WO2011068479A1

WO2011068479A1 - Fluorescence excitation-emission matrices (eem) spectroscopy system and method

Info

Publication number: WO2011068479A1
Application number: PCT/SG2010/000451
Authority: WO
Inventors: Zhi Wei Huang
Original assignee: Agency For Science, Technology And Research
Priority date: 2009-12-01
Filing date: 2010-12-01
Publication date: 2011-06-09

Abstract

A fluorescence excitation-emission matrices (EEM) spectroscopy system and method. The system comprises an acousto-optic excitation filtering module for filtering excitation light prior to incidence on a sample; and an acousto-optic emission filtering module for filtering emission light received from the sample; wherein the excitation filtering module comprises means for suppressing side-ripple artefacts, and wherein the emission filtering module comprises means for generating multiple filtered emission light signals for emission wavelength detection.

Description

FLUORESCENCE EXCITATION-EMISSION MATRICES (EEM) SPECTROSCOPY SYSTEM AND METHOD

FIELD OF INVENTION

The present invention broadly relates to a fluorescence excitation-emission matrices (EEM) spectroscopy system and method.

BACKGROUND

Fluorescence spectroscopy has been studied intensively for non- or minimally-invasive tissue diagnosis and characterization. Typically, fluorescence depends on exogenous or endogenous fluorophores in the tissue, which may undergo a change associated with disease transformation. The fluorophores change may be detected as an alteration in the spectral profile and intensity of fluorescence emission. Different excitation wavelengths can induce different types of tissue fluorophores to fluoresce, resulting in different spectral profiles and intensities of tissue spectra with different diagnostic abilities for disease detection.

It has been noted that fluorescence excitation-emission matrices (EEMs) spectroscopy is a powerful technique for comprehensively investigating fluorescence properties of specific fluorophores in tissues for better diagnosis and characterization. In typical existing EEM systems, four different types of tunable excitation light modules are usually used: (i) an arc lamp coupled with a monochromator or interference bandpass filters, (ii) an arc lamp coupled with double monochromators, (iii) a nitrogen-pumped dye laser, and/or (iv) an optical parametric oscillator (OPO) tunable laser. However, all these conventional types of tunable light sources use stepper motor-type mechanisms for rotating gratings or filter wheels, or crystals for tuning the excitation light wavelengths. Such mechanical movement imposes limitations on the speed of tuning from one wavelength to another, resulting in slow EEM data acquisitions (up to minutes or hours), which are unsuitable for e.g. in-vivo biomedical applications.

To tackle this problem, it has been proposed that an acousto-optic tunable filter (AOTF) can be employed to electronically tune various wavelengths of e.g. a xenon arc lamp with a high-throughput (>90% diffraction efficiency) within milliseconds without moving parts by varying the radio frequency (RF) of the acoustic wave propagating through the crystal. To date, most EEM studies have focused on excitation wavelengths ranging between ultraviolet (UV) and shorter wavelength visible light, which are not optimized for deep tissue imaging applications. However, the proposed AOTF technique suffers from side-ripple artefacts.

A need therefore exists to provide a fluorescence excitation-emission matrices (EEM) spectroscopy system and method that seek to address at least some of the above problems.

SUMMARY

In accordance with a first aspect of the present invention, there is provided a fluorescence excitation-emission matrices (EEM) spectroscopy system, comprising: an acousto-optic excitation filtering module for filtering excitation light prior to incidence on a sample; and

an acousto-optic emission filtering module for filtering emission light received from the sample;

wherein the excitation filtering module comprises means for suppressing side-ripple artefacts, and wherein the emission filtering module comprises means for generating multiple filtered emission light signals for emission wavelength detection.

The excitation filtering module may comprise a first acousto-optic tunable filter (AOTF) and the means for suppressing side-ripple artefacts may comprise a second AOTF, wherein polarization axes of the first and second AOTFs may be substantially orthogonal to each other. The second AOTF may be disposed for coupling of diffracted emission light from the first AOTF.

The first and second AOTFs may be alternately disposed between three polarizers.

Polarization axes of adjacent polarizers may be orthogonally oriented with respect to each other.

The means for generating the multiple filtered emission light signals for emission wavelength detection may comprise third and fourth AOTFs, wherein polarization axes of the third and fourth AOTFs may be substantially parallel to each other.

