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Optics Express

Optics Express

  • Editor: Andrew M. Weiner
  • Vol. 21, Iss. 13 — Jul. 1, 2013
  • pp: 15475–15489
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Quantitative fluorescence and elastic scattering tissue polarimetry using an Eigenvalue calibrated spectroscopic Mueller matrix system

Jalpa Soni, Harsh Purwar, Harshit Lakhotia, Shubham Chandel, Chitram Banerjee, Uday Kumar, and Nirmalya Ghosh  »View Author Affiliations


Optics Express, Vol. 21, Issue 13, pp. 15475-15489 (2013)
http://dx.doi.org/10.1364/OE.21.015475


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Abstract

A novel spectroscopic Mueller matrix system has been developed and explored for both fluorescence and elastic scattering polarimetric measurements from biological tissues. The 4 × 4 Mueller matrix measurement strategy is based on sixteen spectrally resolved (λ = 400 - 800 nm) measurements performed by sequentially generating and analyzing four elliptical polarization states. Eigenvalue calibration of the system ensured high accuracy of Mueller matrix measurement over a broad wavelength range, either for forward or backscattering geometry. The system was explored for quantitative fluorescence and elastic scattering spectroscopic polarimetric studies on normal and precancerous tissue sections from human uterine cervix. The fluorescence spectroscopic Mueller matrices yielded an interesting diattenuation parameter, exhibiting differences between normal and precancerous tissues.

© 2013 OSA

1. Introduction

Mueller matrix polarimetry has received considerable attention for the characterization of a wide range of optical media for numerous practical applications [1

1. D. S. Kliger, J. W. Lewis, and C. E. Randall, Polarized Light in Optics and Spectroscopy (Academic Press, 1990).

6

6. N. Ghosh and I. A. Vitkin, “Tissue polarimetry: concepts, challenges, applications, and outlook,” J. Biomed. Opt. 16(11), 110801 (2011). [CrossRef] [PubMed]

]. The Mueller matrix (a 4 × 4 matrix) is the transfer function of an optical system in its interactions with polarized light and the polarization properties of the medium are encoded in its various elements [2

2. W. S. Bickel and W. M. Bailey, “Stokes vectors, Mueller matrices, and polarization of scattered light,” Am. J. Phys. 53(5), 468–478 (1985). [CrossRef]

,6

6. N. Ghosh and I. A. Vitkin, “Tissue polarimetry: concepts, challenges, applications, and outlook,” J. Biomed. Opt. 16(11), 110801 (2011). [CrossRef] [PubMed]

]. There are three principle characteristics of interest that a Mueller matrix can encode: depolarization, diattenuation and retardance [3

3. R. A. Chipman, “Polarimetry,” Handbook of Optics, 2nd ed., M. Bass, Ed. (McGraw-Hill, 1994) chap. 22, pp. 22.1–22.37

,6

6. N. Ghosh and I. A. Vitkin, “Tissue polarimetry: concepts, challenges, applications, and outlook,” J. Biomed. Opt. 16(11), 110801 (2011). [CrossRef] [PubMed]

]. Motivated by the fact that extraction and quantification of these three medium polarization characteristics can potentially serve as useful biological metric, there has been significant recent interest in exploring quantitative polarimetry for biological tissue characterization, assessment and diagnosis [4

4. V. V. Tuchin, L. Wang, and D. À. Zimnyakov, Optical Polarization in Biomedical Applications, (Springer-Verlag, 2006).

14

14. N. Ghosh, M. F. G. Wood, S. H. Li, R. D. Weisel, B. C. Wilson, R. K. Li, and I. A. Vitkin, “Mueller matrix decomposition for polarized light assessment of biological tissues,” J Biophotonics 2(3), 145–156 (2009). [CrossRef] [PubMed]

]. However, unlike optically clear medium, quantitative polarimetry in complex turbid medium like biological tissue is severely confounded by multiple scattering effects and simultaneous occurrence of several polarization and scattering events [6

6. N. Ghosh and I. A. Vitkin, “Tissue polarimetry: concepts, challenges, applications, and outlook,” J. Biomed. Opt. 16(11), 110801 (2011). [CrossRef] [PubMed]

]. Multiple scattering within tissue leads to strong depolarization of light, creating a large depolarized source of noise which can inhibit the detection of the weak remaining information-carrying polarization signal. Thus, many traditional polarimetry systems are poorly suited for these applications. Moreover, complicated nature of the polarization effects in tissue, including simultaneous multiple effects in presence of multiple scattering, contribute in a complex interrelated way to the Mueller matrix elements, hindering analysis, quantification and unique interpretation of the tissue polarimetry data [6

6. N. Ghosh and I. A. Vitkin, “Tissue polarimetry: concepts, challenges, applications, and outlook,” J. Biomed. Opt. 16(11), 110801 (2011). [CrossRef] [PubMed]

]. The challenges are thus to maximize measurement sensitivity to obtain polarization signals in presence of strong depolarization, and to decouple the individual contributions of simultaneously occurring polarization effects. A number of researchers are thus pursuing innovative solutions to these challenges [4

4. V. V. Tuchin, L. Wang, and D. À. Zimnyakov, Optical Polarization in Biomedical Applications, (Springer-Verlag, 2006).

6

6. N. Ghosh and I. A. Vitkin, “Tissue polarimetry: concepts, challenges, applications, and outlook,” J. Biomed. Opt. 16(11), 110801 (2011). [CrossRef] [PubMed]

, 10

10. N. Ghosh, A. Banerjee, and J. Soni, “Turbid medium polarimetry in biomedical imaging and diagnosis,” Eur. Phys. J. Appl. Phys. 54(3), 30001 (2011). [CrossRef]

]. Quantitative tissue polarimetry research has therefore two major directions – (i) to develop highly sensitive experimental Mueller matrix polarimetry system and (ii) to develop novel inverse analysis methods for extraction and quantification of the constituent intrinsic medium polarimetry characteristics from the ‘lumped’ system Mueller matrix. With regard to the second issue, various kinds of Mueller matrix decomposition methods (product decompositions including the polar decomposition and its other variants, differential matrix decomposition etc.) have been developed and validated in complex tissue-like turbid medium [14

14. N. Ghosh, M. F. G. Wood, S. H. Li, R. D. Weisel, B. C. Wilson, R. K. Li, and I. A. Vitkin, “Mueller matrix decomposition for polarized light assessment of biological tissues,” J Biophotonics 2(3), 145–156 (2009). [CrossRef] [PubMed]

21

21. S. Kumar, H. Purwar, R. Ossikovski, I. A. Vitkin, and N. Ghosh, “Comparative study of differential matrix and extended polar decomposition formalisms for polarimetric characterization of complex tissue-like turbid media,” J. Biomed. Opt. 17(10), 105006 (2012). [CrossRef] [PubMed]

]. Some inroads have also been made to address the first issue, to develop highly sensitive experimental Mueller matrix polarimetry systems [6

6. N. Ghosh and I. A. Vitkin, “Tissue polarimetry: concepts, challenges, applications, and outlook,” J. Biomed. Opt. 16(11), 110801 (2011). [CrossRef] [PubMed]

,12

12. N. Ghosh, M. F. G. Wood, and I. A. Vitkin, “Polarized light assessment of complex turbid media such as biological tissues using Mueller matrix decomposition,” Handbook of Photonics for Biomedical Science, Valery V. Tuchin, Ed. (Taylor and Francis Publishing, 2010) chap. 9, pp. 253 – 282.

, 22

22. J. S. Baba, J. R. Chung, A. H. DeLaughter, B. D. Cameron, and G. L. Coté, “Development and calibration of an automated Mueller matrix polarization imaging system,” J. Biomed. Opt. 7(3), 341–349 (2002). [CrossRef] [PubMed]

30

30. F. Stabo-Eeg, “Development of instrumentation for Mueller matrix ellipsometry,” PhD dissertation, Norwegian University of Science and Technology, (2009).

