartment such as for instance a cell and/or tissue is
easily enhanced by administration of 5-aminolevulinic acid (ALA), a
precursor of the haem biosynthetic pathway. Additionally, or
alternatively, the level of PpIX in a compartment is enhanced by
administration of PpIX. A study of Chantrell et al. reports that
PpIX dimethyl ester does not show measurable phosphorescence in the
visible range (Chantrell et al., 1977). Therefore, this molecule
was not expected to be useful for monitoring a concentration of a
substance like oxygen. However, according to the present invention,
protoporphyrin IX emits delayed fluorescence after excitation.
Protoporphyrin IX possesses an excited triplet state that is
quenched by a substance like for instance oxygen, making its
lifetime dependent on said substance. After excitation of PpIX,
delayed fluorescence is observed. Moreover, triplet-triplet
absorption is measurable. A use of a porphyrin or a functional
part, derivative and/or analogue thereof for determining a
concentration of a substance in a compartment is therefore also
herewith provided. Said porphyrin preferably comprises
protoporphyrin IX. In one preferred embodiment said porphyrin
comprises a clinically used photodynamic agent, preferably (but not
limited to) photofrin, which is currently used for photodynamic
therapy against, amongst other things, tumor cells. This provides
the advantage that oxygen concentration measurements is possible
during therapy with a method of the invention using the therapeutic
agent itself.
[0034] Without being bound to theory, a working model for state
transitions, quenching and measurement modes for PpIX is shown in
the Jablonski diagram in FIG. 4. Most often the population of the
triplet state is achieved through excitation of the molecule from
the ground state S.sub.0 into an excited singlet state (S.sub.1 or
higher), followed by intersystem crossing from S.sub.1 to T.sub.1.
Because the spontaneous T.sub.1.fwdarw.S.sub.0 transition is
spin-disallowed, the rate of occurrence is much less than the
spin-allowed S.sub.1.fwdarw.S.sub.0 transition. This results in
relatively long triplet state lifetimes in the order of .mu.s to
ms. Molecular oxygen, a molecule of which the ground state is a
triplet state, is a quencher of an excited triplet state. If a
molecule while it is in the T.sub.1 state collides with an oxygen
molecule, the oxygen absorbs the energy from the excited molecule.
This event results in a relaxation of the excited molecules at a
rate higher than the rate of spontaneous relaxation. At
sufficiently low concentrations of excited molecules, the
relationship between the T.sub.1 lifetime and the oxygen
concentration is given by the Stern-Volmer relationship:
1 .tau. = 1 .tau. 0 + k q [ O 2 ] ( 2 ) ##EQU00001##
where .tau. is the T.sub.1 lifetime, .tau..sub.0 is the T.sub.1
lifetime in the absence of oxygen and k.sub.q is the rate constant
of quenching by oxygen. Quantitative oxygen concentration
measurements are possible by means of T.sub.1-lifetime
measurements.
[0035] In general, T.sub.1-lifetimes are determined in several
ways. In FIG. 4 three different modes of T.sub.1-lifetime
measurements are shown: phosphorescence, triplet-triplet absorption
and delayed fluorescence. In the case of exogenous phosphorescent
dyes, phosphorescence lifetimes are measured by measuring the decay
of the emitted light after pulsed excitation. However, PpIX does
not show measurable phosphorescence (Chantrell et al., 1977).
Triplet-triplet absorption relies on the measurement of the
transient increase in absorption after photo excitation and
population of the triplet state. Triplet-triplet absorption is also
suitable for measuring a triplet lifetime of PpIX. One embodiment
therefore provides a method of the invention wherein measuring said
transient absorption lifetime comprises measuring triplet-triplet
absorption.
[0036] In view of the fact that PpIX does not show measurable
phosphorescence, PpIX was not considered in the art to be suitable
for monitoring a concentration of a substance like for instance
oxygen. However, according to the present invention, PpIX is
nevertheless suitable since it shows delayed fluorescence and
triplet-triplet absorption with an oxygen dependent lifetime. In
contrast to phosphorescence, delayed fluorescence is not
red-shifted compared to the prompt fluorescence. Delayed
fluorescence of PpIX has not been described in the art.
[0037] In vitro studies by the present inventors have shown that
PpIX shows a type of delayed fluorescence with a decay time
comparable to the decay of the T.sub.1 state. The decay of the
T.sub.1 state was determined by measurement of the light
transmission through the sample, the transmission being the reverse
of the Triplet-Triplet absorption (FIGS. 3A and 3B). The sample
consisted of a solution of PpIX bound to albumin. Moreover,
experiments showed that the decay time of the delayed fluorescence
is dependent on the oxygen concentration (FIG. 3C). FIG. 4 shows a
working model for state transitions, quenching and measurement
modes.
[0038] PpIX is the final precursor in the synthesis of haem used
for haemoglobin, myoglobin and cytochromes, all key substances in
the transport and/or utilization of oxygen. This makes the use of
PpIX as oxygen sensor even more attractive because it provides a
unique method for measurement of an oxygen concentration at the
place where the availability of oxygen is the most important (i.e.
intracellular and inside the mitochondria). Moreover, delayed
fluorescence measurements are easier to implement in vivo and in
clinical use than absorption measurements. With a method of the
invention it has become possible to measure the amount of a
substance like oxygen directly in a cell and/or organelle, without
the need of addition of exogenous, toxic compounds to an organism
and without the need to indirectly deduce said concentration from
for instance the concentration of said substance in the bloodstream
or in an intercellular environment.
[0039] In one aspect a method of the invention is provided wherein
an endogenous compound or a functional part, derivative, analogue
and/or precursor thereof is photo-excited. This is a usual way of
exciting a compound and a lot of equipment for photo-exciting is
available in the art. An example of a photo-exciting device is
described in Shonat et al, 1997, incorporated herein by reference.
However, in other embodiments an endogenous compound or a
functional part, derivative, analogue and/or precursor thereof is
excited by other means, like for instance electromagnetic
radiation.