The fourth AOTF may be disposed for coupling of un-diffracted excitation light from the third AOTF and such that diffracted excitation light from the third AOTF may be substantially not coupled into the fourth AOTF.

The third and fourth AOTFs may be disposed between two polarizers.

Polarization axes of the two polarizers may be orthogonally oriented with respect to each other.

Diffracted excitation light from the third and from the fourth AOTF may be passed through one of the two polarizers.

The AOTFs may be configured for near infrared (NIR) light.

The system may further comprise a bifurcate probe optically coupled between the excitation filtering module and the emission filtering module for transmitting excitation light to the sample and receiving emission light from the sample. The system may further comprise a detector optically coupled to the emission filtering module for fluorescence detection.

The system may further comprise a computing device for EEM data acquisition and processing.

In accordance with a second aspect of the present invention, there is provided an acousto-optic excitation filtering module for fluorescence excitation- emission matrices (EEM) spectroscopy, comprising means for suppressing side- ripple artefacts.

The module may comprise a first acousto-optic tunable filter (AOTF) and the means for suppressing side-ripple artefacts may comprise a second AOTF, wherein polarization axes of the first and second AOTFs may be substantially orthogonal to each other.

In accordance with a third aspect of the present invention, there is provide an acousto-optic emission filtering module for fluorescence excitation-emission matrices (EEM) spectroscopy, comprising means for generating multiple filtered emission light signals for emission wavelength detection.

In accordance with a fourth aspect of the present invention, there is provided a fluorescence excitation-emission matrices (EEM) spectroscopy method, the method comprising the steps of:

acousto-optically filtering excitation light prior to incidence on a sample; and acousto-optically filtering emission light received from the sample;

wherein the acousto-optically filtering of the excitation light comprises suppressing side-ripple artefacts, and the acousto-optically filtering of the emission light comprises generating multiple filtered emission light signals for emission wavelength detection.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will be better understood and readily apparent to one of ordinary skill in the art from the following written description, by way of example only, and in conjunction with the drawings, in which:

Figure 1 shows a schematic diagram of a fluorescence EEM spectroscopy system according to an example embodiment.

Figure 2a shows graphs comparing transmission spectra of the excitation light using the cascaded AOTF-NIR EEM design of the example embodiment and the conventional single AOTF-NIR EEM design.

Figure 2b shows graphs comparing performance of three example embodiments of the system of Figure 1.

Figure 3 shows graphs of fluorescence intensity and signal to noise ratio against concentration, respectively, using the system of Figure 1.

Figure 4 shows an example two-dimensional (2-D) EEM map of three NIR fluorescent dyes using the system of Figure 1.

Figure 5 shows a flow chart illustrating a fluorescence EEM spectroscopy method according to an example embodiment.

DETAILED DESCRIPTION

The example embodiments describe an acoustic-optic tunable filter (AOTF) - based near-infrared (NIR) fluorescence excitation-emission matrices (EEMs) spectroscopy system and method for rapid multi-fluorophores analysis and characterization. In the example embodiments, the excitation and emission AOTF filtering modules coupled with polarizers are incorporated into the EEM system to effectively remove the side-ripple artifacts of AOTFs, as well as to improve the collection efficiency for high-quality fluorescence EEM measurements.

Figure 1 shows a schematic diagram of a fluorescence EEM spectroscopy system 100 according to an example embodiment. System 100 comprises a light source 1 in the form of e.g. a xenon arc lamp, an excitation filtering module 6 for electronic tuning of excitation light wavelengths received from the light source 1 , a bifurcate fiber-optic probe 7 for excitation light delivery (from the excitation filtering module 6 to a sample 14) and fluorescence light collection (from the sample 14), an emission filtering module 9 for fluorescence emission received from the probe 7, and a detector 12 optically coupled the emission filtering module 9.

In the example embodiment, system 100 further comprises a lens 3, a hot mirror 4 and a lens 5, respectively, disposed between the light source 1 and the excitation filtering module 6; a lens 8 disposed between the probe 7 and the emission filtering module 9; and a lens 10 and an optic fibre 11 disposed between the emission filtering module and the detector 12. The detector 12 in the example embodiment is coupled to a computer 13 for e.g. data acquisition and processing.

