Method for improving fluorescence image contrast

Abstract

There is provided an improved method for enhancing fluorescence images of an object, such as a biological tissue, by selectively eliminating or reducing unwanted fluorescence from fluorophores other than the fluorophore of interest. The method is based on the measurement of the lifetime of fluorophores while preserving information related to the fluorescence intensity of the fluorophore of interest.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This is the first application filed for the present invention.

TECHNICAL FIELD

The present invention relates to a method for reducing and/or eliminating unwanted fluorescence signals in optical images based on fluorescence lifetime of fluorophores.

BACKGROUND OF THE INVENTION

The monitoring of pharmacokinetics, genetic, cellular, molecular or other types of events in vivo is of great interest to monitor drug or gene therapy efficacy as well as disease status or progression in small laboratory mammals and in the human body. In this respect, fluorescence imaging, both in vitro and in vivo, has been used extensively to generate anatomical and functional information from within cells and organisms.

Fluorescence imaging of internal parts of animals (including humans) for anatomical or functional purposes often involves the injection of an extrinsic fluorophore, typically chemically coupled with another molecule, that distributes within the animal and accumulates preferentially in cells and organs of interest. Images are then acquired by detecting the fluorescence and mapping the signal relative to the anatomy of the animal. However, the excitation and emission spectra of such extrinsic fluorophores often overlap with those of intrinsic fluorophores such that the fluorescence signal is a combination of the signals from each fluorophore. Furthermore, such studies are often conducted using more than one extrinsic fluorophores which may have overlapping spectra. As a result, fluorescence images often contain undesirable signals that obscure the signal from the fluorophore of interest.

Methods commonly used to attenuate or eliminate unwanted fluorescence signals are based on spectral differences of the fluorescence emission, fluorescence lifetime differences (e.g. FLIM), or frequency domain hardware techniques. All of them have limitations. Methods based on spectral difference are limited to fluorophores having emission spectra that do not significantly overlap thereby allowing acquisition of fluorescence at a non-overlapping wavelength which is specific for a particular fluorophore. Methods based on fluorescence lifetime help distinguish signals from different fluorophores but do not retain the information related to fluorophore intensity and consequently information related to concentration of the fluorophore is lost. Frequency domain hardware techniques require multiple image acquisition at a plurality of phase delays to suppress unwanted fluorescence and are therefore time consuming.

Accordingly, it would be desirable to be provided with a fluorescence imaging method overcoming the above mentioned deficiencies.

SUMMARY OF THE INVENTION

The present invention provides an improved method for enhancing contrast and specificity of fluorescence images of an object, such as a biological tissue, by selectively eliminating or reducing unwanted fluorescence from fluorophores other than the fluorophore of interest. The method is based on the generation of intensity images weighted as a function of measured lifetime in which the intensity information is conserved and hence information related to the concentration of the fluorophore of interest.

Thus in one embodiment there is provided a method for optical imaging of an object containing two or more fluorophore species in which a fluorescence signal is acquired, using time domain or frequency domain, for one or more region of interest (ROI) of the object using an excitation and an emission wavelength compatible with detection of at least one of the two or more fluorophore species. A fluorescence intensity and a fluorescence lifetime are calculated from the fluorescence signals for each of the pixels and the fluorescence intensity is multiplied by a weighting factor. The weighting factor is a function of the calculated fluorescence lifetime and one or more predetermined fluorescence lifetime of the fluorophore species and is used to generate a weighted fluorescence intensity for each pixel of the ROI from which a weighted fluorescence intensity image can be obtained.

In a further embodiment, the method also provides for a adjustment of the fluorescence intensity to account for the relative contribution of each fluorophore. Thus when the fluorescence signal comprises contribution from two or more fluorophore species a contribution fraction is derived for at least one of the fluorophore species and the weighted fluorescence intensity is multiplied by the contribution fraction. The contribution fraction can be determined, for example, by fitting a temporal point spread function (TPSF) of the fluorescence signal with an exponential decay function.