[0040] Since protoporphyrin IX is naturally present within cells,
it has become possible to determine a concentration of a substance,
such as for instance oxygen, within a cell. A method of the
invention is therefore provided wherein said compartment comprises
a cell. In one embodiment said compartment comprises an organelle.
Even more preferably said compartment comprises a mitochondrion,
since protoporphyrin IX is naturally present in mitochondria.
Hence, a method of the invention is particularly suitable for
determining oxygen concentration in mitochondria. This is a
preferred application of the invention since the availability of
oxygen in mitochondria is a measure of tissue bioenergetics. Since
mitochondria normally consume oxygen, a low concentration of oxygen
within mitochondria is indicative for tissue bioenergetics. Tissue
bioenergetics is therefore preferably assessed by determining a
mitochondrial oxygen concentration with a method of the present
invention. One preferred embodiment of the present invention
involves determining mitochondrial oxygen concentration after a
period of tissue dysoxia in order to determine whether tissue cells
are still viable or whether these cells are prone to apoptosis. If
a mitochondrial oxygen concentration appears to be low, it
indicates that bioenergetics still take place and that cells are
still viable. If however mitochondrial oxygen concentrations appear
to be high, bioenergetics hardly--if at all--take place indicating
that cells are prone to apoptosis. Preferably, 5-aminolevulinic
acid is administered to cells, resulting in accumulation of
protoporphyrin IX inside the mitochondria. According to this
embodiment, luminescence and/or transient absorption lifetime of
said accumulated PpIX is measured in order to determine
mitochondrial oxygen concentration. Afterwards, a more diffuse
fluorescence and/or transient absorption is observed in the cytosol
and oxygen concentration throughout the cell is preferably
measured. In one preferred embodiment oxygen concentration in
mitochondria of a cell is determined within four hours, more
preferably within two hours, even more preferably within one hour
after administration of 5-aminolevulinic acid to said cell, because
during this period PpIX primarily accumulates inside mitochondria.
In one embodiment oxygen concentration is determined in other parts
of said cell after four hours.
[0041] A method of the invention is suitable for determining the
concentration of a substance such as oxygen in a tissue or organ,
or in a certain part of a tissue or organ. Important applications
are for instance measurements of oxygen concentration in the heart,
the brain and/or the retina of the eye, preferably during surgery.
Oxygen concentration in the brain and/or heart is for instance
measured in order to determine whether a stroke and/or myocardial
infarction has occurred. In one embodiment oxygen concentrations in
several different parts of a certain tissue or organ are determined
in order to obtain an overall impression, and/or to measure a
pO.sub.2 gradient. A method of the invention is therefore provided
wherein said compartment comprises at least part of a tissue.
[0042] Another application of a method of the invention is
determination of oxygen concentration at a tumor site. In this
embodiment oxygen concentration within a tumor is determined.
Information about oxygen concentration at a tumor site is for
instance required for determining whether a certain kind of
treatment, such as irradiation and/or photodynamic therapy, is
suitable. For instance, little oxygen is present at a solid tumor
site. Irradiation is therefore not likely to be effective at such
site. Therefore, once it is determined with a method of the
invention that an individual is suffering from a solid tumor with
little oxygen, irradiation therapy is preferably not applied.
Instead, alternative treatment is preferred. Hence, therapy is
adapted to information about oxygen concentration, which
information is obtained by a method of the invention. In a further
embodiment, oxygen concentration at a tumor site and/or around a
tumor site is monitored with a method of the invention in order to
monitor progress of disease and/or therapy.
[0043] In one embodiment a concentration of a substance such as for
instance oxygen at a location of interest is measured by providing
an organism with an endogenous compound, and/or with a precursor
thereof, which is coupled to a moiety capable of specifically
binding said location of interest. Said moiety for instance
comprises an antibody or a functional part, derivative and/or
analogue thereof. For instance, if the oxygen concentration in a
tumor is to be measured, an endogenous compound or a precursor
thereof is preferably coupled to an antibody capable of
specifically binding a tumor-specific antigen. A tumor-specific
antigen is an antigen that is present on a tumor cell while it is
less (preferably not) present on normal cells. Said endogenous
compound or precursor coupled to a tumor-specific antibody will
accumulate in and/or around said tumor. This results in an
increased concentration of said endogenous compound and/or
precursor in and/or around said tumor, facilitating oxygen
concentration measurement in and/or around said tumor. Likewise, in
other embodiments the concentration of a substance is specifically
measured at any location of interest, using an endogenous compound
and/or precursor thereof that is coupled to a moiety capable of
specifically binding said location of interest.
[0044] In yet another aspect an endogenous compound such as a
porphyrin, preferably protoporphyrin IX, is administered to the
circulation of an individual. Alternatively, or additionally, a
precursor is administered which is converted in vivo into at least
one metabolite that is essentially the same kind as--preferably
identical to--an endogenous compound and which, if excited,
exhibits a luminescence and/or transient absorption of which the
life time is dependent on the concentration of a given substance.
In one embodiment 5-aminolevulinic acid is administered, which is
metabolized into protoporphyrin IX in vivo. When an endogenous
compound or a precursor thereof is administered to the circulation
of an individual, said molecule is in one embodiment bound, for
instance to albumin, to form a large molecular complex that remains
confined, at least for a certain time, inside the circulation. Said
administered compound which is essentially the same kind as an
endogenous compound, and/or whose metabolite is essentially the
same kind as an endogenous compound, is not or to a lesser extent
toxic as compared to exogenous compounds such as for instance
palladium-porphyrin. Administration of said compound is therefore
not, or to a lesser extent, involved with (harmful) side reactions.
In one embodiment a method of the invention is therefore provided
wherein said compartment comprises the (micro)circulation.