].

One possible method for improving the sensitivity of the measurement procedure is the use of polarization modulation with synchronous detection [6

6. N. Ghosh and I. A. Vitkin, “Tissue polarimetry: concepts, challenges, applications, and outlook,” J. Biomed. Opt. 16(11), 110801 (2011). [CrossRef] [PubMed]

, 12

12. N. Ghosh, M. F. G. Wood, and I. A. Vitkin, “Polarized light assessment of complex turbid media such as biological tissues using Mueller matrix decomposition,” Handbook of Photonics for Biomedical Science, Valery V. Tuchin, Ed. (Taylor and Francis Publishing, 2010) chap. 9, pp. 253 – 282.

, 23

23. E. Compain, S. Poirier, and B. Drevillon, “General and self-consistent method for the calibration of polarization modulators, polarimeters, and Mueller-matrix ellipsometers,” Appl. Opt. 38(16), 3490–3502 (1999). [CrossRef] [PubMed]

27

27. C.-Y. Han and Y.-F. Chao, “Photoelastic modulated imaging ellipsometry by stroboscopic illumination technique,” Rev. Sci. Instrum. 77(2), 023107 (2006). [CrossRef]

]. Many sensitive detection schemes using polarization modulator have therefore been explored [6

6. N. Ghosh and I. A. Vitkin, “Tissue polarimetry: concepts, challenges, applications, and outlook,” J. Biomed. Opt. 16(11), 110801 (2011). [CrossRef] [PubMed]

]. Snapshot Mueller matrix polarimeter, which employs wavelength polarization coding and decoding for highly sensitive, instantaneous measurement of all the sixteen Mueller matrix elements, is another important development in modulation-based polarimetry [26

26. M. Dubreuil, S. Rivet, B. Le Jeune, and J. Cariou, “Snapshot Mueller matrix polarimeter by wavelength polarization coding,” Opt. Express 15(21), 13660–13668 (2007). [CrossRef] [PubMed]

]. While the modulation based approaches yield the desired high sensitivity (capable of detecting very weak polarization retaining signal transmitted / backscattered from tissue), these are poorly suited for spectroscopic (at multiple wavelengths) applications and applications involving large area imaging, since they usually involve synchronous detection [6

6. N. Ghosh and I. A. Vitkin, “Tissue polarimetry: concepts, challenges, applications, and outlook,” J. Biomed. Opt. 16(11), 110801 (2011). [CrossRef] [PubMed]

]. Yet, in many applications involving tissue characterization, the wavelength dependence of the polarization parameters (depolarization, diattenuation and retardance) or their spatial maps are extremely useful. Therefore there has been growing need to develop alternative (non-modulation-based) approaches for sensitive, spectrally and spatially (imaging) resolved Mueller matrix measurements. Indeed, dc measurement approaches involving sequential measurements with different combinations of polarization state generator (PSG) and polarization state analyzer (PSA) units have been explored for this purpose [22

22. J. S. Baba, J. R. Chung, A. H. DeLaughter, B. D. Cameron, and G. L. Coté, “Development and calibration of an automated Mueller matrix polarization imaging system,” J. Biomed. Opt. 7(3), 341–349 (2002). [CrossRef] [PubMed]

]. For example, polarimetric systems based on this principle using liquid crystal variable retarders have been developed for imaging applications [28

28. B. Laude-Boulesteix, A. De Martino, B. Drévillon, and L. Schwartz, “Mueller polarimetric imaging system with liquid crystals,” Appl. Opt. 43(14), 2824–2832 (2004). [CrossRef] [PubMed]

, 29

29. A. De Martino, E. Garcia-Caurel, B. Laude, and B. Drévillon, “General methods for optimized design and calibration of Mueller polarimeters,” Thin Solid Films 455−456, 112-119 (2004).

].

In this paper, we present a novel spectral Mueller matrix system for both fluorescence and elastic scattering polarimetric measurements from depolarizing scattering medium such as tissues. In this approach, four required elliptical polarization states are sequentially generated and analyzed by using a PSG unit (comprising of a fixed linear polarizer with its axis oriented at horizontal position followed by a rotatable achromatic quarter wave retarder) and a PSA unit (similar arrangement of fixed linear polarizer with its axis oriented at vertical position and rotatable achromatic quarter wave retarder, but positioned in a reverse order). Sixteen spectrally resolved measurements are combined to construct the sample Mueller matrix. Eigenvalue calibration [28

28. B. Laude-Boulesteix, A. De Martino, B. Drévillon, and L. Schwartz, “Mueller polarimetric imaging system with liquid crystals,” Appl. Opt. 43(14), 2824–2832 (2004). [CrossRef] [PubMed]

30

30. F. Stabo-Eeg, “Development of instrumentation for Mueller matrix ellipsometry,” PhD dissertation, Norwegian University of Science and Technology, (2009).

] of the system was performed to yield the exact values of the system PSG and PSA matrices over a broad wavelength range (λ = 400 - 800 nm), which also ensured high accuracy of Mueller matrix measurement. With some modifications, the system was successfully used to record fluorescence spectroscopic Mueller matrices in addition to elastic scattering spectral Mueller matrices. To the best of our knowledge, this is the first report on fluorescence spectroscopic Mueller matrix measurement from biological tissue. Initial exploration of such measurements yielded interesting differences in both the fluorescence and elastic scattering spectral diattenuation parameters (derived from respective Mueller matrices) between normal and precancerous tissue sections of human uterine cervix.

The paper is organized as follows. In Section 2, we provide specifics of the signal analysis strategy for construction of sample Mueller matrix. The Eigenvalue calibration method is briefly summarized in this section. A very brief account of the polar decomposition of Mueller matrix (which has been used to analyze the Mueller matrices) is also provided in this section. Section 3 describes the experimental Mueller matrix polarimetry systems. Both the elastic scattering and fluorescence spectroscopic Mueller matrix systems for either forward or backward scattering geometry are presented in this section. The results of the eigenvalue calibration of the spectral Mueller matrix system, and the results of initial exploration of fluorescence and elastic scattering spectral Mueller matrix measurements and analysis on human cervical tissue sections, are presented in Section 4. The paper concludes with a discussion on the salient advantages of the developed spectral Mueller matrix system, and on the potential of fluorescence spectroscopic Mueller matrix polarimetry for tissue characterization.

2. Theory

2.1. Strategy for constructing 4 × 4 spectral Mueller matrix

Our Mueller matrix measurement strategy is based on sixteen measurements performed by sequentially generating and analyzing four elliptical polarization states using a polarization state generator (PSG, comprising of a fixed linear polarizer with its axis oriented at horizontal position, followed by a rotatable quarter wave retarder) and a polarization state analyzer (PSA, similar arrangement of fixed linear polarizer with its axis oriented at vertical position and rotatable quarter wave retarder, but positioned in a reverse order) unit respectively. For the combination of a horizontal linear polarizer and a linear retarder (having retardance δand orientation angle of fast axis θiwith respect to the horizontal axis) in the PSG unit, the output Stokes vector can be written as [6

6. N. Ghosh and I. A. Vitkin, “Tissue polarimetry: concepts, challenges, applications, and outlook,” J. Biomed. Opt. 16(11), 110801 (2011). [CrossRef] [PubMed]

]
W(θi)=(10000Cθi2+Sθi2cosδSθiCθi(1cosδ)Sθisinδ0SθiCθi(1cosδ)Sθi2+Cθi2cosδCθisinδ0SθisinδCθisinδcosδ)×(1100110000000000)×(1000)=(1Cθi2+Sθi2cosδSθiCθi(1cosδ)Sθisinδ)
(1)
Here, Ci=cos2θi;Sθi=sin2θi;