In yet a further embodiment, the method provides for a primary weighting step which can substantially reduce background fluorescence signal from intrinsic fluorophore species. Thus the fluorescence intensity signal can be multiplied by a primary weighting factor prior to the step of multiplying the fluorescence intensity by a weighting factor, the primary weighting factor being a function of the calculated fluorescence lifetime and two predetermined fluorescence lifetimes of two or more fluorophore species that are being imaged.

BRIEF DESCRIPTION OF THE DRAWINGS

Further features and advantages of the present invention will become apparent from the following detailed description, taken in combination with the appended drawings, in which:

FIG. 1A is a schematic representation of the generation of an intensity image from fluorescence signal from a region of interest (ROI) of an object;

FIG. 1B is a schematic representation of the generation of a lifetime image from fluorescence signal from a region of interest (ROI)of an object;

FIG. 2 is a flow chart of an embodiment of the invention in which a weighted intensity image is obtained from a raw intensity image;

FIG. 3 is a flow chart of an embodiment of the invention in which a contribution fraction adjusted weighted intensity image is obtained from a raw intensity image;

FIG. 4 is a flow chart of an embodiment of the invention in which a primary weighting is applied to the raw intensity image;

FIG. 5(a-i) is a raw fluorescence intensity (integration over time of the TPSF in each pixel) image of a [55%:45%] mixture of two fluorophore species namely Cy 5.5 and Atto 680;

FIG. 5(a-ii) is an effective lifetime image generated by fitting the fluorescence TPSF from the dual-dye mixture in each pixel with a mono-exponential decay model.

FIG. 5(a-iii) exhibits a processed intensity image (I_new) obtained by performing a preliminary weighting on the raw intensity image FIG. 1(a-i);

FIG. 5(b-i) exhibits a fluorescence lifetime image of Cy5.5 calculated by dual exponential fitting of the fluorescence TPSF of the dual-dye mixture in each pixel;

FIG. 5(b-ii) exhibits an intensity fraction image of Cy 5.5 calculated by dual exponential fitting of the fluorescence TPSF of the dual-dye mixture in each pixel;

FIG. 5(b-iii) exhibits a weighted fluorescence intensity image (I¹_w) of Cy 5.5 obtained by the method of the invention; At each pixel, the fluorescence intensity is related to the concentration of Cy5.5 at that location;

FIG. 5(c-i) exhibits a fluorescence lifetime image of Atto680 calculated by dual exponential fitting of the fluorescence TPSF of the dual-dye mixture in each pixel;

FIG. 5(c-ii) exhibits an intensity fraction image of Atto680 calculated by dual exponential fitting of the fluorescence TPSF of the dual-dye mixture in each pixel;

FIG. 5(c-iii) exhibits a weighted fluorescence intensity image (I²_w) of Atto680 obtained by the method of the invention; At each pixel, the fluorescence intensity is related to the concentration of Atto680 at that location;

FIG. 6(i) is a raw fluorescence intensity (integration over time of the TPSF in each pixel) image of Cy 5.5 and Atto680; on the left is Atto680; On the right is Cy5.5; One can not distinguish the fluorescence by fluorescence intensity only;

FIG. 6(ii) is a fluorescence lifetime image of the fluorophores Atto680 and Cy5.5; One can distinguish the two fluorophores by their fluorescence lifetime; This is the mechanism behind the fluorescence lifetime image; However, fluorescence intensity (and thus concentration) information is lost in this image;

FIG. 6(iii) is a fluorescence intensity image of Cy5.5 extracted from FIG. 6(i) using the method of the invention; and

FIG. 6(iv) is a fluorescence intensity image of Atto 680 extracted from FIG. 6(i) using the method of the invention.

It will be noted that throughout the appended drawings, like features are identified by like reference numerals.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention provides an improved method for enhancing fluorescence images of an object, such as a biological tissue, by selectively eliminating or reducing unwanted fluorescence from fluorophores other than the fluorophore of interest. The method is based on the measurement of the fluorescence intensity and lifetime of fluorophores. The resulting image preserves information related to the fluorescence intensity (and thus the concentration) of the fluorophore of interest. It will be appreciated that the method may be applied to different techniques such as optical imaging, time-resolved fluorescence microscopy and the like.