[0045] Another application of a method of the invention is the use
of an endogenous compound or a functional part, derivative,
analogue and/or precursor thereof, for determining the
concentration of a substance in a culture medium. In one embodiment
a certain kind of tissue, cell and/or organism is cultured in a
culture medium. In order to determine the concentration of a
substance within said tissue, cell and/or organism, an endogenous
compound of said tissue, cell and/or organism or a functional part,
derivative, analogue and/or precursor thereof is excited.
Subsequently, the lifetime of luminescence and/or transient
absorption is measured. In one embodiment said culture medium
comprises a cell suspension.
[0046] It is possible to administer to said culture medium a
suitable compound which, when excited, displays a luminescence
and/or transient absorption, the lifetime of which is dependent on
the concentration of a certain substance, or a compound which is
converted in vivo into at least one metabolite that is essentially
the same kind as--preferably identical to--an endogenous compound.
Said administered compound for instance comprises a compound which
is essentially the same kind as--preferably identical to--an
endogenous compound. However, said compound need not be naturally
present in said cultured tissue, cell and/or organism. In one
embodiment a porphyrin, preferably protoporphyrin IX, or a
precursor thereof such as for instance 5-aminolevulinic acid is
administered to a culture medium, such as a bioreactor, in order to
monitor oxygen concentration with a method of the invention,
comprising exciting said porphyrin and measuring the lifetime of
delayed fluorescence. Preferably, said oxygen concentration is
measured at several time points, such that the availability of
oxygen is monitored over time.
[0047] In a preferred embodiment the lifetime of luminescence
and/or transient absorption is compared with a reference. A
reference curve (also called a calibration curve) is for instance
generated, from which kq and .tau..sub.0 are derived. Once kq and
.tau..sub.0 are determined, a luminescence lifetime is correlated
with the concentration of a substance, preferably by the
Stern-Volmer relationship. Additionally, or alternatively, a
reference curve is preferably generated in order to correlate the
lifetime of transient absorption to the concentration of a given
substance. In one embodiment, a compartment is successively
provided with various concentrations of a substance in order to
generate a reference curve. Additionally, or alternatively, several
similar compartments are provided with various concentrations of a
substance. According to this embodiment, luminescence and/or
transient absorption lifetime is determined at various
concentrations of said substance. Many alternative methods of
generating a reference curve are known in the art, which are
suitable for a method of the present invention.
[0048] In order to generate a reference curve, a luminescence
and/or transient absorption lifetime is preferably determined at
least two concentrations of said substance. Preferably however,
luminescence and/or transient absorption lifetime is determined at
least three concentrations of said substance, more preferably at
least four concentrations of said substance. The more luminescence
and/or transient absorption lifetime vs substance concentration
values are measured, the more accurate a reference curve will be. A
reference curve is for instance generated by plotting luminescence
lifetime and/or transient absorption lifetime versus concentration
of a substance. Of course, said reference curve need not to be
physically plotted. It is for instance also possible to store
measured reference values, for instance in a (computer) database. A
formula representing a reference curve is for instance calculated.
In one embodiment a measured luminescence lifetime and/or transient
absorption lifetime is entered into said database, after which an
algorithm calculates and discloses the correlated substance
concentration.
[0049] Preferably, a calibration curve is generated using the same
kind of compartment(s) as the compartment(s) wherein the
concentration of at least one substance is to be measured.
Moreover, said reference curve is preferably generated using the
same kind of substance(s) as the substance(s) whose
concentration(s) is/are to be measured. Once a calibration curve is
generated, it is preferably used to correlate a measured
luminescence and/or transient absorption lifetime with a
concentration of a substance. In one embodiment, a calibration
curve is generated before the concentration of a substance in a
compartment is determined. However, once a calibration curve has
been generated, it is not necessary to generate another calibration
curve each time before a concentration of a substance is
determined. For instance, once kq and .tau..sub.0 have been
determined it is preferably repeatedly used for correlating said
lifetime to the concentration of a certain substance.
[0050] In one aspect of the invention a luminescence lifetime is
measured in the time-domain, meaning that said lifetime is measured
after a pulse of light. In another aspect said lifetime is measured
in the frequency-domain, meaning that continuous excitation takes
place. The phase-shift between a modulated excitation source and
the emitted luminescence is measured. For instance, phosphorescence
and/or delayed fluorescence is capable of being measured in the
frequency domain. Measurement of said lifetime in the frequency
domain is usually cheaper. On the other hand, measurement of said
lifetime in the time domain is possible with a higher intensity of
light.
[0051] A method of the invention is suitable for being performed
with single-photon excitation. However, a preferred embodiment
provides a method of the invention wherein multi-photon excitation
is applied, such as for instance two-photon, three-photon or
four-photon excitation. Multi-photon excitation involves excitation
with multiple photons instead of one. The multiple photons for
instance have one half of the energy of a single photon (in case of
two-photon excitation). The multiple photons have one third of the
energy of a single photon (in case of three-photon excitation), or
one fourth of the energy of a single photon (in case of four-photon
excitation), and so on. Multi-photon excitation is preferred
because it allows for deeper tissue penetration and a more precise
and confined selection of an excitation volume as compared to
single-photon excitation, due to the non-linear multi-photon
effect. Hence, with multi-photon excitation inner parts of a
compartment, such as for instance inner parts of a tissue or organ,
are more easily examined. Multi-photon excitation facilitates
determination of a concentration gradient, for instance from an
outer surface of a tissue until an inner part of such tissue or
vice versa. Moreover, since multi-photon excitation allows for a
more precise and confined selection of an excitation volume, damage
to surrounding tissue is more easily avoided.
[0052] In one preferred embodiment a method of the invention is
used for an "optical biopsy". This means that a certain part of
interest, such as a small part of a certain tissue, is investigated
but not excised. A characteristic such as for instance an oxygen
concentration of said part of interest is determined using a method
of the invention specifically directed to said part of interest,
while said part of interest remains at its original site. For
instance, at least part of a tissue of an organism is investigated
while said part remains in said organism. This is preferably
performed using multi-photon excitation because multi-photon
excitation allows for a precise selection of an excitation
volume.