The four required elliptical polarization states can thus be generated by sequentially changing the orientation angle of the retarder (θi1, θi2, θi3, θi4) and the corresponding PSG output can be represented by W, a 4 × 4 matrix whose column vectors are the four generated Stokes vectors W(θi) (of Eq. (1)) incident on the sample. Similarly, after sample interactions, the PSA results can also be described by a transformation matrix for the combination of a general linear retarder (retardanceδ, orientation angleθo) and a fixed linear polarizer (vertical) as
PSA(θo)=(1100110000000000)×(10000Cθo2+Sθo2cosδSθoCθo(1cosδ)Sθosinδ0SθoCθi(1cosδ)Sθo+Cθo2cosδCθosinδ0SθosinδCθosinδcosδ)=(1(Cθo2+Sθo2cosδ)SθoCθo(1cosδ)Sθosinδ1(Cθo2+Sθo2cosδ)SθoCθo(1cosδ)Sθosinδ00000000)
(2)
where once againCθo=cos2θo;Sθo=sin2θo;

Note that the PSA unit is followed by an intensity based detector, which records the total intensity falling on it (the first element of the Stokes vector coming out of the PSA system). Thus, only the first row of the matrix of Eq. (2), is required to represent the PSA unit basis states
A(θo)=[1,(Cθo2+Sθo2cosδ),SθoCθo(1cosδ),Sθosinδ]
(3)
Just like PSG unit generated four input elliptical polarization states, in the PSA unit also, one needs to analyze four output polarization states (by sequentially changing the orientation angle of the retarder - θo1, θo2, θo3, θo4). The corresponding 4 × 4 analysis matrix A can be formed by writing the four basis states (A(θo) of Eq. (3) for four different values of θo) as the row vectors [28

28. B. Laude-Boulesteix, A. De Martino, B. Drévillon, and L. Schwartz, “Mueller polarimetric imaging system with liquid crystals,” Appl. Opt. 43(14), 2824–2832 (2004). [CrossRef] [PubMed]

30

30. F. Stabo-Eeg, “Development of instrumentation for Mueller matrix ellipsometry,” PhD dissertation, Norwegian University of Science and Technology, (2009).

].

Note that the choice of the four sets of orientation angles of the retarders (θi and θo) in the PSG and the PSA units are important for recording stable sample Mueller matrix. Specifically, optimum selection of the basis states of W and A matrices are required to have minimal propagation of errors in the determined Mueller matrix. Theoretical optimization of θi and θo values was therefore performed. One basic criterion for choosing the polarization basis states, is to maximize the value of the determinant of the 16 × 16 matrix Q, since Q is required to be invertible [6

6. N. Ghosh and I. A. Vitkin, “Tissue polarimetry: concepts, challenges, applications, and outlook,” J. Biomed. Opt. 16(11), 110801 (2011). [CrossRef] [PubMed]

]. Note that in practice, the invertability of the matrix Q is not binary (i.e., either invertible or not), but rather presents a continuum in which some matrices are more invertible than others. Optimization of Q was therefore performed by maximizing its determinant for varying orientation angles of the quarter wave retarders (achromatic retarders with δ = π/2).Thus, optimized orientation angles for the two retarders were found to be 35°, 70°, 105° and 140°. For simplicity, the orientation angles of the retarders at the PSG and the PSA unit were kept the same (θi = θo) in the optimization process. A more general criterion for having minimal error propagation has previously been suggested to be based on optimization of the so-called ‘condition number’, defined as the ratio of the smallest to the largest singular values of the individual square matrices W and A [29

29. A. De Martino, E. Garcia-Caurel, B. Laude, and B. Drévillon, “General methods for optimized design and calibration of Mueller polarimeters,” Thin Solid Films 455−456, 112-119 (2004).

]. The above chosen angles were therefore verified further with this more rigorous approach based on singular value decomposition [29

29. A. De Martino, E. Garcia-Caurel, B. Laude, and B. Drévillon, “General methods for optimized design and calibration of Mueller polarimeters,” Thin Solid Films 455−456, 112-119 (2004).

]. Note that the actual forms of the W and A matrices of the experimental set-up might differ from the theoretical ones derived above. Specifically, they might also show some wavelength variations, even though the quarter wave retarders used in the experimental set-up are achromatic (over λ = 400 – 800 nm). The experimentally determined W and A matrices (determined via Eigenvalue calibration method, discussed subsequently) were thus tested to meet the optimization criteria discussed above.

2.2. Eigenvalue calibration method

Calibration of the spectral Mueller matrix polarimeter is an essential issue to ensure accuracy of acquiring sample Mueller matrices. This is particularly important for measurement over a broad wavelength range, since the actual polarization state generator (W) and the analyzer (A) matrices of the experimental system may differ from the theoretical ones, and additionally they might also exhibit wavelength response. Fortunately, there exists a method to address this issue, the so-called Eigenvalue calibration method (ECM), which can determine the exact nature of the system W and A matrices and their wavelength response [28

28. B. Laude-Boulesteix, A. De Martino, B. Drévillon, and L. Schwartz, “Mueller polarimetric imaging system with liquid crystals,” Appl. Opt. 43(14), 2824–2832 (2004). [CrossRef] [PubMed]

30

30. F. Stabo-Eeg, “Development of instrumentation for Mueller matrix ellipsometry,” PhD dissertation, Norwegian University of Science and Technology, (2009).

]. This involves measurements on a set of chosen reference or calibrating samples. The details of this method can be found elsewhere [28

28. B. Laude-Boulesteix, A. De Martino, B. Drévillon, and L. Schwartz, “Mueller polarimetric imaging system with liquid crystals,” Appl. Opt. 43(14), 2824–2832 (2004). [CrossRef] [PubMed]

, 30

30. F. Stabo-Eeg, “Development of instrumentation for Mueller matrix ellipsometry,” PhD dissertation, Norwegian University of Science and Technology, (2009).

]. Here, we provide an outline of the various steps of this method. Usually the calibration is done on samples having Mueller matrices (M) of the form of diattenuating retarder. Let us call these set of measurements as B. The ECM method requires another set of measurements without any sample (blank), denoted by Bo. The essential steps of ECM are as follows.

The above sets of measurements will yield
B=AMW,B0=AW
(7)
From Eq. (7), another two sets of matrices C and C/ are constructed such that one of them is independent of A and the other is independent of W
C=C01B=W1MW,C'=B01=AMA1
(8)
Clearly, the matrices C, C/ and M has the same eigenvalues. Thus the eigenvalues of the Mueller matrix M can be determined from the eigenvalues of either of the matrices C and C/. The actual Mueller matrix M of the reference sample can then be constructed using the determined four set of eigenvalues, through a series of algebraic manipulation, as detailed in references [28

28. B. Laude-Boulesteix, A. De Martino, B. Drévillon, and L. Schwartz, “Mueller polarimetric imaging system with liquid crystals,” Appl. Opt. 43(14), 2824–2832 (2004). [CrossRef] [PubMed]

, 30

30. F. Stabo-Eeg, “Development of instrumentation for Mueller matrix ellipsometry,” PhD dissertation, Norwegian University of Science and Technology, (2009).

].

A=B0W1
(10)

The ECM was used to determine the exact nature of the system W and A matrices and their wavelength response. This was performed using four sets of measurements for two different types of calibrating reference samples, Glen Thompson linear polarizer (as diattenuator, d = 1, over λ = 400 – 800 nm) and quarter waveplate (δ = π/2 at 632.8 nm, as retarder). Measurements from the linear polarizer were taken for two different orientations of polarization axis - 28°, 73°, and from the quarter waveplate for two different orientations of the fast axis - 23°, 68° respectively. These sets of orientation angles of the diattenuator and retarder have previously been shown to be optimum for eigenvalue calibration. Since, ECM provides the actual experimental W and A matrices, without any prior modeling of the PSG and PSA units, many effects which can modify W and A values (misalignment in orientation or configurations, positioning artifacts, beam divergence, non-ideal optical characteristics of the polarizing optics etc.), are automatically taken care off. Moreover, the ratio of the smallest to the largest eigenvalue (λminmax) of the 16 × 16 matrix K, is an indicator of the accuracy or sensitivity of the system, which was thus determined for our spectral Mueller matrix polarimeter. The performance of the system was further tested by quantitatively determining the medium polarization parameters, diattenuation d, linear retardance δ (and their wavelength response) of standard optical elements. The results of the eigenvalue calibration and the initial exploration of the spectral Mueller matrix polarimeter to record spectral Mueller matrices from biological tissues are presented subsequently, in Section 4.

