A technique for correction of attenuations in synchronous fluorescence spectroscopy

Journal of Photochemistry and Photobiology B: Biology 151 (2015) 1–9

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Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol

A technique for correction of attenuations in synchronous fluorescence spectroscopy Seema Devi a, Nirmalya Ghosh b, Asima Pradhan a,c,⇑ a

Department of Physics, Indian Institute of Technology, Kanpur 208016, India Indian Institute of Science Education and Research Kolkata, Mohanpur Campus, 741252, India c Center for Lasers and Photonics, Indian Institute of Technology, Kanpur 208016, India b

a r t i c l e

i n f o

Article history: Received 21 November 2014 Received in revised form 7 June 2015 Accepted 24 June 2015 Available online 25 June 2015 Keywords: Synchronous fluorescence Fluorophore Absorber Scatterer Offsets Tissue Extracted synchronous fluorescence Turbid phantoms

a b s t r a c t Synchronous fluorescence spectroscopy is an efficient technique for decoupling fluorophores which are masked in fluorescence spectroscopy due to overlapping of dominant fluorophores. By choosing appropriate offsets between excitation and emission wavelengths during signal acquisition from turbid samples, responses of individual fluorophores are highlighted as sharp peaks by using this technique. Some of the peaks may, however, still be missed due to wavelength dependent absorption and scattering effects. In this study a correction technique is used to extract such hidden signatures. The technique is validated using tissue phantoms with known concentrations of fluorophores, absorbers and scatterers. On the basis of validation studies on single and combination of two fluorophores, it is found that lower offsets display better recovery due to minimal influence of absorption by blood. Among the different offsets, 55 nm is found to be optimal for investigation of cervical precancers. Ó 2015 Elsevier B.V. All rights reserved.

1. Introduction Fluorescence spectroscopy is routinely used to detect cancerous and precancerous in-vitro and in-vivo lesions since the past couple of decades [1–11]. It has the ability to monitor the various metabolic and pathological changes related to the abnormal cells growth. Fluorescence spectroscopy is a highly sensitive and cost effective technique for detecting subtle changes in native fluorophore concentrations or the local environment in the tissues [5–11]. These native fluorophores in turbid tissue include structural proteins, amino acids, coenzymes, vitamins, lipids and porphyrins, whose contribution to the spectrum depends upon the choice of excitation and emission wavelengths [6]. For human tissue samples, overlapping emission bands of multiple tissue fluorophores contribute to a single broad band fluorescence spectrum and mask signatures of certain fluorophores. So, vital information of individual fluorophore is difficult to extract and often missed. Synchronous fluorescence spectroscopy technique was first introduced by Lloyd [12] and the basic theory was provided by ⇑ Corresponding author at: IIT Kanpur, Department of Physics, SL-111, Kanpur 208016, India. E-mail address: [email protected] (A. Pradhan). http://dx.doi.org/10.1016/j.jphotobiol.2015.06.019 1011-1344/Ó 2015 Elsevier B.V. All rights reserved.

Vo-Dinh [13,14]. It offers a simple way to decouple the activity of individual fluorophore in a single acquisition with the selection of an appropriate offset wavelength between excitation and emission wavelength. Such a spectrum is recorded by simultaneous scanning of the excitation and emission wavelengths, while keeping a constant wavelength offset between them throughout recording of the spectrum [14]. This technique was initially applied for rank determination of coals by Hans von der Dick et al. [15]. Peuravuori et al. used synchronous fluorescence spectroscopy for differentiating different humic-solute aggregates from water sample [16]. In recent years, it has been applied in cancer diagnosis [17–29]. Diagaradjane et al. explored the potential of this technique to discriminate the sequential tissue transformation in DMBA-TPA induced tumor model in mouse skin [30]. Liu et al. used this technique for multicomponent analysis in tissue [26]. Extensive studies have been performed by Alfano et al., to understand the mechanism of synchronous fluorescence spectroscopy in tissue samples at various offset wavelengths for relevant fluorophores [21–25]. Intrinsic fluorescence in turbid samples such as tissue is generally affected by absorption and scattering. Different combinations of fluorescence and diffuse reflectance spectra have been utilized by several groups to extract intrinsic fluorescence [1,31–35]. It is noticed that synchronous fluorescence spectra are similarly

