Development of methods for quantification of cellular association and patterns in growing bacterial colony is of considerable current interest, not only to help understand multicellular behavior of a bacterial species but also to facilitate detection and identification of a bacterial species in a given space and under a given set of condition(s). We have explored quantitative spectral light scattering polarimetry for probing the morphological and structural changes taking place during colony formations of growing Bacillus thuringiensis bacteria under different conditions (in normal nutrient agar representing favorable growth environment, in the presence of 1% glucose as an additional nutrient, and 3 mM sodium arsenate as toxic material). The method is based on the measurement of spectral 3×3 Mueller matrices (which involves linear polarization measurements alone) and its subsequent analysis via polar decomposition to extract the intrinsic polarization parameters. Moreover, the fractal micro-optical parameter, namely, the Hurst exponent H , is determined via fractal-Born approximation-based inverse analysis of the polarization-preserving component of the light scattering spectra. Interesting differences are noted in the derived values for the H parameter and the intrinsic polarization parameters (linear diattenuation d , linear retardance δ , and linear depolarization Δ coefficients) of the growing bacterial colonies under different conditions. The bacterial colony growing in presence of 1% glucose exhibit the strongest fractality (lowest value of H ), whereas that growing in presence of 3 mM sodium arsenate showed the weakest fractality. Moreover, the values for δ and d parameters are found to be considerably higher for the colony growing in presence of glucose, indicating more structured growth pattern. These findings are corroborated further with optical microscopic studies conducted on the same samples.
Development of methodologies for quantification/unique interpretation of the intrinsic polarimetry characteristics of biological tissues are important for various applications involving tissue characterization/diagnosis. A detailed comparative evaluation of the polar decomposition and the differential matrix decomposition of Mueller matrices for extraction/quantification of the intrinsic polarimetry characteristics (with special emphasis on linear retardance δ , optical rotation Ψ and depolarization Δ parameters was performed, because these are the most prominent tissue polarimetry effects) from complex tissue-like turbid media exhibiting simultaneous scattering and polarization effects. The results suggest that for media exhibiting simultaneous linear retardance and optical rotation polarization events, the use of retarder polar decomposition with its associated analysis which assumes sequential occurrence of these effects, results in systematic underestimation of δ and overestimation of Ψ parameters. Analytical relationships between the polarization parameters (δ , Ψ ) extracted from both the retarder polar decomposition and the differential matrix decomposition for either simultaneous or sequential occurrence of the linear retardance and optical rotation effects were derived. The self-consistency of both decompositions is validated on experimental Mueller matrices recorded from tissue-simulating phantoms (whose polarization properties are controlled, known a-priori, and exhibited simultaneously) of increasing biological complexity. Additional theoretical validation tests were performed on Monte Carlo-generated Mueller matrices from analogous turbid media exhibiting simultaneous depolarization (Δ ), linear retardance (δ ) and optical rotation (Ψ ) effects.
We present a novel spectral Mueller matrix measurement system for both elastic and inelastic scattering (fluorescence)
polarimetric measurements. The system comprises of a Xenon lamp as excitation source, a polarization state generator
(PSG) and a polarization state analyzer (PSA) unit to generate and analyze polarization states required for 4 x 4 sample
Mueller matrix measurements, coupled to a spectrometer for spectrally resolved (λ ~ 400 - 800 nm) signal detection.
The PSG unit comprises of a fixed linear polarizer (polarization axis oriented at horizontal position) followed by a
rotatable broadband quarter wave plate. The sample-scattered light is collected and collimated using an assembly of
lenses, then passes through the PSA unit, and is finally recorded using the spectrometer. The PSA unit essentially
consists of a similar arrangement as that of the PSG, but positioned in reverse order, and with the axis of the linear
polarizer oriented at vertical position. A sequence of sixteen measurements are performed by changing the orientation of
the fast axis of the quarter wave plates of the PSG unit (for generating the four required elliptical polarization states) and
that of the PSA unit (for analyzing the corresponding polarization states). The orientation angles
(35°, 70°, 105° and 140°) were chosen based on optimization of the PSG and PSA matrices to yield most stable system
Mueller matrices. The performance of the polarimeter was calibrated using Eigenvalue calibration method which also
yielded the actual values of the system PSG and PSA matrices at each wavelength. The system has been automated and
is capable of Mueller matrix measurement with high accuracy over the entire spectral range 400 - 800 nm (elemental
error < 0.01). For recording the elastic scattering Mueller matrix of sample, the PSG and PSA matrices for each
wavelength are used, while for fluorescence Mueller matrix measurements, the PSG for the excitation wavelength
(chosen to be 405 nm) and PSA for varying emission wavelengths (450 - 800 nm) are used. The developed spectral
Mueller matrix system has been initially used to record both elastic scattering and fluorescence Mueller matrices from
normal and cancerous cervical tissues.
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