In the present disclosure by fluorophore species it is meant fluorophores having different fluorescence lifetime. Thus fluorophore species may refer to different fluorophore molecules but it may also refer to the same fluorophore molecule in different environments with each environment conferring the fluorophore a different fluorescence lifetime. For example, conditions such as pH, viscosity, temperature and the like are known to affect the lifetime of fluorophores. The environment may also refer to the molecular environment of the fluorophore. For example a fluorophore that is free typically exhibits a different lifetime than the same fluorophore bound to another molecule. The term fluorophore may refer to small molecules or to macromolecules such as proteins that may comprise molecular electronic configurations capable of emitting fluorescent light when excited.

In one embodiment of the present invention the lifetime of a fluorophore species and intensity of the fluorescence are obtained using time domain (TD) data acquisition. A time resolved fluorescence image can be obtained by exciting a fluorophore of interest with a pulsed light source at a fluorescence excitation wavelength and by collecting the fluorescence signal at a fluorescence emission wavelength using a time-resolved photo detector. The pulsed light source can be any type of pulsed laser (e.g. diode laser, solid state laser, gas laser etc.) or other pulsed light sources (e.g. pulsed lamp). The time-resolved photo detector can be a photo multiplier tube (PMT), avalanche photodiodes (APD), PIN coupled with time correlated single photon counting (TCSPC), a streak camera, or a gated intensified charged coupled device (ICCD).

The fluorescence image can be generated by direct imaging of the fluorescent object using a camera or by raster scanning the fluorescent object using a point detector and reconstructing the image using information from each detection point (pixel). An example of the latter modality is employed by the explore Optix™ imager described in international patent application WO 2004/044562 A1 which is incorporated herein by reference.

While the embodiments of the invention will be described using time domain as an exemplary modality of data acquisition, it will be appreciated that the method of the invention may also be applied using frequency domain data acquisition. In frequency domain, one can obtain fluorescence intensity and lifetime by measuring modulation of amplitude and phase shift. Such measurements are well known in the art (Hawrysz and Sevick-Muraca, Neoplasia vol. 2 (5), 2000 p. 388-417 which is incorporated herewith by reference).

As shown in FIG. 1A, each signal acquisition corresponds to a temporal point spread function (TPSF) 10 of the fluorescence signal emitted by the object at a specific detection point 12. By time integrating, either completely or partially, the TPSF at each pixel, one can get a fluorescence intensity image 14, denoted by I_ij which can provide information on the concentration and the localization of the fluorophores. Furthermore by mathematically fitting the TPSF, one can obtain the fluorescence lifetime which can be used to generate a lifetime image 18, denoted by τ_ij (FIG. 1B). If the fluorescence signal is from more than one fluorophore species, a multi-exponential decay model can be used to fit the TPSF and to derive lifetimes for each fluorophore species and contribution fractions of the species.

In most practical cases a TPSF measured at a given point is usually composed of several TPSFs due to the various endogenous and exogenous fluorophore species present in the system. The measured TPSF may then be written as, $\begin{array}{cc}{\mathrm{TPSF}}_{\mathrm{meas}}=\sum _{k=1}^n{f}_{\mathrm{ij}}^k\times {\mathrm{TPSF}}_k& \left(1\right)\end{array}$

where TPSF_k is the fluorescence TPSF of k^th component (fluorophore species) considered in the n-component analysis, and f^k is its contribution fraction. Generally, as mentioned above, curve fitting methods are required to resolve the measured TPSF into its component constituents. By convolving the system Impulse Response Function (IRF) with the modeled TPSFs of the components, an approximation of the measured TPSF may be made. With the use of numerical curve fitting methods, estimates of the lifetimes and/or relative fractional contribution of each of the n components can be obtained.

For example, it is possible to model the contributions of different fluorophore species with the following expression (example provided for two fluorophore species):
f¹_ij exp(−t/τ₁)+f²_ij exp(−t/τ₂)   (2)
with f_ij¹+f_ij²=1

where t is the time, and f_ij¹ and f_ij² are contribution fractions (amplitude factors) and exp(−t/τ₁) and exp(−t/τ₂) model the falling slope of each of the two individual TPSFs. The amplitude factors are related to the concentrations of the species and their fluorescent efficiencies (e.g. quantum yields, extinction coefficients, excitation laser wavelength, fluorescence spectrum, filter window, etc.).