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affected. Following the concept of our previous work to calculate intrinsic fluorescence [31], we have now developed a protocol using a combination of synchronous fluorescence and elastic scattering measurements to eliminate the effects of absorbers and scatterers. In this paper, we present the method of enhancing the information of fluorophores by decoupling synchronous fluorescence spectra from the interfering effects of absorbers and scatterers. The objective of the present technique is to extract the intrinsic nature of endogenous fluorophores from tissue mimicking liquid phantoms prepared with a single fluorophore and combination of two fluorophores. Validation of the technique along with preliminary study of human cervical tissue is reported. 2. Experimental 2.1. Sample preparation Commercially available biological fluorophores Nicotinamide Adenine Dinucleotide (NADH, N4505) and Flavin Adenine Dinucleotide (FAD, F6625) were obtained from Sigma Aldrich, USA. The different concentrations of FAD used to make phantoms of single fluorophore were 10 lM, 20 lM, 30 lM, 40 lM and 50 lM, while for two fluorophores studies concentrations of FAD and NADH were fixed at 10 lM and 300 lM respectively to maintain the fluorescence intensities at similar orders of magnitude. Human blood was used as the absorber and preserved in PBS with anticoagulant EDTA. It was diluted with distilled water to get absorption coefficients of 0.74 mm1 and 1.64 mm1 at a wavelength of 410 nm as shown in Fig. 1(c), which represents the typical absorption coefficient range in biological tissue [36–38]. Polystyrene microspheres (PS03N, Bangs Laboratories, Inc.) with mean diameter of 0.96 lm were used as the scatterer. Variations of la and g with wavelengths for this scatterer are calculated using online available Mie calculator and are shown in Fig. 1(e). Turbid liquid phantoms were prepared with these fluorophores, scatterers and absorbers in distilled water. The combinations for one fluorophore phantoms are listed in Table 1.

Fresh cervical tissue samples used in experiment were provided by the G.S.V.M. Medical College and measurements were made within 2 h of excision of tissue. The samples were sent in two separate vials labeled as normal and abnormal and dimensions were approximately 2  5  2 mm. The samples were rinsed with saline water to remove superficial blood before performing the experiment. After completing the signal acquisition, these tissue samples were sent back to medical college for histopathology. 2.2. Data acquisition Relevant regions of absorption of fluorophores and human blood were determined from the UV–visible spectra using a UV/vis spectrophotometer (Perkin-Elmer Lambda 35).The synchronous fluorescence spectra of the tissue mimicking liquid phantoms and fresh cervical tissue specimens were recorded with different offset wavelengths using a commercial spectro-fluorometer (SPEX, Fluorolog 3, Model FL3-22). The spectra of phantoms were recorded by placing them in a quartz cuvette and measurements were made in the right angle mode with respect to the incident unpolarized light. Synchronous fluorescence acquisition was done with 40 nm, 55 nm, 70 nm, 100 nm and 120 nm offset wavelengths with integration time of 0.2 s and with wavelength interval of 1 nm. In addition, corresponding elastic scattering spectra were also recorded with zero offset wavelengths between excitation and emission monochromators. 2.3. Proposed technique The synchronous fluorescence (SF) spectral intensity is the combination of both the excitation and emission intensity distributions [13,14]. When light with given excitation wavelength is incident on a turbid sample, it get absorbed and scattered before interacting with the fluorophores distributed (uniformly) inside the medium. Following excitation of fluorescence, the isotropically emitted fluorescence light (at the emission wavelengths) travels through the medium. The resulting wavelength-dependent fluorescence intensity at the emission wavelengths (the line shape of which is the

Fig. 1. Typical plots of (a) scattering coefficient versus wavelength, (b) anisotropy parameter versus wavelength for polystyrene microspheres, (c) absorption coefficient of blood, (d) M1 and M2 corresponding to absorption coefficients b1 and b2 used for correction, and (e) different reduced scattering coefficient of scatterer used to make phantoms.

