Early detection of stripe rust infection in wheat using light-induced fluorescence spectroscopy.

In the current study, the application of fluorescence spectroscopy along with the advanced statistical technique and confocal microscopy was investigated for the early detection of stripe rust infection in wheat grown under field conditions. The indigenously developed Fluorosensor fitted with LED, emitting monochromatic light was used that covered comparatively larger leaf area for recording fluorescence data thus presenting more reliable current status of the leaf. The examined leaf samples covered the entire range of stripe rust disease infection from no visible symptoms to the complete disease prevalence. The molecular changes were also assessed in the leaves as the disease progresses. The emission spectra mainly produce two fluorescence emission classes, namely the blue-green fluorescence (400-600 nm range) and chlorophyll fluorescence (650-800 nm range). The chlorophyll fluorescence region showed lower chlorophyll bands both at 685 and 735 nm in the asymptomatic (early diseased) and symptomatic (diseased) leaf samples than the healthy ones as a result of partial deactivation of PSII reaction centers. The 735 nm chlorophyll fluorescence band was either slight or completely absent in the leaf samples with lower to higher disease incidence and thus differentiate between the healthy and the infected leaf samples. The Hydroxycinnamic acids (caffeic and sinapic acids) showed decreasing trend, whereas the ferulic acid increased with the rise in disease infection. Peak broadening/shifting has been observed in case of ferulic acid and carotenes/carotenoids, with the increase in the disease intensity. While using the LEDs (365 nm), the peak broadening and the decline in the chlorophyll fluorescence bands could be used for the early prediction of stripe rust disease in wheat crop. The PLSR statistical techniques discriminated well between the healthy and the diseased samples, thus showed promise in early disease detection. Confocal microscopy confirmed the early prevalence of stripe rust disease infection in a susceptible variety at a stage when the disease is not detectable visually. It is inferred that fluorescence emission spectroscopy along with the chemometrics aided in the effective and timely diagnosis of plant diseases and the detected signatures provide the basis for remote sensing.

Synthesis and photophysical investigations of pyridine-pyrazolate bound boron(III) diaryl complexes.

This study presents the design and synthetic pathway of unsymmetric ligands based on pyridine-pyrazolate scaffold with Donor-Acceptor (D-A) molecular arrays and their boron complexes to achieve a large Stokes shift. Intermolecular charge transfer (ICT) triggered by the uneven molecular charge distribution from electronically dense pyrazolate (donor) part of the ligands to electron-deficient boron centre (acceptor) resulted in a mega Stokes shift up to 263 nm for selected compounds while retaining the characteristic quantum efficiency and chemical stability. The photophysical properties of derivatization of pyrazolate group in the pyridine-pyrazolate scaffold of diaryl boron complexes were explored based on UV-Visible, steady-state and time-resolved fluorescence spectroscopy. An interesting dual emission along with quenching behaviour was also observed for 2-(6-methoxynaphthelene) 5-(2-pyridyl) pyrazolate boron complex (P5) due to the formation of a twisted intermolecular charge transfer (TICT) state from a locally excited (LE) state rendering it a potential candidate for sensing applications based on H-Bond quenching. In addition, the extended excited state lifetime of the reported compounds compared to classical boron-dipyrromethene (BODIPY) makes them suitable as potential probes for analytical applications requiring a longer excited state lifetime.

Quantitative auto-fluorescence quenching of free and bound NADH in HeLa cell line model with Carbonyl cyanide-p-Trifluoromethoxy phenylhydrazone (FCCP) as quenching agent.