2.3. Inverse analysis of Mueller matrix using polar decomposition

Polar decomposition of Mueller matrix, originally developed by Lu and Chipman [15

15. S. Y. Lu and R. A. Chipman, “Interpretation of Mueller matrices based on polar decomposition,” J. Opt. Soc. Am. A 13(5), 1106–1113 (1996). [CrossRef]

], has been widely explored for inverse analysis of Mueller matrices to extract the constituent polarization properties from any unknown system. This approach involves sequential factorization of a given Mueller matrix M into the product of three ‘basis’ matrices
MMΔMR.MD
(11)
Here, the matrix MD contains information on the effects of linear and circular diattenuation, MR accounts for linear and circular retardance (or optical rotation) effects, and MΔ includes the effect of any depolarization present in the medium [6

6. N. Ghosh and I. A. Vitkin, “Tissue polarimetry: concepts, challenges, applications, and outlook,” J. Biomed. Opt. 16(11), 110801 (2011). [CrossRef] [PubMed]

, 15

15. S. Y. Lu and R. A. Chipman, “Interpretation of Mueller matrices based on polar decomposition,” J. Opt. Soc. Am. A 13(5), 1106–1113 (1996). [CrossRef]

]. This product decomposition and its other variants have recently been validated in complex scattering media such as tissues, which exhibit simultaneously polarization and scattering effects [15

15. S. Y. Lu and R. A. Chipman, “Interpretation of Mueller matrices based on polar decomposition,” J. Opt. Soc. Am. A 13(5), 1106–1113 (1996). [CrossRef]

21

21. S. Kumar, H. Purwar, R. Ossikovski, I. A. Vitkin, and N. Ghosh, “Comparative study of differential matrix and extended polar decomposition formalisms for polarimetric characterization of complex tissue-like turbid media,” J. Biomed. Opt. 17(10), 105006 (2012). [CrossRef] [PubMed]

]. Once decomposed, the four individual polarization medium properties, namely, diattenuation (d), depolarization coefficients (Δ), linear retardance (δ), and circular retardance or optical rotation (ψ, circular retardance = 2 × optical rotation), can be determined from the corresponding basis matrices as [6

6. N. Ghosh and I. A. Vitkin, “Tissue polarimetry: concepts, challenges, applications, and outlook,” J. Biomed. Opt. 16(11), 110801 (2011). [CrossRef] [PubMed]

, 15

15. S. Y. Lu and R. A. Chipman, “Interpretation of Mueller matrices based on polar decomposition,” J. Opt. Soc. Am. A 13(5), 1106–1113 (1996). [CrossRef]

]
d=1MD(1,1)MD(1,2)2+MD(1,3)2+MD(1,4)2
(12a)
Δ=1|tr(MΔ1)|3,0Δ1
(12b)
δ=cos1(MR(2,2)+MR(3,3))2+(MR(3,2)MR(2,3))21
(12c)
Ψ=tan1(MR(3,2)MR(2,3)MR(2,2)+MR(3,3))
(12d)
The above analysis was applied to the spectral Mueller matrices acquired using our polarimetry system. Specifically, the wavelength variation of the polarization parameters for the calibrating reference samples, fluorescence and elastic scattering polarimetry parameters (and their wavelength variation) of normal and precancerous human cervical tissue sections, were derived using Eqs. (12a)-(12d).

3. Experimental methods

We have developed two separate experimental systems based on the measurement scheme described in Section 2.1. A schematic of the first one, for angle resolved elastic scattering spectral Mueller matrices, is shown in Fig. 1(a)
Fig. 1 Schematic of the experimental systems (a) for angle resolved elastic scattering spectral Mueller matrix measurement (set-up 1) and (b) for elastic scattering and fluorescence spectral Mueller matrix measurement in the exact backscattering configuration (set-up 2). P1 and P2 are linear polarizers; QWP1 and QWP2 are rotatable achromatic quarter wavw retarders; L1, L2, L3, L4, L5 are lenses, A1, A2, A3 are variable apertures; BS1, BS2 are beam splitters; MO is microscope objective (in set-up2). In both the set-ups, P1, QWP1 form the PSG unit and P2, QWP2 form the PSA unit. The Xe-lamp is used as excitation source for recording elastic scattering spectral Mueller matrices (in both the set-ups), whereas the 405 nm line of a diode laser is used as excitation source for recording fluorescence spectral Mueller matrices (in set-up 2).
. The second one is capable of Mueller matrix measurement in the exact backscattering configuration (Fig. 1(b)). In addition to recording elastic scattering spectral Mueller matrices, this set-up was also used to record fluorescence spectroscopic Mueller matrices in the exact backscattering configuration (results are presented subsequently in Section 4). Either of the systems comprise of a Xe lamp (USB4000, Ocean Optics, USA) as excitation source (for elastic scattering spectral measurements), a polarization state generator (PSG) and a polarization state analyzer (PSA) unit to generate and analyze the required polarization states, coupled to a spectrometer for spectrally resolved (wavelength λ = 400 - 800 nm) signal detection. The PSG unit comprises of a fixed linear polarizer (P1, LPVIS100, Thorlabs, USA) with its axis oriented at horizontal position, followed by a rotatable achromatic quarter wave retarder (Q1, AQWP05M-600, Thorlabs, USA) mounted on a computer controlled rotational mount (PRM1/M-27E, Thorlabs, USA). The retarders used in the system are achromatic quarter waveplates over λ = 400 – 800 nm, has a retardance accuracy of ~λ/60. Note that these sets of achromatic retarders used in the PSG and the PSA units, are different from the other type of quarter wave retarder (WPQ10M-633, δ = π/2, at λ = 632.8 nm, retardance accuracy of ~λ/300) used in the eigenvalue calibration as a reference sample (discussed in the section 2.2).

The sample-scattered light collected and collimated using an assembly of lenses, then passes through the PSA unit, and is finally recorded using a spectrometer. The PSA unit essentially consists of a similar arrangement of fixed linear polarizer (P2, oriented at vertical position) and a rotatable achromatic quarter wave retarder (Q2), but positioned in a reverse order. A series of sixteen measurements are performed by sequentially changing the orientation of the fast axis of the quarter wave retarders of the PSG unit and that of the PSA unit, to the four optimized angles 35°, 70°, 105° and 140° (as discussed in Section 2.1). The spectra corresponding to the sixteen combinations of the PSG and PSA are recorded using a fiber optic spectrometer (HR2000, Ocean Optics, USA) in set-up - 1 and using a CCD spectrometer (Shamrock imaging spectrograph, SR-303i-A, ANDOR technology, USA) in set-up-2.

In order to record fluorescence spectroscopic Mueller matrices, the 405 nm line of a diode laser (PE.BDL.405.50, Pegasus, Shanghai, China) is used as excitation source in set-up 2 (exact backscattering configuration). The fluorescence spectra (λem = 450 – 800 nm) are recorded for sixteen combinations of the PSG and PSA. The set-up was completely automated using Labview so that sixteen spectrally resolved measurements can be performed with relative ease.