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S. Devi et al. / Journal of Photochemistry and Photobiology B: Biology 151 (2015) 1–9 Table 1 Details of the phantom sets (1, 2 . . . etc.) used in the legends of Fig. 3, representing the different concentrations of FAD, absorbers and scatterers used to make tissue mimicking liquid phantoms. Here, designations (10, 20, 30, 40, 50), (b1, b2) and (SC2, SC3, SC4, SC5) refer to FAD concentrations in lM, absorption coefficients (shown in Fig. 1c) and reduced scattering coefficients (shown in Fig. 1e), respectively. One fluorophore phantoms set #

Wavelength offsets (Dk) = 120 nm & 100 nm

Wavelength offsets (Dk) = 70 nm & 55 nm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

10 + b1 + SC5 20 + b1 + SC5 30 + b1 + SC5 40 + b1 + SC5 50 + b1 + SC5 10 + b2 + SC5 20 + b2 + SC5 30 + b2 + SC5 40 + b2 + SC5 50 + b2 + SC5

10 + b1 + SC3 20 + b1 + SC3 30 + b1 + SC3 30 + b1 + SC4 40 + b1 + SC3 40 + b1 + SC4 50 + b1 + SC2 50 + b1 + SC3 50 + b1 + SC4 10 + b2 + SC4 10 + b2 + SC5 20 + b2 + SC4 20 + b2 + SC5 30 + b2 + SC4 30 + b2 + SC5 40 + b2 + SC3 40 + b2 + SC4 40 + b2 + SC5 50 + b2 + SC4 50 + b2 + SC5

Table 2 Values of correlation coefficient between the spectra of dilute solutions of fluorophores and the corresponding (a) extracted spectra and (b) uncorrected measured spectra for different sets of phantom. (a) Conc. of FAD(lM)

10 20 30 40 50

Correlation coefficient at different wavelength-offsets (Dk)for extracted spectra

Dk = 120 nm Mean ± std

Dk = 100 nm Mean ± std

Dk = 70 nm Mean ± std

Dk = 55 nm Mean ± std

0.986 ± 0.002 0.981 ± 0.013 0.986 ± 0.005 0.986 ± 0.003 0.979 ± 0.003

0.987 ± 0.004 0.993 ± 0.002 0.994 ± 0.002 0.991 ± 0.003 0.990 ± 0.001

0.985 ± 0.013 0.981 ± 0.016 0.989 ± 0.007 0.979 ± 0.011 0.973 ± 0.012

0.972 ± 0.026 0.979 ± 0.013 0.987 ± 0.005 0.979 ± 0.008 0.972 ± 0.011

(b) Conc. of FAD (lM)

10 20 30 40 50

Correlation coefficient at different wavelength-offsets (Dk) for uncorrected (measured) spectra

Dk = 120 nm Mean ± std

Dk = 100 nm Mean ± std

Dk = 70 nm Mean ± std

Dk = 55 nm Mean ± std

0.187 ± 0.248 0.179 ± 0.280 0.314 ± 0.204 0.262 ± 0.290 0.299 ± 0.281

0.592 ± 0.124 0.597 ± 0.140 0.667 ± 0.105 0.653 ± 0.136 0.673 ± 0.130

0.951 ± 0.019 0.949 ± 0.016 0.958 ± 0.012 0.955 ± 0.013 0.957 ± 0.011

0.983 ± 0.077 0.979 ± 0.071 0.979 ± 0.061 0.973 ± 0.087 0.973 ± 0.087

intrinsic characteristic of the fluorophores), once again gets modulated by the wavelength-dependent scattering and absorption properties of the turbid medium. Note that the elastically scattered light with the same wavelengths (as that of the fluorescence emission wavelengths) should in principle bear similar wavelength dependent modulation of scattering and absorption properties [31]. Hence, the fluorescence spectrum divided by the corresponding elastic scattering spectrum is expected to remove the modulation of the wavelength dependent scattering and absorption properties on the intrinsic fluorescence spectrum. This is the main rationale of implementing Eq. (1). Initially, it is assumed that, on an average, the path traveled by the excitation light (which suffers series of elastic scattering events) before interacting with fluorophore is half of the total path, other half of the path is that of

the fluorescence at the emission wavelength which also eventually suffer series of elastic scattering events before escaping from the medium [32]. To eliminate the effects of wavelength dependent absorption and scattering in the turbid samples, the measured synchronous fluorescence spectrum is accordingly divided by the corresponding elastically scattering spectrum:

F EXTRACTED ðkÞ ¼ SYN

F MEASURED ðkÞ SYN 0:5

fRðkÞ  RMðkþDkÞ ðk þ DkÞg

ð1Þ

Here FMEASURED(k) represents synchronous fluorescence intensities at each excitation wavelength (k). This intensity of synchronous fluorescence spectrum is the combination of both excitation (at wavelength k) as well as fluorescence emission (at wavelength k + Dk) intensities at each wavelength (at wavelength k). Here R(k) represents photon weight corresponding to the elastic scattering at excitation wavelength and R(k + Dk) is the corresponding photon weight of the fluorescence photons at the emission wavelength, which also suffer series of elastic scattering events following one isotropic fluorescence event. The factor M(k + Dk) is introduced to account for the differences in the paths between the elastically scattered photons at the emission wavelength and that of the fluorescence photons at the same wavelength. Note that the path length of the photons inside a turbid medium is primarily controlled by the anisotropy parameter g. This difference arises due to the fact that in case of the fluorescence photons, there is one isotropic scattering event (fluorescence emission) followed by a sequence (say N1) of anisotropic scattering events (determined by g at that wavelength), as opposed to the elastic scattering at the same wavelength, where the incident photon eventually comes out of the medium following a series (N-number) of anisotropic scattering events. The factor M(k + Dk) (=NAV(k + Dk)FLUORESCENCE /NAV(k + Dk)ESS) is determined based on the above principle. In light-tissue interaction model, an incident photon is either scattered or absorbed and reemitted as fluorescence. Path traversed by each incident photon is determined by the anisotropy parameter (g) and characterized by the escape probability (fN(g)) and photon weight (aN = [ls/(la + ls)]N, where a = albedo). Here, expression for escape probability function (fN(g)) has been taken from the reference [39] and this function describes the escape probability of an incident photon from the tissue sample after going through n scattering events with anisotropy coefficient g. Albedo, a is calculated using scattering coefficient and absorption coefficient of polystyrene microspheres as shown in Figs. 1(a) & (c), respectively. For an incident photon which undergoes N anisotropic scattering events, diffuse reflectance can be expressed as R(k) = R aN fN(g) [39]. So average number of anisotropic scattering events (NAV) suffered by the incident photon can be calculated as:

P N a Nf ðgÞ NAV ¼ P N N a f N ðgÞ

ð2Þ

The path traveled by the incident photon depends upon the anisotropy parameter g. But in case of fluorescence, each isotropically emitted photon is followed by N1 anisotropic scattering events. So the value of the effective anisotropy parameter is reduced ESS to geff = (NESS for the case of fluorescence [36]. AV  1)g/NAV NAV(k + Dk)FLUORESCENCE and NAV (k + Dk)ESS are then calculated using the values of anisotropy parameter geff(k + Dk) and g(k + Dk), respectively in the expression of escape probability (fN(g)) and above Eq. (2) at each emission wavelength averaged for a range of scattering events (N) from 1 to 300. Finally, corresponding M(k + Dk) are calculated and plots are displaying in Fig. 1(d) for tissue mimicking phantoms.

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For human tissue, value of anisotropy parameter g is taken as 0.9 due to weak dependence on wavelengths to calculate average number of anisotropic scattering events (NAV). Hence, M is calculated to be 0.84 for tissue, using albedo a = 0.9, g = 0.9 and geff = 0.81. M is assumed to be constant for tissue as it does not vary significantly with wavelength as evident in Fig. 1(d) for turbid phantoms. The synchronous fluorescence spectra of turbid phantom systems are measured at 120 nm, 100 nm, 70 nm, 55 nm and 40 nm offsets along with corresponding elastic scattering spectra measured at zero offset for turbid phantom systems prepared with a single fluorophore and with a mixture of two fluorophores. Intrinsic synchronous fluorescence spectra of the above phantoms are obtained using above Eq. (1) and this has been referred to as ‘‘extracted spectra’’ in this paper. Similarly, measured and extracted synchronous fluorescence spectra with elastic scattering spectra at different offset wavelengths have been acquired for cervical tissue samples.

3. Results and discussion Figs. 1(a) & (b) show the scattering coefficient and anisotropy parameters of polystyrene microspheres used to calculate M for two different concentrations of blood shown in Fig. 1(d). Variations of M1 and M2 with wavelengths as well as with one another are not significant. Figs. 1(c, e) show the absorption spectra and reduced elastic scattering spectra, respectively, for the different