The autofluorescence of endogenous biomolecules (Nicotinamide adenine dinucleotide (NAD, its reduced form NADH and the phosphorylated form NAD(P)H take part in cellular metabolic pathways and has vital importance for in vivo and ex vivo photo diagnostic applications of biological tissues. We present a detailed quenching analysis of Carbonyl cyanide-p-Trifluoromethoxy phenylhydrazone (FCCP) 50-1000 µM and analyzed the fluorescence signal from NADH/ NAD(P)H in vitro (in solution) and in vivo (HeLa cell suspension).The in vitro samples of pure NADH/ NAD(P)H were excited at λ=340±1 nm while the fluorescence signal was collected in the range of 400-550 nm. The quenching process was characterized using excitation emission matrix (EEM) fluorescence spectroscopy and Stern- Volmer plots. The experimental results illustrated maximum fluorescence emission for the control NADH samples (i.e., no FCCP), while the fluorescence signal from the solution progressively decreased with the increasing concentration of the FCCP, until it reaches the base line (i.e., no fluorescence signal) at 1000 µM of FCCP. In vitro study shows that the fluorescence quenching of free NADH was found to be lower than the bound NAD(P)H with similar diminishing trend. The quenching of bound NAD(P)H in cells is attenuated compared to solution quenching possibly due to a contribution from the metabolic/antioxidant response in cells and fluorescence exponential decay curve lies between plated and suspended HeLa cells. A two-fold increase in the fluorescence intensity of NAD(P)H was observed after the bond formation with L-Malate Dehydrogenase (L-MDH, Sigma Aldrich #10127248001) protein This work has applications for sharp tumor demarcation during sensitive surgical procedures as well as to enhance fluorescence based diagnosis of biological tissues.

Simulated Annealing-Based Image Reconstruction for Patients With COVID-19 as a Model for Ultralow-Dose Computed Tomography.

The proposed algorithm of inverse problem of computed tomography (CT), using limited views, is based on stochastic techniques, namely simulated annealing (SA). The selection of an optimal cost function for SA-based image reconstruction is of prime importance. It can reduce annealing time, and also X-ray dose rate accompanying better image quality. In this paper, effectiveness of various cost functions, namely universal image quality index (UIQI), root-mean-squared error (RMSE), structural similarity index measure (SSIM), mean absolute error (MAE), relative squared error (RSE), relative absolute error (RAE), and root-mean-squared logarithmic error (RMSLE), has been critically analyzed and evaluated for ultralow-dose X-ray CT of patients with COVID-19. For sensitivity analysis of this ill-posed problem, the stochastically estimated images of lung phantom have been reconstructed. The cost function analysis in terms of computational and spatial complexity has been performed using image quality measures, namely peak signal-to-noise ratio (PSNR), Euclidean error (EuE), and weighted peak signal-to-noise ratio (WPSNR). It has been generalized for cost functions that RMSLE exhibits WPSNR of 64.33 ± 3.98 dB and 63.41 ± 2.88 dB for 8 × 8 and 16 × 16 lung phantoms, respectively, and it has been applied for actual CT-based image reconstruction of patients with COVID-19. We successfully reconstructed chest CT images of patients with COVID-19 using RMSLE with eighteen projections, a 10-fold reduction in radiation dose exposure. This approach will be suitable for accurate diagnosis of patients with COVID-19 having less immunity and sensitive to radiation dose.

A review of the medical hyperspectral imaging systems and unmixing algorithms' in biological tissues.

Hyperspectral fluorescence imaging (HFI) is a well-known technique in the medical research field and is considered a non-invasive tool for tissue diagnosis. This review article gives a brief introduction to acquisition methods, including the image preprocessing methods, feature selection and extraction methods, data classification techniques and medical image analysis along with recent relevant references. The process of fusion of unsupervised unmixing techniques with other classification methods, like the combination of support vector machine with an artificial neural network, the latest snapshot Hyperspectral imaging (HSI) and vortex analysis techniques are also outlined. Finally, the recent applications of hyperspectral images in cellular differentiation of various types of cancer are discussed.

Mitosis detection in breast cancer histopathology images using hybrid feature space.