Note that an important advantage of constructing Mueller matrix according to this scheme is that, this is independent of the source and detector polarization responses since the two linear polarizers P1 and P2 are always fixed at horizontal and vertical polarization states respectively. This is important particularly, for spectrally resolved (employing a spectrometer) Mueller matrix measurements, since usually spectrometers are associated with complex polarization response (owing to the presence of grating). Never-the-less, eigenvalue calibration was performed following the process discussed in Section 2.2, to yield the exact nature of the system polarization state generator W(λ) and polarization state analyzer A(λ) matrices. These along with the sixteen spectrally resolved measurements (Mi(λ)) were used to construct the spectral Mueller matrices M(λ) (Section 2.1 Eqs. (4)-(6)). For constructing the elastic scattering spectral Mueller matrices, the W(λ)andA(λ)matrices for varying excitation and emission wavelengths are used (λex, λem = 400 - 800 nm). For constructing fluorescence spectroscopic Mueller matrices (in set-up 2), generator matrix W(λ) for a fixed excitation wavelength (λex = 405 nm) and analyzer matrix A(λ)for varying emission wavelengths (λem = 450 – 800 nm) are used.

The tissues were histopathologically characterized (CIN or dysplasia Grade I, II and III) biopsy samples of human cervical tissues, obtained from G.S.V.M. Medical College and Hospital, Kanpur, India. The unstained tissue sections (thickness ~5μm, lateral dimension ~4 mm × 6 mm) were prepared on glass slides. The standard method employed was tissue dehydration, embedding in wax, vertical sectioning (containing both epithelial and connective tissue regions) under a rotary microtome and subsequent de-waxing. The normal counterparts were obtained from the adjacent normal area of the resected tissues.

4. Results and discussion

4.1. Results of eigenvalue calibration

For eigenvalue calibration, we used separately a linear polarizer (for two different orientations of polarization axis - 28°, 73°) as diattenuator calibration sample and a quarter waveplate (at 632.8 nm, for two different orientations of the fast axis - 23°, 68°) as the retarder calibrating samples. The derived system polarization state generator W(λ)and polarization state analyzerA(λ) matrices and their wavelength variation are shown in Fig. 2(a)
Fig. 2 The Eigenvalue calibration-derived wavelength variation of the individual elements of the (a) polarization state generator W(λ) matrices and (b) polarization state analyzer A(λ) matrices (for set-up 2, operating in exact backscattering configuration). The results are shown here for the wavelength range λ = 475–800 nm.
and 2(b) respectively.

In order to evaluate the overall system performance of the spectral Mueller matrix set-up, the Mueller matrices corresponding to the series of calibrating samples and those without any sample (blank) were determined using the W(λ)andA(λ)matrices. A useful way to test the accuracy of the system is to look at the expected null elements (which are expected to vanish) of the recorded Mueller matrix of any reference sample [28

28. B. Laude-Boulesteix, A. De Martino, B. Drévillon, and L. Schwartz, “Mueller polarimetric imaging system with liquid crystals,” Appl. Opt. 43(14), 2824–2832 (2004). [CrossRef] [PubMed]

, 29

29. A. De Martino, E. Garcia-Caurel, B. Laude, and B. Drévillon, “General methods for optimized design and calibration of Mueller polarimeters,” Thin Solid Films 455−456, 112-119 (2004).

]. The off-diagonal elements of Mueller matrix for blank (no sample) and the expected null elements of the calibrating diattenuator (polarizer) and retarder (quarter waveplate) were indeed found to be quite small (~0.01).Typical illustrative example of such measurement is shown in Fig. 3(a)
Fig. 3 Wavelength variations of (a) expected null elements (M12, M13 and M14 elements) of the Mueller matrix for a calibrating quarter wave retarder sample (fast axis oriented at 23°). The inset shows the element M44, which ideally should vanish at λ = 632.8 nm (M44~0.01, noted inside the figure). The results are shown here for λ = 475 – 800 nm. (b) Diattenuation d (λ) (left axis, dotted line) and linear retardanceδ (λ) (right axis, solid line) for the calibrating linear polarizer (orientation of polarization axis - 28°) and the quarter wave retarder (δ = π/2 at 632.8 nm, orientation of fast axis - 23°) samples respectively (shown for λ = 500 – 800 nm). The value of d for the linear polarizer is close to unity over the entire wavelength range (mean value ~0.984). The determined value for δ of the quarter wave retarder is 1.57 radian at 632.8 nm. (c) The ratio of the smallest to the largest eigenvalue (λminmax) of the 16 × 16 matrix K (shown for λ = 475 – 800 nm).
, where the wavelength variations of the expected null elements (M12, M13 and M14 elements) of the Mueller matrix for a quarter wave retarder (orientation angle of fast axis from the horizontal −23°) are shown. The inset shows the element M44, which ideally should vanish at λ = 632.8 nm (M44 = cos δ, δ = π/2 at 632.8 nm). The small value of the M44 (~0.01) element at 632.8 nm, and the other elements over the entire spectral range (~0.01), ensured reasonably high accuracy of the spectral Mueller matrix system.

In order to further illustrate the efficacy of the Mueller matrix system for quantitative characterization of the spectral polarimetric properties, the wavelength variation of the polarization parameters, diattenuation d (λ), linear retardance δ (λ), optical rotation ψ (λ) and depolarization coefficients Δ (λ), were determined from polar decomposition (following Section 2.3, Eqs. (11) and (12)) of Mueller matrices recorded from the calibrating diattenuator and the retarder samples. Typical wavelength variation of the extracted d (λ) and δ (λ) for the calibrating linear polarizer (orientation of polarization axis - 28°) and the quarter wave retarder (orientation of fast axis - 23°) sample are shown in Fig. 3(b). As can be seen from the figure, the derived values are in agreement with that expected for an ideal polarizer (d ~1) over the spectral range 500 nm – 800 nm. The values for the other derived polarization parameters (δ (λ),ψ (λ) and Δ (λ)), which are expected to be zero for an ideal polarizer, were also quite negligible (≤ 0.01). Similarly, the determined value for linear retardance δ = 1.57 radian at 632.8 nm reasonably agrees with the expected value (δ = π/2), and the corresponding wavelength variation also exhibits the expected 1/λ behavior. Finally, as discussed in Section 2.2, the ratio of the smallest to the largest eigenvalue (λminmax) of the 16 × 16 matrix K, is an indicator of the accuracy or sensitivity of the system. The wavelength variation of λminmax is shown in Fig. 3(c). The considerably small magnitudes of the parameter λminmax(~10−3) confirm high accuracy of the system in acquiring Mueller matrices over a broad wavelength range.

The results of Eigenvalue calibration presented above are for the Mueller matrix system operating in the exact backscattering configuration (set-up 2, Fig. 1(b)). Similar calibration studies were also conducted on the system operating in the forward detection geometry (set-up 1, Fig. 1(a), results not shown here), which also yielded similar accuracy of spectral Mueller matrix measurements. Following successful calibration (results presented above and other results on a series of calibrating samples), the developed set-ups were initially explored for biomedical tissue polarimetry - for quantitative fluorescence and elastic scattering spectral Mueller matrix polarimetry on normal and precancerous human cervical tissues, as illustrated below.