concentrations of absorbers and scatterers used to prepare tissue mimicking phantoms. The values of absorption coefficients and reduced elastic scattering coefficients used in this study are typically similar to biological tissue in the UV and visible wavelength regions [36–38]. Symbols used in legends of Figs. 1(c, e), represent the different concentrations of absorbers and scatterers used to make turbid phantoms. All spectra are divided by the lamp spectrum to remove the wavelength dependence of the excitation source. Elastic scattering spectra mentioned everywhere refer to the denominator of Eq. (1). The ability of the technique described by Eq. (1) to extract the intrinsic synchronous fluorescence from liquid turbid phantoms of two fluorophores is demonstrated in Fig. 2(b) for 70 nm offset. The emitted photons which are absorbed by blood around 350 nm in measured synchronous fluorescence profile are recovered in the extracted spectrum and its profile matches well with the synchronous fluorescence spectrum of pure NADH + FAD for 70 nm offset. Area normalized synchronous fluorescence spectra (only fluorophore, series of fluorophore + scatterer + absorber) for single fluorophore (FAD 20 lM) phantoms are shown in Figs. 2(c, d) and those for two fluorophores (FAD 10 lM & NADH 300 lM) phantoms are shown in Figs. 2(e, f) at different wavelength offsets with area normalized absorption spectrum of blood. It is clearly evident from Figs. 2(c, e) that absorption spectrum of blood affects different regions of SF spectra at different offsets. This produces dips in the same region at corresponding wavelength offsets of the measured SF spectra as shown in Figs. 2(d) & (f) for single fluorophore and two fluorophores phantoms, respectively.

Fig. 2. (a) & (b) Typical measured synchronous fluorescence spectrum with its elastic scattering and extracted synchronous fluorescence spectrum for 70 nm offset for liquid turbid phantoms of single fluorophore (FAD) and mixture of two fluorophores (FAD & NADH), respectively. Area normalized synchronous fluorescence spectra of (c) pure FAD, (d) FAD + scatterer + absorber, mixture of two fluorophores (e) NADH + FAD, and (f) NADH + FAD + scatterer + absorber for wavelength offsets of 40 nm, 55 nm, 70 nm, 100 nm and 120 nm with area normalized absorption spectrum of blood.

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Fig. 3.1. Typical spectra of phantoms with single fluorophore (Measured synchronous fluorescence, elastic-scattering, extracted synchronous fluorescence) with different wavelength offsets of 120 nm (a, b, c) and 100 nm (d, e, f). Area normalized SF spectra of extracted and pure FAD at 120 nm and 100 nm offsets are shown in (g) and (i), respectively. Variations of mean SF intensity at excitation wavelength of (h) 414 nm and (j) 430 nm with different concentrations of FAD for both extracted and measured fluorescence are shown at 120 nm and 100 nm offsets, respectively. Here, error bar represents the standard deviation of SF intensity of phantoms consisting same concentration of fluorophore but different composition of scatterer and absorber. Designations of numeric symbols in legends are given in Table 1.

Figs. 3.1(a, d) and 3.2(a, d) show the synchronous fluorescence spectra of phantoms with various combinations of FAD, scatterer and absorber of different concentrations measured at 120 nm, 100 nm, 70 nm and 55 nm offsets, respectively. Figs. 3.1(b, e), 3.2(b, e) and Figs. 3.1(c, f), 3.2(c, f) show the corresponding elastic scattering spectra and extracted synchronous fluorescence spectra, respectively for all the above mentioned offsets. Detailed descriptions of the numeric symbols used in legends of Fig. 3 that represent different compositions of tissue mimicking phantoms are given in Table 1. Significant absorption dips around 410 nm are observed in the profiles of measured synchronous fluorescence spectra of the phantoms as well as in the elastic scattering spectra for 120 nm offset corresponding to the absorption spectrum of blood. One may note that the synchronous fluorescence spectral profile are red shifted as the wavelength offsets decrease from 120 nm to 40 nm as shown in Fig. 2(c). Hence the absorption dip around 410 nm gradually loses its significance as the wavelength offsets decrease from 120 nm to 40 nm leading to lesser deviation from intrinsic spectra and hence better recovery. The extracted synchronous fluorescence spectra recover

the intensity information which varies consistently with increasing concentrations of fluorophores as well as spectral shape after eliminating the effects of absorbers and scatterers. Figs. 3.1(h, j) and 3.2(h, j) show the mean of SF intensity with error bar (standard deviation) for both measured and extracted synchronous fluorescence for all wavelength offsets. It is clearly demonstrated that mean intensity of extracted synchronous fluorescence varies linearly with increasing concentrations of fluorophores for all wavelength offsets. The error bars (standard deviations) representing the SF intensity variations among the spectra of same concentrations of fluorophore but different compositions of scatterer and absorber, are also small in case of extracted synchronous fluorescence compared to measured one. Correlation coefficients are calculated between extracted and pure fluorophore synchronous fluorescence spectra to measure the extent of recovery of spectral line shape. These coefficients are defined as the covariance of the variables (here extracted and pure fluorophore synchronous fluorescence spectra are variables) divided by the product of their standard deviations [40]. These coefficients measure the strength and direction of relationship between two