Referring to FIG. 2 and assuming that the fluorophore species of interest (i.e. from which the image is to be reconstructed) has a fluorescence lifetime τ, the intensity image 14 can be multiplied at 20 by a weighting factor which is a function of the fluorescence lifetime τ and the measured (effective) fluorescence lifetime τ_ij, and used to generate a weighted fluorescence intensity image at 22 which is representative of the distribution and the concentration of the fluorophore species of interest.

In one embodiment, the weighting factor is determined by an Indicator (or Rect) function defined by a range of user determined lifetimes that encompasses the measured lifetime τ_ij at a particular pixel. Pixels exhibiting lifetimes outside the predetermined range can be weighted accordingly or simply eliminated.

In a preferred embodiment the error Δτ derived from the fitting of the TPSF to calculate the effective lifetime can be used to determine the range. Thus one can generate a logical image map L_ij by the following criteria: $\begin{array}{cc}{L}_{\mathrm{ij}}=\{\begin{array}{ccc}1& \mathrm{if}& \tau -\mathrm{\Delta \tau }<{\tau }_{\mathrm{ij}}<\tau +\mathrm{\Delta \tau }\\ 0& & \mathrm{otherwise}\end{array}& \left(3\right)\end{array}$

By element-wise multiplying this matrix to the raw intensity image I_ij, one could get a weighted intensity image I_w, in which unwanted fluorescence and/or noise are suppressed. It will be noted that this treatment of the fluorescence signal retains the intensity information of the fluorescence signal.

In the case where two (or more) fluorophore species are contributing to the TPSF, one can obtain the fluorescence lifetimes τ_ij¹ and τ_ij² (more generally τⁿ_ij, and the contribution fractions ƒ_ij¹ and ƒ_ij² (more generally ƒ_ijⁿ) where f_ij¹+f_ij²=1 of the two fluorophores by fitting the TPSF by using a dual exponential decay model.

Referring now to FIG. 3, by element-wise multiplying matrix ƒ_ijⁿ to I_w at 30, one can get a new intensity image Iⁿ_w at 32, which is proportional to the intensity of fluorophore species n. If only 2 fluorophore species are present image I²_w can be obtained by the method summarized in FIG. 3 or by simply subtracting I¹_w from I_w. It will be appreciated that the contribution fractions can be multiplied to the raw intensity image I before performing the weighting step.

Referring now to FIG. 4, it may be advantageous to “clean” the intensity image, prior to weighting, by performing a preliminary weighting at 40 based on the lifetimes of at least two fluorophore species. For example, in an object comprising two fluorophore species with fluorescence lifetimes τ₁ and τ₂, and where τ₁<τ₂, then the measured fluorescence lifetime τ satisfies τ₁<τ<τ₂ if a single exponential decay model is used. Further assuming the fitting error is Δτ, then one can generate a logical image map by the following criteria $\begin{array}{cc}{L}_{\mathrm{ij}}=\{\begin{array}{ccc}1& \mathrm{if}& {\tau }_1-\mathrm{\Delta \tau }<{\tau }_{\mathrm{ij}}<{\tau }_2+\mathrm{\Delta \tau }\\ 0& & \mathrm{otherwise}\end{array}& \left(4\right)\end{array}$

By element-wise multiplying this matrix to the intensity image I_ij, one can generate a new intensity image I_new at 42 which is background suppressed. The background may comprise, for example, fluorescence from intrinsic molecules. I_new can then be used in the process described in FIGS. 2 and 3 to obtain I_w, I¹_w, etc.

It will be appreciated that the method described above can be extended to multi-fluorophore species using a multi-exponential decay model for fluorescence lifetime fitting instead of dual exponential decay model.

It will also be appreciated that the ranges of lifetime on which the weighting is based can be defined by the user according to the desired fluorescence information. In a preferred embodiment, the ranges are defined by the expected (τⁿ) lifetime of the fluorophore species.