[0053] In a preferred embodiment, two-photon excitation is applied.
The principles and advantages of two-photon excitation are outlined
in (Mik, 2004), which is incorporated herein by reference. In
contrast to single-photon excitation, two-photon excitation is a
non-linear optical process in which a compound is excited by two
photons instead of a single photon with a double energy (or half
the wavelength). By considering the excitation as the rate-limiting
step in a chemical reaction consisting of a single-step
termolecular process involving one molecule and two photons, one
derives the rate of production of excited-state molecules,
R.sub.TPE:
R TPE = .delta. 2 I A CP 2 ( 3 ) ##EQU00002##
[0054] where .delta. is the two-photon cross-section, l the
path-length, A the cross-sectional area of the beam (multiplying l
by A defines the interaction volume), C the molar concentration of
the excitable compounds and P the power of the excitation beam. In
phosphorescence measurements, the intensity of the signal is
proportional to R.sub.TPE, therefore equation (3) can be rewritten
in terms of signal intensity versus excitation power:
I.sub.0.varies.CP.sup.2 (4)
[0055] where I.sub.0 is the measured phosphorescence intensity at
time zero, i.e. directly after the excitation pulse. In equation 4,
constants influencing the absolute value of I.sub.0, like the
molecular constants, excitation geometry and detection efficiency
are omitted. These constants are intensity independent so that the
proportionality sign describes the relation between I.sub.0 and
P.sup.2. The non-linear behavior of TPE provides a means of
selective excitation within a 3-dimensional space, and the
quadratic dependence of emission intensity versus excitation power
is regarded as proof of the two-photon nature of the studied
phenomena.
[0056] The invention furthermore provides a device for determining
a concentration of a substance in a compartment comprising: [0057]
means for exciting an endogenous compound or a functional part,
derivative, analogue and/or precursor thereof, wherein said
compound, part, derivative, analogue and/or precursor, if excited,
exhibits a luminescence and/or transient absorption of which the
lifetime is dependent on said substance, and [0058] means for
measuring said lifetime.
[0059] Preferably, equipment for optical spectroscopy comprises an
illumination light source, an optical system (for instance
comprising filters, mirrors and lenses) and a detection unit. The
detector for instance comprises a sensitive CCD camera,
photomultiplier tube and/or spectrophotometer. Several descriptions
of optical systems are described in the literature (Carlsen et al,
2002; Baxter et al, 1997; Green et al, 1988). An example of a
frequency domain phosphorescence lifetime measurement device is
described in Shonat et al, 1997, incorporated herein by reference.
Non-limiting examples of a device of the invention are outlined in
the Examples. In a preferred embodiment a combination of a prism
and a bandpassfilter is used, at least partly preventing a high
amount of excitation light to reach the filters in order to avoid
possible disturbance of a delayed fluorescence signal as a result
of fluorescence and/or phosphorescence of the filters themselves.
In a further preferred embodiment a device of the invention
comprises a fast shutter in front of a PMT, preferably a pockel
cell, in order to prevent distortion of the first 20 to 30 .mu.s of
a signal which would otherwise occur if a PMT is gated by switching
the voltages of the second and third dynodes during the laser
pulse. Alternatively a semi-conductor device, preferably an
avalanche-photodiode is used, which is cheaper.
[0060] In one preferred embodiment a device according to the
invention comprising an imaging device capable of oxygen mapping,
preferably a CCD camera and/or a diode array, is used in order to
allow imaging of a specific location.
[0061] Reference measurements are preferably performed for
quantitative measurements, in order to take account of possible
influences of tissue optical properties on the signal.
[0062] The invention is further explained in the following
examples. The examples do not limit the scope of the invention;
they merely serve to exemplify the invention.
EXAMPLES
Example 1
[0063] The spectra of prompt and delayed luminescence were recorded
using a LS50B luminescence spectrometer (Perkin-Elmer, Wellesley,
Mass., USA). Prompt fluorescence was measured using the
fluorescence mode with excitation source correction. Delayed
luminescence was recorded in the phosphorescence mode, using
varying delay times with respect to the excitation flash and a gate
width of 100 .mu.s. The measurements were made at room temperature.
Excitation and emission wavelengths and slit widths will be
specified in the results section. Spectra were recorded with either
air-saturated samples or samples containing zero oxygen. Adding a
sufficient amount of ascorbic acid (20 .mu.l of 200 mM solution) to
the already ascorbate oxidase containing samples (1 unit ascorbate
oxidase per ml, 3 ml total sample volume) created the zero-oxygen
conditions. This method of reducing oxygen levels is explained in
more detail below. The amounts of ascorbate oxidase and ascorbic
acid used did not interfere with the readings of the spectra.
[0064] The experiments concerning comparison of triplet-triplet
absorption kinetics with delayed fluorescence lifetimes, and the
measurement of transient absorption spectra, were performed using a
LFDL-3/Remote flash lamp pumped dye laser (Candela Laser
Corporation, Wayland, Mass.). This system provided pulses with a
duration of approximately 1 .mu.s at 505 nm at a repetition
frequency of 10 Hz. The output of the laser was directly focussed
on the sample, consisting of a quartz cuvette containing the PpIX
solution. The used detector was a R928 (HAMAMATSU, Hamamatsu City,
Japan) photomultiplier tube (PMT) with a C1392-09 (HAMAMATSU,
Hamamatsy City, Japan) gated socket. The detector was coupled to a
monochromator (Oriel 77320) in order to select the emission
wavelength of interest. The output of the PMT was fed into an
oscilloscope (Tektronix 2440, TEKTRONIX INC., Beaverton Oreg., USA)
and transferred to a computer by the serial bus. The
wavelength-dependent transient absorption was measured using a
white light source and scanning of the monochromator. These
experiments were carried out at room temperature (20.degree.
C.).