4.2. Results of fluorescence and elastic scattering spectroscopic Mueller matrices from tissue sections

Polarization properties of auto-fluorescence (fluorescence from endogenous tissue fluorophores) have previously been explored for discriminating between normal and cancerous tissues [4

4. V. V. Tuchin, L. Wang, and D. À. Zimnyakov, Optical Polarization in Biomedical Applications, (Springer-Verlag, 2006).

]. These studies have shown that measurement of depolarization of auto fluorescence from tissue can provide additional diagnostic information [31

31. S. K. Mohanty, N. Ghosh, S. K. Majumder, and P. K. Gupta, “Depolarization of autofluorescence from malignant and normal human breast tissues,” Appl. Opt. 40(7), 1147–1154 (2001). [CrossRef] [PubMed]

]. These studies were based on conventional measurement of fluorescence polarization anisotropy (ratio of the polarized fluorescence component to the total intensity, a measure of depolarization) with linear polarization excitation [32

32. J. Lackowicz, “Principles of Fluorescence Spectroscopy,” New York, (Plenum Press, 1983).

]. The fluorescence polarization anisotropy is known to arise due to several causes like, the random orientation of the fluorophore molecules, rotational diffusion of fluorophores, the radiation less energy transfer among fluorophores etc., and accordingly this carries useful information on the fluorophore and its local environment [32

32. J. Lackowicz, “Principles of Fluorescence Spectroscopy,” New York, (Plenum Press, 1983).

]. In tissue, depolarization of fluorescence is additionally influenced by multiple scattering effects, encoding the signature of the medium scattering properties in the measured fluorescence polarization anisotropy [4

4. V. V. Tuchin, L. Wang, and D. À. Zimnyakov, Optical Polarization in Biomedical Applications, (Springer-Verlag, 2006).

, 31

31. S. K. Mohanty, N. Ghosh, S. K. Majumder, and P. K. Gupta, “Depolarization of autofluorescence from malignant and normal human breast tissues,” Appl. Opt. 40(7), 1147–1154 (2001). [CrossRef] [PubMed]

]. Note that when the emitting fluorophores exhibit all the three basic polarimetry effects, namely, diattenuation, depolarization and retardance, the resulting fluorescence polarization anisotropy determined using the conventional two orthogonal linear polarization state measurements, should reflect ‘lumped’ polarization effects. The analysis / interpretation get considerably simplified in case of isotropic fluorescence emission from randomly oriented fluorophores, where the fluorescence polarization anisotropy can be interpreted as mainly due to the depolarization effects (arising due to randomization of the excitation and the emission dipoles) [32

32. J. Lackowicz, “Principles of Fluorescence Spectroscopy,” New York, (Plenum Press, 1983).

, 33

33. O. Arteaga, S. Nichols, and B. Kahr, “Mueller matrices in fluorescence scattering,” Opt. Lett. 37(14), 2835–2837 (2012). [CrossRef] [PubMed]

]. However, when the fluorescence emission takes place from fluorophores having anisotropically oriented / organized molecular structures, the other polarimetry characteristics may also have significant contributions [33

33. O. Arteaga, S. Nichols, and B. Kahr, “Mueller matrices in fluorescence scattering,” Opt. Lett. 37(14), 2835–2837 (2012). [CrossRef] [PubMed]

]. In such situation, measurement and analysis of the complete 4 × 4 fluorescence spectral Mueller matrix may yield additional structural information on the organization, orientation of the fluorophore molecules. Note that collagen is known to be the major fluorophore in connective tissue (stroma), which possess such anisotropically oriented / organized molecular structures [34

34. N. Ramanujam, “Fluorescence spectroscopy of neoplastic and non-neoplastic tissues,” Neoplasia 2(1-2), 89–117 (2000). [CrossRef] [PubMed]

]. The anisotropic structure primarily arises due to the cross-linking between individual collagen molecules, which results in formation of micro-fibrils and fibers. It is well recognized that progression of epithelial precancers and cancers involves defective interactions between epithelial cells and the underlying stroma, and alterations in stromal biology may precede and stimulate neoplastic progression in pre-invasive disease [35

35. D. Arifler, I. Pavlova, A. Gillenwater, and R. Richards-Kortum, “Light scattering from collagen fiber networks: micro-optical properties of normal and neoplastic stroma,” Biophys. J. 92(9), 3260–3274 (2007). [CrossRef] [PubMed]

]. Quantification of intrinsic fluorescence polarimetry characteristics (from fluorescence Mueller matrix) of collagen present in stroma may thus prove to be useful for probing defective epithelial-stromal interactions, as signature of precancer.

In order to explore this possibility, fluorescence spectral Mueller matrices (with 405 nm excitation) were recorded from human cervical tissue sections (vertically sectioned tissue slices, thickness ~5 μm) having different grades of precancers. The spectral Mueller matrices were recorded from the tissue sections in the exact backscattering configuration using set-up 2. Figure 4
Fig. 4 The 4 × 4 fluorescence spectroscopic (λem = 475 – 800 nm) Mueller matrix recorded (in the exact backscattering configuration using set-up 2) with 405 nm excitation, from the connective tissue region (stroma) of a typical Grade I precancerous tissue section.
shows typical fluorescence spectral Mueller matrix recorded from the connective tissue region (stroma) of a Grade I precancerous tissue section. Several observations are at place. The M11(λ) element of the Mueller matrix represent the unpolarized fluorescence spectra. As noted, this is characterized by broad spectral feature with a peak around 500 nm, which is characteristic of collagen emission with 405 nm excitation [34

34. N. Ramanujam, “Fluorescence spectroscopy of neoplastic and non-neoplastic tissues,” Neoplasia 2(1-2), 89–117 (2000). [CrossRef] [PubMed]

, 36

36. Y. Wu and J. Y. Qu, “Autofluorescence spectroscopy of epithelial tissues,” J. Biomed. Opt. 11(5), 054023 (2006). [CrossRef] [PubMed]

]. The spectral intensities of the diagonal elements, M22(λ),M33(λ) and M44(λ) are considerably weaker as compared to M11(λ). Among these, the M44(λ) element appears to be the weakest. The considerably weak magnitude of M44(λ) is characteristics of fluorescence emission from molecules exhibiting very weak optical activity (circular anisotropy) [33

33. O. Arteaga, S. Nichols, and B. Kahr, “Mueller matrices in fluorescence scattering,” Opt. Lett. 37(14), 2835–2837 (2012). [CrossRef] [PubMed]

]. In general, the diagonal elements of a pure depolarization Mueller matrix represent the polarization preserving components for horizontal/ vertical, + 45° / −45°linear polarization and circular polarizations (left and right) respectively [6

6. N. Ghosh and I. A. Vitkin, “Tissue polarimetry: concepts, challenges, applications, and outlook,” J. Biomed. Opt. 16(11), 110801 (2011). [CrossRef] [PubMed]

]. Low magnitudes of all these elements thus underscore the overall strong depolarizing nature of fluorescence emission from tissue. Indeed, the values of depolarization coefficient Δ(λ) (derived using Eq. (12b) of Section 2.3) were found to be quite high (Δ ≥ 0.9) over the entire spectral range (not shown here). Such high values of Δ are primarily due to the intrinsic causes of depolarization of fluorescence (random orientation, rotational diffusion and radiation-less energy transfers among fluorophores etc.) [32

32. J. Lackowicz, “Principles of Fluorescence Spectroscopy,” New York, (Plenum Press, 1983).

]. Even though, multiple scattering is not expected to be a significant issue for thin tissue sections (thickness ~5 μm), some additional contribution of depolarization of fluorescence due to scattering at the excitation and the emission wavelengths, is unavoidable [4

4. V. V. Tuchin, L. Wang, and D. À. Zimnyakov, Optical Polarization in Biomedical Applications, (Springer-Verlag, 2006).

, 31

31. S. K. Mohanty, N. Ghosh, S. K. Majumder, and P. K. Gupta, “Depolarization of autofluorescence from malignant and normal human breast tissues,” Appl. Opt. 40(7), 1147–1154 (2001). [CrossRef] [PubMed]

]. Indeed, the corresponding elastic scattering spectroscopic Mueller matrices recorded from the same tissue sections showed non-zero value of Δ (not shown here), indicating non-negligible contribution of scattering-induced depolarization effect for the backscattering collection geometry. Quantitative description of scattering-induced depolarization in tissue would necessitate use of vector radiative transfer theory or its other variants [4

4. V. V. Tuchin, L. Wang, and D. À. Zimnyakov, Optical Polarization in Biomedical Applications, (Springer-Verlag, 2006).

6

6. N. Ghosh and I. A. Vitkin, “Tissue polarimetry: concepts, challenges, applications, and outlook,” J. Biomed. Opt. 16(11), 110801 (2011). [CrossRef] [PubMed]

, 10

10. N. Ghosh, A. Banerjee, and J. Soni, “Turbid medium polarimetry in biomedical imaging and diagnosis,” Eur. Phys. J. Appl. Phys. 54(3), 30001 (2011). [CrossRef]

, 37

37. D. Bicout, C. Brosseau, A. S. Martinez, and J. M. Schmitt, “Depolarization of Multiply Scattered Waves by Spherical Diffusers: Influence of the Size Parameter,” Phys. Rev. E Stat. Phys. Plasmas Fluids Relat. Interdiscip. Topics 49(2), 1767–1770 (1994). [CrossRef] [PubMed]

]. However, since the main finding and emphasis of the present study is the observation of an interesting fluorescence spectral diattenuation effect (as illustrated in greater details below), such quantitative modeling of scattering-induced depolarization has not been attempted here.