Typically, the light source 1 comprises an adjustable parabolic reflector 2 as shown in Figure 1. An example suitable light source 1 comprises a 300 Watts (W) xenon arc lamp, emission spectrum from 300-1000 nanometres (nm), manufactured by Newport Inc., Stratford, Connecticut. As can be seen from the enlarged portion of the excitation filtering module 6, in the example embodiment, the excitation filtering module 6 comprises a pair of cascaded NIR AOTFs 63, 65 (e.g. model no. MD 21152-9201 , manufactured by Brimrose Corp., Sparks, Maryland) coupled with three orthogonally oriented polarizers 62, 64, 66 (to be described in detail below). Additionally, the emission filtering module 9 in the example embodiment comprises a pair of AOTFs 93, 95 arranged in series coupled with two orthogonally oriented polarizers 92, 94 (to be described in detail below). Further, the detector 12 comprises an avalanche photodiode integrated with a single-photon counting module (SPCM) (e.g. model no. SPC -AQR, manufactured by PerkinElmer Inc., California) for fluorescence detection. The computer 13 comprises e.g. Labview-based software for real-time EEM data acquisition and processing (e.g. synchronization of AOTFs for excitation and emission wavelengths tuning, SPCM dark-noise subtraction, wavelength calibration, system spectral response calibration, wavelength-dependent excitation power calibration and normalization, etc.).

As described, the excitation filtering module 6 in the example embodiment comprises the cascaded AOTFs 63, 65 coupled with three polarizers 62, 64, 66. In this excitation filtering design, the orientations of polarization axes of the adjacent polarizers 62, 64, 66 as well as of the two AOTF modules 63, 65 are orthogonally oriented with each other to optimize the coupling of the incident light among the different optical components.

In the example embodiment as shown in Figure 1 , the approximately collimated (i.e. nearly parallel) incident light of the xenon lamp 1 passes through the first polarizer 62 oriented in the vertical direction, and the resulting vertically polarized incident light is optimally coupled into the first AOTF 63 such that the excitation light can be spectrally filtered out by the first AOTF 63 along its first order diffraction direction which is well separated from the undiffracted zero order beam. The spectrally filtered light from the first AOTF 63 is horizontally polarized (the polarization direction of light changes 90 degrees after the first AOTF 63), and passes through the second polarizer 64 for further purification of the polarization status of the incident light along the horizontal direction. Then, the horizontally polarized light is coupled into the second AOTF 65 for further spectral filtering and another 90° polarization rotation. A third polarizer 66 oriented in vertical polarization is placed after the second AOTF 65 to block the residual horizontally polarized light while allowing the doubly spectral filtered incident light in vertical polarization to exit from the excitation filtering module 6 for fluorescence excitation.

As will be understood by a person skilled in the art, the side-ripple suppression level, S^ , of an excitation filtering module comprising a single AOTF is the ratio of the maximum intensity of the first side-robe band I_s to the primary band transmission intensity :

where u is the acoustic-optic (AO) coupling coefficient, and δ is the smallest positive non-zero solution of equation tan£ = δ .

Without a loss of generality, by assuming that the coupling coefficients u of the two AOTFs are the same, the side-ripple suppression level, S_c , of the excitation filtering module 6 comprising cascaded AOTFs 63, 65 in the example embodiment can be expressed as.

Hence, compared to e.g. the single AOTF filtering design, the cascaded AOTF filtering module design in the example embodiment provides approximately 2- fold improvements in side-ripple suppression of the spectrally filtered excitation light. Additionally, the orthogonal polarization settings between the cascaded AOTFs 63, 65 and the polarizers 62, 64, 66 ensure both the polarization and spatial separations of the spectrally filtered light beam (i.e. first order diffraction) from the non-diffracted light beam (i.e. zero order) to be about twice as compared to the single AOTF filtering module, thereby further reducing the out-of-band light leakage.