[0065] Calibration experiments with varying oxygen concentrations
were performed with a different set-up. A XeCl excimer laser
(Lambda Physik LPX 110i, Gottingen, Germany), operated at 10 Hz and
producing 50 mJ pulses was used to pump a dye laser (Lambda Physik,
LPD 3002) operating at 405 nm. The output of the dye laser was
focussed on a quartz optical fiber with a core of 0.6 mm (Ensign
Bickford Optics, Avon, Conn.) using a 3 cm F/1.2 quartz lens. The
fiber was coupled to the reaction vessel (described below) used for
the calibration experiments. The detector was the same R928 PMT
with C1392-09 socked, switched off during 5 .mu.s gate width. The
detector was coupled to the reaction vessel by a VIS-type liquid
light guide with a 5 mm optical core (Oriel, Stratford, USA).
Instead of the monochromator three 630 nm long pass glass filters
were used for filtering of the emission light. The laser pulse was
fired 1 .mu.s after off gating of the PMT, the repetition rate was
10 Hz. Per measurement 64 traces were averaged on a digital
oscilloscope (Tektronix TDS-350, Tektronix Inc., Beaverton Oreg.,
USA). Data were transferred to a computer by serial bus and
lifetime analysis was performed using LabView 5.1 graphical
programming software (National Instruments, Austin, Tex., USA).
Mono-exponential fitting was performed using a Marquard-Levenberg
non-linear fit.
[0066] To perform delayed fluorescence lifetime measurements at
varying oxygen concentrations the oxygen concentration in the PpIX
solution was varied using the ascorbate oxidase/ascorbic acid
enzymatic reaction. Calibration experiments, needing precisely
controlled oxygen concentrations, were performed using a specially
made reaction vessel. The vessel had to be airtight, allow
continuous mixing of the PpIX solution, temperature control,
continuous temperature monitoring and physical access to the
content. The latter was necessary to allow injection of aliquots of
ascorbic acid solution but should not go at the expense of an
interfering oxygen back-diffusion into the sample. It consisted of
two glass parts, a bottom part and a top part. Both parts were
interconnected by screw lock. An airtight connection was assured by
a teflon ring surrounding the connection site. The bottom of the
reaction vessel was flat, to allow continuous stirring of the
content by a magnetic stirrer. The top part contained three
capillary entries: one allowing insertion of a small thermocouple,
one for the insertion of the light guide from the excitation source
and the latter for injection of ascorbic acid. The capillaries had
a length of 2 cm and a lumen of 1 mm diameter. The diffusion
barrier was large enough to prevent measurable oxygen back
diffusion within an hour, an adequate time span for calibration
experiments. This was checked by oxygen dependent quenching of
phosphorescence of Pd-meso-tetra(4)-carboxyphenyl porphine starting
at varying oxygen concentrations below 40 .mu.M. The reaction
vessel was mounted in a temperature-controlled water jacked on top
of a magnetic stirring device. The total content of the reaction
vessel, after insertion of the magnetic stirrer, was 30.7 ml.
Injection of 10 .mu.l of a 200 mM solution of ascorbic acid
resulted therefore in 32.5 .mu.M oxygen steps (PO.sub.2 steps of
approximately 20 mmHg). Prior to the experiments the reaction
vessel was filled with pre-heated, room-air equilibrated PpIX
solution. Special care was taken to remove all air bubbles from the
solution. Calibration experiments were performed at 22.degree. C.
and 37.degree. C.
[0067] Chemicals
[0068] Pd-meso-tetra(4)-carboxyphenyl porphine was purchased from
Porphyrin Products (Porphyrin Products Inc., Logan, Utah, USA).
Protoporphyrin IX disodium salt (PpIX) was purchased from Sigma
(Sigma Chemical CO., St. Louis, Mo., USA). Two regimens of creating
PpIX solutions were used. In the first regimen, 8 mg/ml PpIX was
dissolved in distilled water brought at a pH of 8.0 by titration
with 1M TRIS base. From this solution 0.5 ml was added to 50 ml of
a human albumin solution (40 gr/l) in phosphate buffered saline
(PBS). This mixture was brought to a pH of 7.4 by titration with
HCl. The PpIX is dissolved in an albumin solution to obtain a
complex, mimicking the environmental circumstances in cells and
tissue (Takemura et al., 1991). The experiments concerning
triplet-triplet absorption were performed with PpIX solution
prepared following this protocol. Since dissolving PpIX according
to the protocol above takes rather long (PpIX is usually not
completely dissolved after several hours), during the course of the
study we looked for a more efficient way of preparing the PpIX
solutions. In the second regimen, 4.0 gram of bovine serum albumin
(BSA, Sigma Chemical CO. St. Louis Mo. USA) was dissolved in 200 ml
PBS. To increase the buffer capacity, needed to prevent pH changes
when adding aliquots of ascorbic acid to the solution, 800 mg HEPES
was added. PpIX was dissolved in methanol (6.07 mg PpIX in 10 ml
methanol) and 2 ml of this PpIX solution was immediately added to
the albumin solution, resulting in a final concentration of
approximately 10 .mu.M PpIX. PpIX solutions according to the second
regimen were used for the recording of the shown spectra and
calibration experiments, unless stated otherwise.
[0069] Results
[0070] Metallo-porphyrins used for oxygen concentration
measurements in vivo can usually be effectively excited at several
different wavelengths. For example Pd-porphyrin, the most widely
used phosphorescent dye for in vivo measurements, can be
effectively excited around 400 nm (the Soret maximum) and 530 nm
(the Q-band). Generally, light with a longer wavelength penetrates
deeper into tissue, the reason why usually excitation at 530 nm is
favoured for in vivo measurements, although the excitation
efficiency at 400 nm is much higher. FIG. 5 shows the fluorescence
emission versus the excitation wavelength of PpIX bound to albumin.
Two peak emissions, one around 400 nm and one around 510 nm are
prominently present. The excitation wavelengths of the used lasers
are indicated in the figure for convenience. As will become
apparent, both wavelengths are effective for delayed fluorescence
measurements.