For applications such as diagnosis and pharmacological studies, it is often desirable to have an image that provides information on the concentration and depth of the fluorophore species. However, to assume that the fluorescence intensity signal is proportional to the flurophore concentration can be misleading since the depth of the flurophore will also impact the fluorescence intensity signal. Thus to generate an image that reflects the concentration of the fluorophore species the propagation loss of the fluorescence due to tissue absorption and scattering should be taken in consideration. An example of concentration determination is provided below.

EXAMPLES

Example 1

Equal volumes of 50 nM Cy5.5 and 150 nM Atto680 were mixed together. Fluorescence signal was obtained using eXplore Optix™ with a pulsed diode laser wavelength at 666 nm as the excitation light source. When the quantum yield, extinction coefficient, and fluorescence spectrum and filter window information are taken into account, the fluorescence signal ratio of Cy5.5 and Atto680 from the mixture is about 0.55:0.45.

FIG. 5 illustrates the method described above. Panel(a-i) is a raw fluorescence intensity image of the Cy 5.5 and Atto 680 mixture. A lifetime image (panel (a-ii)) was generated using an effective lifetime (fitting the TPSF with a single exponential). Panel (a-iii) exhibits a processed intensity image (I_new) obtained by performing a preliminary weighting on the raw intensity image. Because only Cy5.5 and Atto 680 are present there is no difference between the raw image and processed image (no background fluorescence). Panels(b-i) and (c-i) exhibit lifetime images based on the lifetime of one fluorophore species only after dual exponential fitting of the TPSF. Panels (b-ii) and (c-ii) exhibit a contribution factor image of the fluorophore species. Both lifetime and fraction are obtained at the same time by direct fitting of the TPSF in each pixel using a dual exponential decay model. Panels (b-iii) and (c-iii) exhibit a weighted fluorescence intensity image (I¹_w,I²_w) obtained by the method described above. In the present example the fluorescence intensity is proportional to the concentration of Cy5.5 or Atto680 since both fluorophore are at the same depth (phantom surface);

Example 2

One hundred nM Cy5.5 and 200 nM Atto680 solution were arranged in two separate locations. Fluorescence signal was obtained using eXplore Optix with a pulsed diode laser wavelength at 666 nm as the excitation light source.

In the particular case where the location of fluorophore species within the object are not overlapping, there is no need for multi-exponential fitting of the TPSF and one can proceed directly with the weighting step of the method. FIG. 6 provides such an example in which the two fluorophores species do not overlap. Panel (i) of FIG. 6 is a measured raw fluorescence intensity image with two fluorophore species, Atto 680 on the left and Cy5.5 on the right. From the intensity image alone, without knowing a priori where the fluorophores are located, one would not be able to identify the fluorophores species. Panel (ii) is the corresponding fluorescence lifetime image obtained by fitting the TPSF of each pixel with a single exponential decay model. While the lifetime image enables the determination of the species of the fluorophore if the lifetimes are known a priori, it does not convey any intensity information. However, when the method of the present invention is used the intensity information is preserved. Thus in the example provided below the weighting function was based on a range of lifetimes determined to be between 0.9 and 1.05 ns for Cy 5.5 and 1.7 and 1.83 ns for Atto 680. using the criteria: $L_{\mathrm{ij}}=\{\begin{array}{ccc}1& \mathrm{if}& 0.9<{\tau }_{\mathrm{ij}}<1.05\\ 0& & \mathrm{otherwise}\end{array}$
for Cy 5.5 one obtains the image displayed in panel (iii) and $L_{\mathrm{ij}}=\{\begin{array}{ccc}1& \mathrm{if}& 1.7<{\tau }_{\mathrm{ij}}<1.83\\ 0& & \mathrm{otherwise}\end{array}$

for Atto 680 one obtains the image displayed in panel (iv). Both images retain the intensity information for the fluorophore of interest. Since they are both at the same depth (phantom surface), the intensity is related to their concentration through quantum yield, extinction coefficient, fluorescent spectrum and filter window.

Example 3

The fluorophore species may be the same fluorophore molecule in different environment. Thus, for example, the object may comprise one fluorophore having a lifetime τ₁ when it is bound to a protein and a lifetime τ₂ when it is free. In this case it is possible to model the TPSF by the following dual exponential:
f exp(−t/τ₁)+(1−f)exp(−t/τ₂)   (5)

where, t is the time, τ₁ and τ₂ are the respective lifetimes of the bound and free states and f is the fraction of fluorophores in the bound state: f=[bound]/([bound]+[free]). The parameters in this model can then be obtained from measured data through multi-variate curve fitting. The dual exponential for free/bound fluorophore species can be used to obtain weighted intensity images as described above.