Breast Cancer grading is a challenging task as regards image analysis, which is normally based on mitosis count rate. The mitotic count provides an estimate of aggressiveness of the tumor. The detection of mitosis is a challenging task because in a frame of slides at X40 magnification, there are hundreds of nuclei containing few mitotic nuclei. However, manual counting of mitosis by pathologists is a difficult and time intensive job, moreover conventional method rely mainly on the shape, color, and/or texture features as well as pathologist experience. The objective of this study is to accept the atypaia-2014 mitosis detection challenge, automate the process of mitosis detection and a proposal of a hybrid feature space that provides better discrimination of mitotic and non-mitotic nuclei by combining color features with morphological and texture features. To exploit color channels, they were first selected, and then normalized and cumulative histograms were computed in wavelet domain. A detailed analysis presented on these features in different color channels of respective color spaces using Random Forest (RF) and Support Vector Machine (SVM) classifiers. The proposed hybrid feature space when used with SVM classifier achieved a detection rate of 78.88% and F-measure of 72.07%. Our results, especially high detection rate, indicate that proposed hybrid feature space model contains discriminant information for mitotic nuclei, being therefore a very capable are for exploration to improve the quality of the diagnostic assistance in histopathology.

Biomedical Applications of Integrating Sphere: A Review.

Integrating sphere remains a valuable tool for biomedical optics research. Specifically, it has been used to determine the optical properties (i.e., absorption coefficient, scattering coefficient and anisotropy factor) of biological tissues. This study presents an overview of the literature on integrating sphere and its biomedical applications. In particular, we start with a brief introduction of tissue optics with emphasis on the optical properties of biological tissues, followed by a detailed discussion of the hardware and related procedures of the integrated sphere system. Both the experimental procedure and subsequent analytical models (i.e., first order scattering, Kubelka Munk, diffusion approximation, Monte Carlo and inverse adding-doubling methods) along with illustrative examples are also outlined. Finally, illustrative examples to explore the optical properties of tissue phantoms and biological samples have been discussed. This survey will provide a ready reference and overview for the applications of integrating sphere in biomedical optics.

Programmable LED-based integrating sphere light source for wide-field fluorescence microscopy.

Wide-field fluorescence microscopy commonly uses a mercury lamp, which has limited spectral capabilities. We designed and built a programmable integrating sphere light (PISL) source which consists of nine LEDs, light-collecting optics, a commercially available integrating sphere and a baffle. The PISL source is tuneable in the range 365-490nm with a uniform spatial profile and a sufficient power at the objective to carry out spectral imaging. We retrofitted a standard fluorescence inverted microscope DM IRB (Leica) with a PISL source by mounting it together with a highly sensitive low- noise CMOS camera. The capabilities of the setup have been demonstrated by carrying out multispectral autofluorescence imaging of live BV2 cells.

Fluorescence quenching of free and bound NADH in HeLa cells determined by hyperspectral imaging and unmixing of cell autofluorescence.

Carbonyl cyanide-p-trifluoro methoxyphenylhydrazone (FCCP) is a well-known mitochondrial uncoupling agent. We examined FCCP-induced fluorescence quenching of reduced nicotinamide adenine dinucleotide / nicotinamide adenine dinucleotide phosphate (NAD(P)H) in solution and in cultured HeLa cells in a wide range of FCCP concentrations from 50 to 1000µM. A non-invasive label-free method of hyperspectral imaging of cell autofluorescence combined with unsupervised unmixing was used to separately isolate the emissions of free and bound NAD(P)H from cell autofluorescence. Hyperspectral image analysis of FCCP-treated HeLa cells confirms that this agent selectively quenches fluorescence of free and bound NAD(P)H in a broad range of concentrations. This is confirmed by the measurements of average NAD/NADH and NADP/NADPH content in cells. FCCP quenching of free NAD(P)H in cells and in solution is found to be similar, but quenching of bound NAD(P)H in cells is attenuated compared to solution quenching possibly due to a contribution from the metabolic and/or antioxidant response in cells. Chemical quenching of NAD(P)H fluorescence by FCCP validates the results of unsupervised unmixing of cell autofluorescence.