[0071] In order to locate an appropriate wavelength for
triplet-triplet absorption measurements, the transient transmission
spectrum was recorded. FIG. 6 shows the transient transmission
spectrum of a 20 .mu.M PpIX solution as function of the
transmission wavelength. The maximum at 400 nm is caused by
depletion of the ground state by the laser pulse, the minimum at
450 nm is due to population of the T.sub.1 level and absorption to
the T.sub.2 level. These results are in good agreement with
previous studies (Chantrell et al., 1977; Bonnett et al., 1983;
Sinclair et al., 1980).
[0072] To identify the type of delayed luminescence that was
observed after pulsed excitation of PpIX solutions, prompt and
delayed luminescence spectra were recorded. FIG. 7 shows the prompt
fluorescence spectrum, with its characteristic peak at 636 nm.
Delayed luminescence spectra, recorded using varying delays after
the excitation flash, are shown in FIG. 8. FIG. 8A shows the
delayed luminescence in an air-saturated sample. Delayed
luminescence is hardly detectable 30 .mu.s after the excitation and
is totally vanished after a delay of 100 .mu.s. In contrast, FIG.
8B shows that under zero oxygen conditions delayed luminescence can
be detected even after a 1 ms delay. From FIG. 8B it is also
evident that the spectrum of the delayed luminescence is
qualitatively the same as the prompt fluorescence spectrum shown in
FIG. 7. Especially the red shift, characteristic for
phosphorescence, is absent. We therefore identify the delayed
luminescence as delayed fluorescence.
[0073] To be useful for quantitative oxygen measurements, the
delayed fluorescence lifetime should be an appropriate
representative of the T.sub.1 lifetime. To test this, the delayed
fluorescence lifetime was compared to the lifetime of transient
Triplet-Triplet absorption in a deoxygenated sample. FIG. 9 shows
the decay of the triplet state measured with both delayed
fluorescence and Triplet-Triplet absorption. Panel A displays the
decay curve measured by delayed fluorescence at 636 nm after pulsed
excitation at 505 nm. The fast decaying first part of the curve is
an artefact introduced by the excitation source. Panel B contains
the corresponding decay trace as measured by Triplet-Triplet
absorption at 470 nm.
[0074] From FIG. 8 it is already noticeable that the lifetimes of
the delayed fluorescence are highly dependable upon the oxygen
concentration in the solution. A quantitative relationship between
the lifetime and the oxygen concentration is mandatory if delayed
fluorescence lifetimes are to be used for oxygen concentration
measurements. To test the applicability of the Stern-Volmer
relationship we measured delayed fluorescence lifetimes at varying
oxygen concentrations. These experiments were performed using the
described reaction vessel. Starting at a high oxygen concentration
(the sample was equilibrated with room air) the oxygen
concentration was lowered in steps of 32.5 .mu.M as described in
the Materials and Methods section. The Stern-Volmer relationship
predicts a linear relationship between the reciprocal lifetime
(1/.tau.) and the oxygen concentration. FIG. 10 shows the measured
values of the reciprocal lifetime versus the oxygen concentration
at 22 and 37.degree. C. By performing a linear fit procedure on
these data, the quenching constant k.sub.q was determined. The
best-fit results are also shown in FIG. 10. At 22.degree. C.
k.sub.q was found to be 243.+-.5 M.sup.-1.mu.s.sup.-1 and at
37.degree. C. this value was 471.+-.7 M.sup.-1.mu.s.sup.-1.
Measured values for the decay time at zero oxygen conditions,
.tau..sub.0, were 1.4.+-.0.1 ms and 1.0.+-.0.1 ms for 22.degree. C.
and 37.degree. C. respectively. From FIG. 10 it is clear that a
good linearity between the reciprocal values of the lifetimes and
the oxygen concentration exists, as is confirmed by correlation
coefficients of 0.9882 and 0.9924 for 22.degree. C. and 37.degree.
C. respectively. Moreover, no significant departure from linearity
could be detected by a Runs test, providing p-values of 0.07 and
0.79 for 22.degree. C. and 37.degree. C. respectively. These
results show that, at least over the tested oxygen concentration
range, the Stern-Volmer is accurate in quantifying the relationship
between the delayed fluorescence lifetimes and oxygen
concentrations. As a check, a calibration experiment with a PpIX
solution prepared according to the first regimen was run. The
result was comparable to the calibrations performed with solutions
according to the second regimen (data not shown), indicating that
the reported phenomena are independent of the followed preparation
procedure.
[0075] Discussion
[0076] The main findings of this study can be summarized as
follows: 1) PpIX shows delayed luminescence besides the already
known prompt fluorescence. 2) The emission spectrum of the delayed
luminescence overlaps the spectrum of the prompt fluorescence and a
red shift is absent, therefore the delayed luminescence is
classified as delayed fluorescence. 3) The lifetime of this delayed
fluorescence is a representative of the lifetime of the first
Triplet state. 4) Oxygen is a known quencher of the Triplet state
of PpIX and this study shows that the delayed fluorescence lifetime
is also oxygen dependent. 5) Moreover, it is shown that the
Stern-Volmer relationship describes quantitatively the dependence
of the delayed fluorescence lifetime on the oxygen concentration.
These findings show that oxygen-dependent quenching of delayed
fluorescence provides an exciting new method to measure oxygen
concentrations, since it allows non-invasive tissue- and
intracellular oxygen concentration measurements by an endogenous
compound such as for instance a porphyrin.
Example 2
[0077] In this example we demonstrate the feasibility of the
proposed method for measuring intramitochondrial oxygen levels in
living cells.