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Fig. 3.2. Typical spectra of phantoms with single fluorophore (Measured synchronous fluorescence, elastic-scattering, extracted synchronous fluorescence) with different wavelength offsets of 70 nm (a, b, c) and 55 nm (d, e, f). Area normalized SF spectra of extracted and pure FAD at 70 nm and 55 nm offsets are shown in (g) and (i), respectively. Variations of mean SF intensity at excitation wavelength of (h) 453 nm and (j) 464 nm with different concentrations of FAD for both extracted and measured fluorescence are shown at 70 nm and 55 nm offsets, respectively. Here, error bar represents the standard deviation of SF intensity of phantoms consisting same concentration of fluorophore but different composition of scatterer and absorber. Designations of numeric symbols in legends are given in Table 1.

variables and value of these being positive one (+1) means that two variables are perfectly (linearly) related in a similar manner (same direction). In our case, profiles of the extracted spectra resemble the line-shape of the dilute FAD solution with small deviations as shown in Figs. 3.1(g, i) & 3.2(g, i) as well as by correlation coefficients in Table 2(a) compared to those of uncorrected (measured) and pure FAD solution SF spectra in Table 2(b). Here, it is seen that effect of distortion due to absorbers on measured SF spectra decreases for lower offsets compared to higher offsets. Measured SF spectra with corresponding elastic scattering and extracted SF spectra at 120 nm and 100 nm offsets for tissue mimicking phantoms of two fluorophores are shown in Figs. 4(a) & (b), respectively. Figs. 4(c, d, e, f), (g, h, i, j) and (k, l, m, n) show the SF spectra (measured, elastic-scattering, extracted, extracted with NADH + FAD) at 70 nm, 55 nm and 40 nm offsets, respectively. Here, extracted SF spectra clearly illustrate that shape recovery is not so efficient for larger offsets (120 nm, and 100 nm), while for smaller offsets (70 nm, 55 nm, 40 nm) line-shape and intensity information are both recovered with minimal variations.

Moreover, extracted SF spectra for smaller offsets (70 nm, 55 nm, 40 nm) recover line-shape (shown in Figs. 4(f, j, n)) and intensity information both for low as well for high concentrations of scatterers. Efficacy of this technique to recover intrinsic SF for turbid phantoms of two fluorophores is illustrated by calculating correlation coefficients between SF spectrum of two fluorophores (FAD + NADH) and extracted SF spectrum. Table 3 shows the mean correlation coefficients with its standard deviations for all phantoms at 120 nm, 100 nm, 70 nm, 55 nm and 40 nm wavelength offsets. It can be noted here that spectra shown in Figs. 4(a) & (b) are for one phantom, while mean correlation coefficients with its standard deviations are calculated after including all phantoms for higher offsets (120 nm & 100 nm). On the basis of single and two fluorophores study, we can conclude that the proposed technique recovers both line-shape and consistent intensity information efficiently for a wide range of concentrations of scatterers, absorbers and fluorophores at lower offsets (70 nm, 55 nm, 40 nm) for single as well as for two fluorophores turbid phantoms. The range of la/l0s over which the

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Fig. 4. All spectra shown here are for a mixture of two fluorophores FAD (10 lM) and NADH (300 lM). Figures (a) and (b), Typical measured synchronous fluorescence (SF) spectrum with its elastic-scattering and extracted synchronous fluorescence at 120 nm and 100 nm wavelength offsets, respectively. Different spectra (Measured SF, elasticscattering, extracted SF and area normalized extracted SF with NADH + FAD SF spectra) are shown for 70 nm (c, d, e, f), 55 nm (g, h, i, j), and 40 nm (k, l, m, n) wavelength offsets.