Next, the off-diagonal elements, M23(λ) / M32(λ), M24(λ) / M42(λ),and M34(λ) / M43(λ) also show low intensities. While the elements M34(M43) and M24(M42) represent linear retardance-δ effects for horizontal/ vertical and + 45° /-45° linear polarizations respectively,

Since, the fluorescence process is associated with strong de-phasing (randomization of phase), the resulting emission does not exhibit any appreciable phase retardation effects [33

33. O. Arteaga, S. Nichols, and B. Kahr, “Mueller matrices in fluorescence scattering,” Opt. Lett. 37(14), 2835–2837 (2012). [CrossRef] [PubMed]

], and accordingly, the derived values of δ (λ) and ψ(λ) (Eq. (12c) and (12d)) were also found to be quite negligible (δ,ψ ≤ 0.01 radian, within the experimental errors of the system). Interestingly, the elements M12(λ) / M21(λ),and M13(λ) / M31(λ) show appreciable intensity values and they also preserve the spectral characteristics of the fluorescence emission. The magnitudes of the remaining M41(λ) / M41(λ) elements, on the other hand, are relatively weaker. Analogous to the retardation effects, while the elements M12(M21) and M13 (M31) represent linear diattenuation effect for horizontal/ vertical and + 45° /-45° linear polarizations respectively, the elements M41(M41) represent circular diattenuation (between left and right circular polarization states). Accordingly, the values for the diattenuation parameter d(λ) (derived using Eq. (12a) of Section 2.3) were found to be appreciable (subsequent results shown in Fig. 5
Fig. 5 The Mueller matrix-derived (a) fluorescence diattenuation parameter dFL(λ) and (b) elastic scattering diattenuation parameter dES(λ) as a function of wavelength (λ = 475 – 800 nm) for normal (open circle) and precancerous (open square) tissue sections. Results are shown for the connective tissue region (stroma) of the tissue sections. Mean values of three normal tissue samples and five precancerous tissue samples respectively, are shown here.
), with major contribution arising from linear diattenuation (negligible circular diattenuation contribution).

Some qualitative understanding on the origin of such interesting fluorescence spectral diattenuation effects can be gained from a phenomenological approach of modeling fluorescence Mueller matrix as a sequential product of Mueller matrices for each of the contributing processes in the resulting emission as [33

33. O. Arteaga, S. Nichols, and B. Kahr, “Mueller matrices in fluorescence scattering,” Opt. Lett. 37(14), 2835–2837 (2012). [CrossRef] [PubMed]

]

MF=MEMMGEMA
(13)

Here, the matrix MA includes the polarizing transfer function of the absorption process at the excitation wavelength (due to the ground molecular state), the matrix MG→E accounts for the effect of ground state to the excited state transformation and the remaining matrix MEM includes the effects of the excited state on the resulting fluorescence emission [33

33. O. Arteaga, S. Nichols, and B. Kahr, “Mueller matrices in fluorescence scattering,” Opt. Lett. 37(14), 2835–2837 (2012). [CrossRef] [PubMed]

]. In case of isotropic molecules, where the dipolar approximation is valid, MG→E of Eq. (13) can be assumed to be a diattenuating depolarizer matrix [32

32. J. Lackowicz, “Principles of Fluorescence Spectroscopy,” New York, (Plenum Press, 1983).

, 33

33. O. Arteaga, S. Nichols, and B. Kahr, “Mueller matrices in fluorescence scattering,” Opt. Lett. 37(14), 2835–2837 (2012). [CrossRef] [PubMed]

]. Whereas, the depolarization effects included in MG→E arise from various processes such as the random orientation of the fluorophore molecules, rotational diffusion of fluorophore, radiation less energy transfer among fluorophores etc., the diattenuation effect is characteristic of dipolar absorption / emission and has geometric origin [32

32. J. Lackowicz, “Principles of Fluorescence Spectroscopy,” New York, (Plenum Press, 1983).

, 33

33. O. Arteaga, S. Nichols, and B. Kahr, “Mueller matrices in fluorescence scattering,” Opt. Lett. 37(14), 2835–2837 (2012). [CrossRef] [PubMed]

]. Note that this diattenuation effect encoded in the M12 / M21elements of the matrix MG→E is maximum at a scattering angle of θ = 90° and vanishes at exact forward or backward scattering angles (θ = 0° / 180°, as should be the case for our experimental geometry of backscattering Mueller matrix measurement - θ = 180°) [33

33. O. Arteaga, S. Nichols, and B. Kahr, “Mueller matrices in fluorescence scattering,” Opt. Lett. 37(14), 2835–2837 (2012). [CrossRef] [PubMed]

]. On the other hand, the matrices MA and MEM may possess intrinsic diattenuation and retardance properties of the ground molecular state and the excited state respectively [33

33. O. Arteaga, S. Nichols, and B. Kahr, “Mueller matrices in fluorescence scattering,” Opt. Lett. 37(14), 2835–2837 (2012). [CrossRef] [PubMed]

].These matrices can be simplified further to represent as pure diattenuator matrices, if one assumes that fluorescence process is associated with strong randomization of the phase information. In such situation, these matrices would carry information on the anisotropic (linear and circular) absorption and emission properties of fluorophores arising from their anisotropically oriented / organized molecular structures [33

33. O. Arteaga, S. Nichols, and B. Kahr, “Mueller matrices in fluorescence scattering,” Opt. Lett. 37(14), 2835–2837 (2012). [CrossRef] [PubMed]

]. Note that in addition to these, for fluorophores embedded in a scattering medium (like tissue), there would be scattering-induced depolarization effects also, which could be incorporated by an additional depolarization matrix. Never-the-less, the resulting fluorescence Mueller matrix MF will possess diattenuation property in addition to depolarization. The observed appreciable intensities of M12(λ) / M21(λ),and M13(λ) / M31(λ) elements of the fluorescence Mueller matrices (and accordingly significant value of linear diattenuation) from tissue are thus manifestation of the intrinsic linear anisotropy (linear dichroism) of the emitting fluorophores (collagen). The weak magnitudes of M41(λ) / M41(λ), on the other hand, indicate that the emitting molecules exhibit very little / or no circular anisotropy (circular dichroism).