Equipment
[0078] In this Example a method of the invention is for instance
performed in the time-domain using pulsed excitation from an
experimental high power tuneable laser. The laser of this Example
consists of a doubled flash-lamp pumped Nd-YAG laser pumping an
optical parametric oscillator (OPO). This results in a tuneable
laser providing 10 mJ pulses of 6 ns duration. The laser is coupled
to a quart cuvette containing the studied samples using a glass
fiber. Perpendicular to the laser beam is a detector consisting of
coupling lens, monochromator and photomultiplier tube (PMT). The
photomultiplier (Hamamatsu R928) is working in photon-counting mode
and is gated during laser excitation by reversing the polarities of
the second and third dynode. The current from the PMT is voltage
converted using a fast-switching integrator (integration time 3.5
.mu.s and reset time 0.5 .mu.s). The voltage is digitised at a
sample rate of 250 kHz using a data-acquisition board in a PC. The
signal of 64 pulses is averaged before applying a mono-exponential
fit procedure to the measured decay curves. The lifetime typically
varies from 20 ms at high oxygen levels to 700 ms at zero-oxygen
conditions.
Results
[0079] First the intracellular distribution of protoporphyrin IX as
a function of time after the administration of 5-aminolevulinic
acid (ALA) was investigated. Therefore neuroblastomacells were
incubated with ALA during varying periods of time. Cells were
observed using a Leica fluorescence microscope with appropriate
filterset. FIG. 11 shows the distribution of the PpIX fluorescence
at three different time points (2, 4 and 8 hours for panel A, B and
C respectively). At least until four hours, the PpIX fluorescence
shows a spotty appearance corresponding to a mitochondrial pattern.
At 8 hours a more diffuse fluorescence is observed located in the
cytosol.
[0080] To demonstrate the ability to measure intramitochondrial
oxygen levels, calibration experiments were performed in
suspensions of neuroblastoma cells (4*10.sup.6 cells/ml) after 4
hours incubation with ALA. Extracellular oxygen levels were
controlled using a rotational cell oxygenator and gas flow
controllers. Intramitochondrial oxygen measurements were performed
before and after administration of rotenone. Rotenone is a blocker
of complex 1 of the mitochondrial respiratory chain and therefore
inhibits mitochondrial oxygen consumption. If the measurement is
indeed mitochondrial of nature, adding rotenone will cause a
decrease of intracellular oxygen gradients until ultimately the
intramitochondrial oxygen level is the same as the extracellular
oxygen level. For the measurement this implies that adding rotenone
will cause an increase in the measured intramitochondrial oxygen
concentration. FIG. 12 shows the results of such a measurement. It
is clear that adding rotenone causes the predicted effect, thus the
PpIX signal is mitochondrial of nature. Moreover, the signal can be
calibrated, making quantitative measurements possible.
[0081] From this example it is concluded that after administration
of ALA a time window exists in which PpIX accumulates inside the
mitochondria. Moreover, it is concluded that quantitative
intramitochondrial oxygen measurements are possible in living
cells.
Example 3
[0082] An example of an experimental two-photon set-up is given in
FIG. 13. In this example, excitation is achieved using a Q-switched
laser operating at 1064 nm (Laser 1-2-3, Schartz Electro-Optics
Inc., Orlando, Fla., USA). The laser provides pulses of
approximately 10 ns duration and an energy ranging from 10 mJ per
pulse for in vitro experiments to 100 mJ per pulse in in vivo
experiments. The bundle diameter of the laser beam is slightly
expanded to a final diameter of 5 mm by a beam expander, before
being directed to the focusing lens by an optical mirror with an
enhanced silver reflection surface (Opto Sigma, Santa Anna, Calif.,
USA). The focusing lens is a single plan-convex lens with a focal
length of 2.0 cm. Based on Gaussian beam optics, the bundle
diameter of 5 mm combined with a lens with a focal length of 2.0 cm
results in a focal spot size of 8 .mu.m and a focus length of 94
.mu.m (in air). Assuming a refractive index in tissue of 1.4, the
measurement volume is approximately a cylinder with diameter of 10
.mu.m and a length of 130 .mu.m. The focusing lens is connected to
a micrometer-screw for manual adjustment of the focal plane,
thereby allowing longitudinal measurements to be made. For in vivo
application, the reading of the micrometer screw is multiplied by
the refractive index of tissue, assumed to be 1.4. Emission light
is collected by the same lens and directed towards the photo
detector by two mirrors. Selection of the phosphorescence light is
achieved by two 700.+-.20 nm band pass filters (Oriel, Stratford,
Conn., USA), positioned in series before the cathode of the
photomultiplier tube (PMT, type R928, Hamamatsu, Hamamatsu City,
Japan). The output of the PMT is voltage-converted by a
current-to-voltage converter with subsequent wide-band amplifier
(30 MHz) and fed into a digital oscilloscope (Tektronix 2440,
Tektronix Inc., Beaverton, Oreg., USA). To increase signal-to-noise
ratio, luminescent traces are averaged on the oscilloscope. For
instance, an average of 32 traces is used. The resulting averaged
traces are transferred to a computer by serial bus for
data-collection and analysis using software, for instance written
in LabView (National Instruments, Austin, Tex., USA).
Example 4
[0083] Luminescence lifetimes can be measured both in the
time-domain as well as in the frequency-domain. In the time-domain
the real decay curve is measured after photo excitation with a
short pulse of light. In the frequency-domain the (continuous)
excitation light is modulated with a known frequency and the
lifetime can be determined from the phase-shift between excitation
and emission light. Both methods have their specific advantages and
disadvantages:
TABLE-US-00001 Time-domain Frequency-domain Pros: Pros: No
disturbance by prompt Lock-in amplification (high fluorescence
S/N-ratio) No influence on oxygen tension Relatively cheap Cons:
Cons: Background light needs Possible disturbance by prompt to be
taken care of fluorescence Expensive Oxygen consumption
[0084] Technical improvements in the time domain are described
below.
Optics:
[0085] A monochromator is preferably used instead of filters in
order to avoid possible disturbance of the delayed fluorescence
signal as a result of fluorescence and/or phosphorescence of the
filters themselves. Unfortunately monochromators have low
transmission efficiency and a gain in performance is achieved by
using a different optical system. A cost-effective solution is a
use of band-pass filters combined with an optical system that at
least partly prevents a high amount of excitation light to reach
the filters. An example of this embodiment is shown in FIG. 14.