Table 3 Values of correlation coefficient between the spectra of dilute solutions of fluorophores and the corresponding extracted spectra for different sets of two fluorophores phantoms. Correlation coefficient between

Extracted & pure spectra

Correlation coefficient at different wavelength-offsets (Dk)

Dk = 120 nm Mean ± std

Dk = 100 nm Mean ± std

Dk = 70 nm Mean ± std

Dk = 55 nm Mean ± std

Dk = 40 nm Mean ± std

0.904 ± 0.017

0.939 ± 0.023

0.973 ± 0.009

0.942 ± 0.013

0.911 ± 0.028

technique is applied and recovery is efficient is 0.04–3.08 for wavelength range of 350–520 nm. For turbid phantoms of single fluorophore, the effect of absorption is very high for higher offsets (120 nm, 100 nm) as evident from the overlapped spectral region of SF spectra at higher offsets and absorption spectrum as shown in Fig. 2(c). Hence the present technique fails to recover both line-shape and intensity information completely at higher offsets. Selection of optimum wavelength offset and quality of SF signal in case of multiple fluorophores depend upon two parameters, namely resolution and magnitude [23,25]. On the basis of these two parameters smaller wavelength offsets are selected as optimum offset to study behavior of multiple components in biological samples [17,18,20–25,28–30]. It is observed that our technique

works efficiently for lower offsets for a wide-range of concentrations of scatterers and absorbers compared to larger one, consistent with the offsets reported in literature [17,18,20–25,28–30]. A preliminary investigation of the efficacy of this technique for 50 human cervical samples is also performed at different offset wavelengths. All samples show consistent results as shown by the mean of area normalized extracted SF spectra of 50 human cervical tissue samples with standard deviation in Fig. 5(e) at 55 nm offset. A typical result has been demonstrated here. Fig. 5(b) shows the area normalized measured synchronous fluorescence spectrum for 55 nm offset wavelength with its corresponding elastic scattering spectrum. Some of the peaks hidden in the measured spectrum appear in the extracted spectrum in addition to the narrowing and

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Fig. 5. (a), (b), (c) and (d) Typical measured synchronous fluorescence (SF) spectrum of a human cervical tissue with its elastic scattering and extracted synchronous fluorescence spectrum for 40 nm, 55 nm, 70 nm and 120 nm wavelength offsets, respectively. Figure (e) shows the mean of area normalized extracted SF spectra of 50 human cervical tissue samples with error bar (standard deviation).

sharpening of the broader peaks. The fluorophores assigned to the significant peaks are collagen, bound NADH, free NADH and FAD [21,41–43]. Measured and extracted synchronous fluorescence spectra with corresponding elastic scattering spectra for 40 nm, 70 nm and 120 nm offset wavelengths are shown in Figs. 5(a, c & d), respectively which clearly illustrate that the given formulation in Eq. (1) unveils information of all the native fluorophores that contribute during the pathological changes of dysplastic cells with narrowing and sharpening of emission peaks of measured spectra. Preliminary studies on cervical tissue have shown well differentiated extracted synchronous fluorescence peaks for monitoring biochemical activities of fluorophores. These narrow and sharp peaks of various endogenous fluorophores may further help in discriminating normal and dysplastic tissue more efficiently. Moreover, hidden peaks such as free or bound NADH & FAD are revealed using this technique and hence can be used efficiently for extraction of individual fluorophore activities for different tissue grades [41–43]. To obtain a statistically significant result a systematic study with larger number of tissue samples is currently in progress.

human blood from liquid tissue mimicking phantoms. The extraction is seen to be more efficient for all concentrations sets of phantoms studied at lower offsets. At higher offsets, the efficiency of extraction is low due to the higher influence of blood absorption. The technique works efficiently for la/l0s equal to 0.04–3.08 in a wavelength range of 350–520 nm. Synchronous fluorescence spectroscopy corrected for the absorption effects by elastic scattering reveals hidden peaks and sharpens broad peaks of the measured synchronous fluorescence spectra for human cervical tissue samples. On the basis of represented results we have shown the potential of this correction technique in diagnosis of precancerous lesions. Acknowledgement The authors would like to acknowledge Dr. Kiran Pandey and Dr. Asha Agarwal of GSVM Medical College, Kanpur for providing well characterized tissues for preliminary studies. Seema Devi acknowledges her colleague Mr. Pankaj Singh for helpful discussions. References

4. Conclusion A correction to synchronous fluorescence spectra has been developed and validated in a systematic study with a large set of turbid phantoms of different concentrations. The results demonstrate that extracted synchronous fluorescence technique successfully eliminates the absorption effect of different concentration of

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