It will be appreciated while more pairs of AOTFs and polarizers may be used for better side-ripple suppression, the intensity level of the excitation light in such cases may drop significantly (every pair of AOTFs used can only give about 80% of transmission of the incident light). Thus, the double-diffraction (i.e. cascaded AOTFs) design of the example embodiment is advantageously capable of providing a proper incident light intensity level while still maintaining a relatively good side- ripple suppression level compared to e.g. the single AOTF design. Using the excitation filtering module 6 of the example embodiment, it is possible to electronically continuously tune the excitation wavelengths of the xenon light source 1 ranging from about 550 to 950 nm with a narrow bandwidth (e.g. full width at half maximum (FWH ) of approximately 5 nm (or even narrower) for each excitation wavelength) at about 10 nm increments or even smaller intervals. The wavelength tuning at different intervals may be carried out, e.g. by changing the RF frequency of the controller electronically, as will be understood by a person skilled in the art.

Furthermore, in the example embodiment as shown in Figure 1 , the emission filtering module 9 comprises a pair of NIR AOTFs 93, 95 arranged in series (i.e. with the same incident axis and polarization orientation with each other) and two orthogonally oriented polarizers 92, 94. In the example embodiment, the polarizer 92 is disposed before the first AOTF 93 and the second polarizer 94 is disposed after the second AOTF 95, respectively. The emission filtering design of the example embodiment ensures that the total diffractive fluorescence intensity, I_em , from both the first and second AOTFs 93, 95 can pass through the second polarizer 94 for fluorescence detection, which can be written as:

where p denotes the cross-talk coefficient between the first and the second AOTFs 93, 95 whereby the spectrally filtered fluorescence light from the first AOTF 93 is collected by the second AOTF 95.

By choosing the separation/disposition between the parallel polarization AOTFs 93, 95, the cross-talk coefficient can be minimized (i.e. Hence, the overall fluorescence intensity / in the example embodiment is rewritten as:

2 -sin' sin = (2 -/₀Wo (4) I₀ = sin (4.1 )

\2j

where I₀ (<1 ) is the maximum diffraction efficiency of a single AOTF emission filtering module.

That is, the double AOTF emission filter module design of the example embodiment advantageously provides a higher diffraction efficiency of fluorescence detection (approximately 2-fold improvement) compared to the single AOTF filtering module. The present design is also able to define/scan the fluorescence emission wavelengths without a mechanical scan.

The inventors have verified the improvements of the side-ripple suppression and the detection efficiency for fluorescent dyes measurements based on the excitation and emission filtering modules of the example embodiment.

Figure 2a shows graphs comparing transmission spectra of the excitation light using the cascaded AOTF-NIR EEM design of the example embodiment and the conventional single AOTF-NIR EEM design. Here, the wavelength of excitation light from the xenon arc lamp is about 730 nm. As can be seen from Figure 2a, the out-of-band rejection in the cascaded AOTF EEM design of the example embodiment (as exemplified by curve 202) is approximately 2 orders of magnitude higher than that of the single AOTF EEM design (curve 204) (i.e. improved from about -20 dB to -40 dB as shown in Figure 2a).

Figure 2b shows graphs comparing performance of three example embodiments of the system of Figure 1. More specifically, Figure 2b shows the comparison of fluorescence spectra of diethylthiatricarbocyanine (DTTC) iodide with a concentration of 1.84X10^"6 M in ethanol solution acquired by using three different fluorescence EEM spectroscopy designs: curve 212 is acquired using a preferred embodiment of the system; curve 214 is acquired using an alternate embodiment of the system in which the excitation filtering module is replaced with a single AOTF module design; and curve 216 is obtained using another embodiment of the system in which the emission filtering module is replaced with a single AOTF module design. In Figure 2b, each fluorescence spectrum is normalised to its excitation power on the sample.

As can be seen from Figure 2b, the side-ripple artefacts of the AOTF peaking at about 830, 885, 910 and 920 nm occur in the fluorescence spectrum measured using the embodiment with the excitation filtering module comprising a single AOTF (as exemplified by curve 214), while these spectral contaminations are effectively removed using embodiments with the excitation filtering module comprising cascaded AOTFs (as exemplified by curves 212 and 216). Furthermore, curve 212 shows a higher peak fluorescence intensity than curve 216. That is, the diffraction efficiency of the preferred embodiment using the emission filtering module with double AOTFs (curve 212) is enhanced compared to that of the embodiment using the emission filtering module with a single AOTF (curve 216).