Detectors:
[0086] Considering the low signal levels PMT's are a good choice.
Due to the high energetic laser pulse and the resulting high amount
of prompt fluorescence the detector and electronics are preferably
protected against damage. In one embodiment gating of the PMT is
performed by switching the voltages of the second and third dynodes
during the laser pulse. This causes distortion of the first 20 to
30 .mu.s of the signal, diminishing adequate measurement of short
lifetimes. A dedicated microchannelplate PMT is therefore a
preferred option. An alternative is using a fast shutter in front
of a standard PMT, e.g. a pockel cell. An even cheaper alternative
is the use of semi-conductor devices like
avalanche-photodiodes.
LEGENDS OF THE FIGURES
[0087] FIG. 1: Schematic example of frequency-domain
phosphorimeter. A sinusoidal voltage (V.sub.excitation) with a
frequency of 2000 Hz is generated by a data acquisition board
(PCI-MIO-16E1, National Instruments). The light output of the green
LED is modulated by V.sub.excitation through a voltage-to-current
converter. The excitation light is filtered by a 530 broadband
bandpass filter (F1) and focussed into a liquid light guide (LLG,
Oriel) by a lens (L1). The emission light returning from the sample
is directed to the detector by a dichroic mirror (M). L2 is a
coupling lens and F2 is a 700 nm bandpass filter. The detector is a
red-sensitive photomultiplier tube (PMT, Hamamatsu R928). The
current from the PMT passes a current-to-voltage converter and is
amplified to generate a signal (V.sub.signal) that can be sampled
by the DAQ-board. The phase-shift between V.sub.excitation and
V.sub.signal is determined by software, for instance written in
LabView (such as version 5.1, National Instruments). The
phosphorescence lifetime (.tau.) is calculated from .DELTA..PHI.,
allowing the calculation of the oxygen tension (pO.sub.2) by the
Stern-Volmer relationship, with .tau..sub.0 the lifetime under
zero-oxygen conditions and k.sub.q the quenching constant.
[0088] FIG. 2: Structure formulas of porphine and protoporphyrin
IX.
[0089] FIG. 3: Panel A: The delayed fluorescence at zero oxygen
conditions measured in a solution of PpIX bound to albumin. Panel
B: The Triplet-Triplet absorption at 470 nm also at zero oxygen,
same sample as A. Panel C: Reciprocal lifetime of delayed
fluorescence as a function of oxygen concentration at two different
temperatures.
[0090] FIG. 4: Jablonski diagram showing, schematically, the energy
states and state-transitions of PpIX and its interaction with
dioxygen. S.sub.0-S.sub.2 denote singlet states. T.sub.0-T.sub.2
denote triplet states. K.sub.q and k.sub.p are the rate-constants
of the occurrence of quenching and phosphorescence respectively. In
the diagram, parentheses and the broken arrow depict the absence of
detectable phosphorescence. K.sub.isc is the rate constant of the
T.sub.1.fwdarw.S.sub.1 intersystem crossing.
[0091] FIG. 5: Emission intensity of PpIX fluorescence as a
function of the excitation wavelength.
[0092] FIG. 6: The transient transmission of 20 .mu.M PpIX in 4%
albumin as function of the transmission wavelength after pulsed
excitation at 505 nm. The maximum at 400 nm is caused by depletion
of the ground state by the laser pulse, the minimum at 450 nm is
due to population of the T.sub.1 level and absorption to the
T.sub.2 level.
[0093] FIG. 7: Prompt fluorescence emission spectrum of PpIX bound
to albumin. Excitation wavelength was 405.+-.2.5 nm. The emission
was detected with a 4 nm slit width of the monochromator.
[0094] FIG. 8: Delayed luminescence spectra of PpIX bound to
albumin. The emission spectra recorded at varying delays after the
excitation flashes are shown. The used gate-width was 100 .mu.s.
All spectra are the result of summation of 10 consecutive runs. The
spectra shown in 8A were recorded in an air-equilibrated sample.
The spectra in 8B show the increase in delayed luminescence after
deoxygenation of the sample (see text for details).
[0095] FIG. 9: Panel A: The delayed fluorescence at zero oxygen
conditions measured in a solution of PpIX bound to albumin. Panel
B: The Triplet-Triplet absorption at 470 nm also at zero oxygen,
same sample as A.
[0096] FIG. 10: Reciprocal lifetime of delayed fluorescence as a
function of oxygen concentration at two different temperatures.
[0097] FIG. 11: Microscopy in ALA incubated neuroblastoma cells.
From up to down the image shows phase-contrast wide field, PpIX
fluorescence and a combination image. Panel A: 2 hours ALA
incubation. Panel B: 4 hours ALA incubation. Panel C: 8 hours ALA
incubation.
[0098] FIG. 12: Intramitochondrial oxygen measurement in a cell
suspension of neuroblastoma cells. Rotenone is a blocker of the
mitochondrial oxygen consumption. After administration of rotenone,
the intramitochondrial PO.sub.2 is assumed to be the same as the
extracellular PO.sub.2.
[0099] FIG. 13: Schematic diagram of the experimental set-up. The
laser provided pulses of 10 ns with a wavelength of 1064 nm at a
repetition rate of 10 Hz. F1 is a 1064 nm laser line bandpass
filter. L1 and L2 form a beam-expander resulting in a beam width of
approximately 5 mm. Mirror M1 is a standard optical mirror with a
central bore-hole for passing of the laser-beam. Mirror M2 has an
enhanced silver surface. L3 is a lens with a focal length of 2 cm.
This distance of this lens to the sample can be varied in the
z-plane (AZ) for adjustment of the measurement depth. Filters F2
and F3 are 700 nm bandpass filters. The detector is a red-sensitive
photomultiplier tube, the output is fed into a digital
oscilloscope.
[0100] FIG. 14: A combination of a prism and a bandpass filter is a
cost-effective alternative to a monochromator and provides even
better transmission efficiency.
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