Example 4

Under certain assumptions such as assuming that the optical properties of the medium are the same at the excitation and emission wavelength, the fluorescence intensity as a function of time can be expressed by the Born approximation: $\begin{array}{cc}\begin{array}{c}\varphi \left(t\right)\cong \sum _{\mathrm{dipoles}}\left(\mathrm{QC}\frac{r_{\mathrm{sp}}+r_{\mathrm{pd}}}{4\pi {\mathrm{Dr}}_{\mathrm{sp}}{r}_{\mathrm{pd}}}v{\left(4\pi \mathrm{Dvt}\right)}^{-3/2}{e}^{-\frac{{\left(r_{\mathrm{sp}}+r_{\mathrm{pd}}\right)}^2}{4\mathrm{Dvt}}-{\mu }_a\mathrm{vt}}\right)*\\ \left(\frac{e^{-\frac{t}{\tau }}}{\tau }\right)*\left(\mathrm{IRF}\right)\end{array}& \left(6\right)\end{array}$

Where:

r_ap is the distance from source s (point on the object at which light is injected) to fluorophore depth position P;

r_pd is the distance from fluorophore depth position p to detector d;

μ_a is the optical absorption coefficient;

D is the optical diffusion coefficient, $D=\frac{1}{3{\mu }_s^{\prime }}$
where; μ_s′ is the reduced optical scatter coefficient;

ν is the speed of light in the medium;

Q is the quantum efficiency;

C is the concentration of the fluorophore;

τ is the lifetime of the fluorophore;

the symbol * refers to the operation of convolution and

IRF is the impulse response function of the instrument used to measure fluorescence.

By setting the first derivative of equation 6 as a function of time equal to zero, the time position of the maximum of the TPSF (t_max) can be found. Under certain approximations (absorption is small at time shorter than t_max, the scatter coefficient is known or can be approximated) and by assuming that r_sp is approximately equal to r_pd, it is found that the following equation can be derived from equation 6: $\begin{array}{cc}{t}_{\max }\cong \frac{d\sqrt{\tau }}{\sqrt{\mathrm{Dv}}}& \left(7\right)\end{array}$

where d is the depth of the fluorophore object.

For a given depth, the intensity I of the emission signal detected at the surface can be related to fluorophore concentration by the optical properties of the medium (absorption and scattering coefficients) and the depth of the fluorophore. $\begin{array}{cc}I\propto C{e}^{-\sqrt{\frac{{\mu }_a}{D}d}}& \left(8\right)\end{array}$

Using time-domain information as described above, the depth d can be determined. Isolating C in equation 8 and knowing signal intensity and depth of the fluorophore, one can thus recover the concentration of fluorophore (i.e. the amount of fluorescent molecules per unit volume) within an accuracy that depends exponentially on the recovered depth accuracy. Thus, in another aspect of the invention, estimates of the relative concentration of the fluorophore, Conc._Relative, can be obtained by determining its depth, d, and normalizing the surface intensity measurement, I, as follows (Equation 9):
Conc._Relative=Id²e^{2d√{square root over (μn/D)}}  (9)

under certain assumptions, equation 9 can be derived from equation 1.

If the fluorophore objects are not at the surface of the tissue, the method described above can be used to obtain their concentration map from the weighted intensity image.

Claims

1. A method for optical imaging of an object containing two or more fluorophore species, said method comprising:

acquiring a fluorescence signal comprising time and amplitude information for one or more pixel of a region of interest (ROI) of said object using an excitation and an emission wavelength compatible with detection of at least one of said two or more fluorophore species;

calculating a fluorescence intensity and a fluorescence lifetime from said fluorescence signal for each of said one or more pixel;

multiplying said fluorescence intensity by a weighting factor, said weighting factor being a function of said calculated fluorescence lifetime and at least one predetermined fluorescence lifetime, to generate a weighted fluorescence intensity for said one or more pixel;

generating an image of said ROI based on said weighted fluorescence intensity of said one or more pixel.