The repeatability and sensitivity of the AOTF-based NIR EEM system of the example embodiment have also been evaluated. Figure 3 shows graphs 302, 304 of fluorescence intensity and signal to noise ratio against concentration, respectively, using the system of Figure 1. In Figure 3, the fluorescence peak intensity of DTTC is measured at wavelength of about 805 nm with the concentration ranging from 5.74 * 10^"8 to 1.84 x 10^"6 M in ethanol solution. The corresponding signal-to-noise ratios (SNR) are measured under the excitation wavelength of 730 nm with incident power of 50 microwatts (uW) on the sample. As can be seen from graph 302 of Figure 3, the variations (standard deviations for 10 measurements at each concentration) of the fluorescence signals detected by the NIR-EEM system of the example embodiment are less than about 8%. Also, the fluorescence intensity observed is approximately proportional to the increased DTTC concentrations (from 5.74 * 10^"8 to 1.84 x 10^"6 M in ethanol solution), and the corresponding signal-to-noise ratios (as exemplified by graph 304) also increase (from 2 dB up to 30 dB) accordingly.

The inventors have also noted that NIR fluorescence EEM spectroscopy (e.g. 41 excitation wavelengths ranging from 550 to 950 nm in 10 nm increments; fluorescence emission from 570 to 1000 nm at 5 nm intervals) can be acquired from multi-fluorophores within 10 seconds (e.g. each data point is integrated with about 10 ms to ensure a good SNR of 30 dB) or even shorter (e.g. in the range of a few seconds or subseconds by using a shorter integration time of 1 ms for each data point) utilizing the system of the example embodiment. Figure 4 shows an example two- dimensional (2-D) EEM map of three NIR fluorescent dyes using the system of Figure 1. The three NIR fluorescent dyes comprise oxazine 750 at 2.13 * 10^"6 M, DTTC iodide at 1.84 ^χ 10^"6 M, and IR 140 dye at 1.28 10^"6 M mixed in ethanol solution. The EEM in this example is acquired in 10 seconds (s) with an SNR of over 20 dB. The excitation-emission maxima of the three dyes, such as Ex=670 nm: Em=695 nm for Oxazine750, Ex=780 nm: Em=800 nm for DTTC iodide, and Ex=810 nm: Em=850 nm for IR140, can be identified using the AOTF-based NIR-EEM system of the example embodiment.

It will be appreciated that the slight shifts of the EEM maxima of each fluorescent dye observed in the mixtures are probably due to the inter-system energy transfers among the different fluorescent dyes in ethanol solution. As such, the AOTF NIR-EEM system of the example embodiment may also be useful for monitoring the relative stability of the fluorescence dyes as well as the extent of energy transfer between different fluorescent dyes in mixtures followed by different light irradiations. For example, the maxima of the excitation-emission pairs in EEM contour plots which correspond to specific fluorophores may be monitored such that any shifts of the maxima, which may indicate the energy transfer among different fluorophores, can be revealed readily in the EEM contour plots.

Figure 5 shows a flow chart 500 illustrating a fluorescence EEM spectroscopy method according to an example embodiment. At step 502, excitation light is acousto-optically filtered prior to incidence on a sample. At step 504, emission light received from the sample is acousto-optically filtered; wherein the acousto-optically filtering of the excitation light comprises suppressing side-ripple artefacts, and the acousto-optically filtering of the emission light comprises generating multiple filtered emission light signals for emission wavelength detection.

The NIR EEM technique as described in the example embodiments may have the advantages of fully electronic tuning abilities of wavelengths without any mechanical scanning parts, thereby enabling fast excitation and emission wavelengths tuning, large sizes of excitation-emission data matrices for multi- fluorophores analysis, as well as high reproducibility of EEM scans in a rapid manner. The excitation and emission filtering modules of the example embodiments may advantageously ensure an effective suppression of side-ripple artifacts of AOTFs and improve the detection efficiency for high quality fluorescence NIR EEM measurements. Preferably, the NIR EEM technique of the example embodiments may also have fast measurement times (in the order of tens of seconds or shorter), making in vivo measurements convenient and efficient, and have the potential to become an effective tool in clinical research and practice in clinical settings (e.g. diagnosis at endoscopy, dermatology, etc).