5. Conclusions

A novel spectral Mueller matrix system has been developed, which is capable of acquiring both fluorescence and elastic scattering Mueller matrices over a broad wavelength range. The performance of the system has been calibrated using Eigenvalue calibration method that also yielded the exact values of the system polarization state generator and polarization state analyzer matrices over the entire wavelength range. The accuracy of the system for measuring sample Mueller matrices were tested on various calibrating reference samples. Following successful evaluation, the system was used to acquire fluorescence and elastic scattering spectroscopic Mueller matrices from biological tissue sections. Initial exploration of the system for quantitative fluorescence spectral polarimetric studies on normal and precancerous human cervical tissue sections has yielded promising results. Specifically, the polar decomposition analysis on fluorescence Mueller matrix from connective tissue region yielded an interesting spectral diattenuation effect, which was attributed to the anisotropic molecular structure of collagen. Note that the results presented here are on thin tissue sections. The potential of this fluorescence spectral diattenuation parameter for probing precancerous alterations in connective tissue however, needs to be rigorously investigated from intact tissues. Our current studies are thus directed towards – (i) development of a more encompassing approach for the analysis / interpretation of fluorescence Mueller matrix and (ii) comprehensive evaluation and exploration of the quantitative fluorescence Mueller matrix polarimetry (and the derived intrinsic fluorescence polarimetry characteristics) for the detection of precancerous changes from intact tissues. The novel spectral Mueller matrix polarimetry system is also being explored for other interdisciplinary applications [38

38. N. Patil, J. Soni, N. Ghosh, and P. De, “Swelling-induced optical anisotropy of thermoresponsive hydrogels based on poly(2-(2-methoxyethoxy)ethyl methacrylate): Deswelling kinetics probed by quantitative Mueller matrix polarimetry,” J. Phys. Chem. B 116(47), 13913–13921 (2012). [CrossRef] [PubMed]

].

Acknowledgments

This work was supported by Indian Institute of Science Education and Research (IISER) - Kolkata, an autonomous teaching and research institute funded by the Ministry of Human Resource Development (MHRD), Govt. of India. The authors thank Prof. Asima Pradhan, Department of Physics, IIT Kanpur, India for fruitful discussions and Dr. Asha Agarwal, G.S.V.M. Medical College and Hospital, Kanpur, India for providing the histopathologically characterized tissue samples.

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A. De Martino, E. Garcia-Caurel, B. Laude, and B. Drévillon, “General methods for optimized design and calibration of Mueller polarimeters,” Thin Solid Films 455−456, 112-119 (2004).

30.

F. Stabo-Eeg, “Development of instrumentation for Mueller matrix ellipsometry,” PhD dissertation, Norwegian University of Science and Technology, (2009).

31.

S. K. Mohanty, N. Ghosh, S. K. Majumder, and P. K. Gupta, “Depolarization of autofluorescence from malignant and normal human breast tissues,” Appl. Opt. 40(7), 1147–1154 (2001). [CrossRef] [PubMed]

32.

J. Lackowicz, “Principles of Fluorescence Spectroscopy,” New York, (Plenum Press, 1983).

33.

O. Arteaga, S. Nichols, and B. Kahr, “Mueller matrices in fluorescence scattering,” Opt. Lett. 37(14), 2835–2837 (2012). [CrossRef] [PubMed]

34.

N. Ramanujam, “Fluorescence spectroscopy of neoplastic and non-neoplastic tissues,” Neoplasia 2(1-2), 89–117 (2000). [CrossRef] [PubMed]

35.

D. Arifler, I. Pavlova, A. Gillenwater, and R. Richards-Kortum, “Light scattering from collagen fiber networks: micro-optical properties of normal and neoplastic stroma,” Biophys. J. 92(9), 3260–3274 (2007). [CrossRef] [PubMed]

36.

Y. Wu and J. Y. Qu, “Autofluorescence spectroscopy of epithelial tissues,” J. Biomed. Opt. 11(5), 054023 (2006). [CrossRef] [PubMed]

37.

D. Bicout, C. Brosseau, A. S. Martinez, and J. M. Schmitt, “Depolarization of Multiply Scattered Waves by Spherical Diffusers: Influence of the Size Parameter,” Phys. Rev. E Stat. Phys. Plasmas Fluids Relat. Interdiscip. Topics 49(2), 1767–1770 (1994). [CrossRef] [PubMed]

38.

N. Patil, J. Soni, N. Ghosh, and P. De, “Swelling-induced optical anisotropy of thermoresponsive hydrogels based on poly(2-(2-methoxyethoxy)ethyl methacrylate): Deswelling kinetics probed by quantitative Mueller matrix polarimetry,” J. Phys. Chem. B 116(47), 13913–13921 (2012). [CrossRef] [PubMed]

OCIS Codes
(120.5410) Instrumentation, measurement, and metrology : Polarimetry
(170.3660) Medical optics and biotechnology : Light propagation in tissues
(170.4580) Medical optics and biotechnology : Optical diagnostics for medicine
(170.6510) Medical optics and biotechnology : Spectroscopy, tissue diagnostics
(170.7050) Medical optics and biotechnology : Turbid media
(260.5430) Physical optics : Polarization
(300.6280) Spectroscopy : Spectroscopy, fluorescence and luminescence
(170.6935) Medical optics and biotechnology : Tissue characterization
(240.2130) Optics at surfaces : Ellipsometry and polarimetry

ToC Category:
Medical Optics and Biotechnology

History
Original Manuscript: February 25, 2013
Revised Manuscript: May 13, 2013
Manuscript Accepted: May 13, 2013
Published: June 21, 2013

Virtual Issues
Vol. 8, Iss. 8 Virtual Journal for Biomedical Optics

Citation
Jalpa Soni, Harsh Purwar, Harshit Lakhotia, Shubham Chandel, Chitram Banerjee, Uday Kumar, and Nirmalya Ghosh, "Quantitative fluorescence and elastic scattering tissue polarimetry using an Eigenvalue calibrated spectroscopic Mueller matrix system," Opt. Express 21, 15475-15489 (2013)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-21-13-15475


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References

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  29. A. De Martino, E. Garcia-Caurel, B. Laude, and B. Drévillon, “General methods for optimized design and calibration of Mueller polarimeters,” Thin Solid Films455−456, 112-119 (2004).
  30. F. Stabo-Eeg, “Development of instrumentation for Mueller matrix ellipsometry,” PhD dissertation, Norwegian University of Science and Technology, (2009).
  31. S. K. Mohanty, N. Ghosh, S. K. Majumder, and P. K. Gupta, “Depolarization of autofluorescence from malignant and normal human breast tissues,” Appl. Opt.40(7), 1147–1154 (2001). [CrossRef] [PubMed]
  32. J. Lackowicz, “Principles of Fluorescence Spectroscopy,” New York, (Plenum Press, 1983).
  33. O. Arteaga, S. Nichols, and B. Kahr, “Mueller matrices in fluorescence scattering,” Opt. Lett.37(14), 2835–2837 (2012). [CrossRef] [PubMed]
  34. N. Ramanujam, “Fluorescence spectroscopy of neoplastic and non-neoplastic tissues,” Neoplasia2(1-2), 89–117 (2000). [CrossRef] [PubMed]
  35. D. Arifler, I. Pavlova, A. Gillenwater, and R. Richards-Kortum, “Light scattering from collagen fiber networks: micro-optical properties of normal and neoplastic stroma,” Biophys. J.92(9), 3260–3274 (2007). [CrossRef] [PubMed]
  36. Y. Wu and J. Y. Qu, “Autofluorescence spectroscopy of epithelial tissues,” J. Biomed. Opt.11(5), 054023 (2006). [CrossRef] [PubMed]
  37. D. Bicout, C. Brosseau, A. S. Martinez, and J. M. Schmitt, “Depolarization of Multiply Scattered Waves by Spherical Diffusers: Influence of the Size Parameter,” Phys. Rev. E Stat. Phys. Plasmas Fluids Relat. Interdiscip. Topics49(2), 1767–1770 (1994). [CrossRef] [PubMed]
  38. N. Patil, J. Soni, N. Ghosh, and P. De, “Swelling-induced optical anisotropy of thermoresponsive hydrogels based on poly(2-(2-methoxyethoxy)ethyl methacrylate): Deswelling kinetics probed by quantitative Mueller matrix polarimetry,” J. Phys. Chem. B116(47), 13913–13921 (2012). [CrossRef] [PubMed]

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