2. The method as claimed in claim 1 wherein said fluorescence signal comprises contribution from two or more fluorophore species, the method further comprising steps of:

deriving a contribution fraction for at least one of said fluorophore species; and

multiplying said weighted fluorescence intensity by said contribution fraction.

3. The method as claimed in claim 2 wherein said contribution fraction is determined by modeling said fluorescence signal with a multi-exponential function.

4. The method as claimed in claim 3 wherein said modeling is applied to a temporal point spread function (TPSF) of said fluorescence signal.

5. The method as claimed in claim 1 wherein said weighting factor is determined by an Indicator function.

6. The method as claimed in claim 5 wherein said Indicator function is defined by boundaries which are function of said predetermined fluorescence lifetime.

7. The method as claimed in claim 6 wherein said boundaries are also function of an error associated with said measured lifetime.

8. The method as claimed in claim 7 wherein said weighting factor is 1 when said measured lifetime is within said boundaries and 0 otherwise.

9. The method as claimed in claim 2 further comprising a step of:

multiplying said fluorescence intensity by a preliminary weighting factor prior to said step of multiplying said fluorescence intensity by said weighting factor or said contribution fraction, said preliminary weighting factor being a function of said calculated fluorescence lifetime and two predetermined fluorescence lifetimes corresponding to expected lifetimes of said two or more fluorophore species.

10. The method as claimed in any one of claim 1-9 wherein said fluorescence signal acquisition is selected from frequency domain and time domain modality.

11. The method as claimed in any one of claim 1-9 wherein said fluorescence species comprises a fluorophore that is distributed between a free state and a bound state.

12. The method as claimed in any one of claim 1-9 wherein said weighted intensity is further processed to yield concentration of at least one of said two or more fluorophore species.

13. A method for optical imaging of an object containing two or more fluorophore species, said method comprising:

acquiring a fluorescence signal, said signal comprising fluorescence from said two or more fluorophore species and comprising lifetime information for one or more pixel of a region of interest (ROI) of said object;

calculating a fluorescence intensity and a fluorescence lifetime from said fluorescence signal for each of said one or more pixel;

deriving a contribution fraction for at least one of said fluorophore species;

multiplying said calculated fluorescence intensity by said contribution fraction to generate a species weighted fluorescence intensity;

multiplying said species weighted fluorescence intensity by a weighting factor, said weighting factor being a function of said calculated fluorescence lifetime and at least one predetermined fluorescence lifetime, to generate a weighted fluorescence intensity for said one or more pixel;

generating an image of said ROI based on said weighted fluorescence intensity of said one or more pixel.

14. The method as claimed in claim 13 wherein said contribution fraction is determined by modeling said fluorescence signal with a multi-exponential function.

15. The method as claimed in claim 14 wherein said modeling is applied to a temporal point spread function (TPSF) of said fluorescence signal.

16. The method as claimed in claim 13 wherein said weighting factor is determined by an Indicator function.

17. The method as claimed in claim 16 wherein said Indicator function is defined by boundaries which are function of said predetermined fluorescence lifetime.

18. The method as claimed in claim 17 wherein said boundaries are also function of an error associated with said measured lifetime.

19. The method as claimed in claim 18 wherein said weighting factor is 1 when said measured lifetime is within said boundaries and 0 otherwise.

20. The method as claimed in claim 13 further comprising a step of:

multiplying said fluorescence intensity by a preliminary weighting factor prior to said step of multiplying said fluorescence intensity by said contribution fraction, said preliminary weighting factor being a function of said calculated fluorescence lifetime and two predetermined fluorescence lifetimes corresponding to expected lifetimes of said two or more fluorophore species.

21. The method as claimed in any one of claim 13-20 wherein said fluorescence signal acquisition is selected from frequency domain and time domain modality.

22. The method as claimed in claim 13-21 wherein said fluorescence species comprises a fluorophore that is distributed between a free state and a bound state.

23. The method as claimed in claim 13-21 wherein said weighted intensity is further processed to yield concentration of at least one of said two or more fluorophore species.