The rapid and high-throughput fluorescence EEM technique of the example embodiments potentially has many other on-iine applications. For example, EEMs can be used to evaluate topical pharmaceuticals, assay and quantify drug delivery in photodynamic therapy, validate the ultraviolet (UV) light protection of sunscreens, and determine epidermal proliferation. This technique can even be used to improve the performance of high-performance iiquid-chromatography (HPLC) instrumentation. The EEM technique of the example embodiments can also be a powerful diagnostic tool for identifying and analyzing the concentrations of components in other materials (e.g. starch, protein, liquid, and fiber concentrations of grains; octane numbers of gasoline; moisture content of chemicals or food; or the iignin content of pulp and paper, etc.).

It will be appreciated by a person skilled in the art that numerous variations and/or modifications may be made to the present invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. For example, the system of the example embodiment has been described with respect to visible/NIR bands where currently available AOTFs have optimum transmission efficiency, it can also be readily modified into an EEM system for UV/visible bands by replacing the visible/NIR AOTFs with high performance UV/visible AOTFs. The present embodiments are, therefore, to be considered in all respects to be illustrative and not restrictive.

Claims

1. A fluorescence excitation-emission matrices (EEM) spectroscopy system, comprising:

an acousto-optic excitation filtering module for filtering excitation light prior to incidence on a sample; and

2. The system as claimed in claim 1 , wherein the excitation filtering module comprises a first acousto-optic tunable filter (AOTF) and the means for suppressing side-ripple artefacts comprises a second AOTF, wherein polarization axes of the first and second AOTFs are substantially orthogonal to each other.

3. The system as claimed in claim 2, wherein the second AOTF is disposed for coupling of diffracted emission light from the first AOTF.

4. The system as claimed in claims 2 or 3, wherein the first and second AOTFs are alternately disposed between three polarizers.

5. The system as claimed in claim 4, wherein polarization axes of adjacent polarizers are orthogonally oriented with respect to each other.

6. The system as claimed in any one of the preceding claims, wherein the means for generating the multiple filtered emission light signals for emission wavelength detection comprises third and fourth AOTFs, wherein polarization axes of the third and fourth AOTFs are substantially parallel to each other.

7. The system as claimed in claim 6, wherein the fourth AOTF is disposed for coupling of un-diffracted excitation light from the third AOTF and such that diffracted excitation light from the third AOTF is substantially not coupled into the fourth AOTF.

8. The system as claimed in claims 6 or 7, wherein the third and fourth AOTFs are disposed between two polarizers.

9. The system as claimed in claim 8, wherein polarization axes of the two polarizers are orthogonally oriented with respect to each other.

10. The system as claimed in claim 9, wherein diffracted excitation light from the third and from the fourth AOTF is passed through one of the two polarizers.

11. The system as claimed in any one of the preceding claims, wherein the AOTFs are configured for near infrared (NIR) light.

12. The system as claimed in any one of the preceding claims, further comprising a bifurcate probe optically coupled between the excitation filtering module and the emission filtering module for transmitting excitation light to the sample and receiving emission light from the sample.

13. The system as claimed in any one of the preceding claims, further comprising a detector optically coupled to the emission filtering module for fluorescence detection.

14. The system as claimed in claim 13, further comprising a computing device for EEM data acquisition and processing.

15. An acousto-optic excitation filtering module for fluorescence excitation-emission matrices (EEM) spectroscopy, comprising means for suppressing side-ripple artefacts.

16. The module as claimed in claim 15, comprising a first acousto-optic tunable filter (AOTF) and the means for suppressing side-ripple artefacts comprises a second AOTF, wherein polarization axes of the first and second AOTFs are substantially orthogonal to each other.

17. An acousto-optic emission filtering module for fluorescence excitation- emission matrices (EEM) spectroscopy, comprising means for generating multiple filtered emission light signals for emission wavelength detection.

18. The module as claimed in claim 17, wherein the means for generating the multiple filtered emission light signals for emission wavelength detection comprises third and fourth AOTFs, wherein polarization axes of the third and fourth AOTFs are substantially parallel to each other.

19. A fluorescence excitation-emission matrices (EEM) spectroscopy method, the method comprising the steps of:

acousto-optically filtering excitation light prior to, incidence on a sample; and acousto-optically filtering emission light received from the sample;