Development and characterization of portable smartphone-based imaging device.

Many diseases in rural areas and developing countries are detected late at an advanced stage when treatment might involve complications and higher cost, resulting in a greater number of fatalities. This study aims to make early disease detection simpler and affordable for people living in remote areas and developing countries. A new age optical microscope with high sensitivity diagnosis can revolutionize this gap in disease detection. Here, a smartphone-based imaging device (SID) using optics and a smartphone interface was developed to speedup the process of diagnosis in areas that do not have easy access to health centers and diagnostic clinics. The device was built using acrylic sheets to make it less bulky and customizable and three-dimensional (3D) printed mechanical parts were used to increase stability. The study includes calibration, and testing the device with various samples to determine its capabilities. Images were acquired using the various types of BLIPS lens integrated onto the smartphone camera lens and compared with optical microscope images. The device can visualize single human blood cell which is 8 µm in size using ultra-BLIPS lens and magnification is comparable to an objective lens used in an optical microscope.

Design and Fabrication of Low-cost Microfluidic Channel for Biomedical Application.

This paper presents the design, simulation and low-cost fabrication of microfluidic channel for biomedical application. Channel is fabricated using soft lithography technique. Printed Circuit Board (PCB) is used to make the master for the channel. Channel pattern is transferred on PCB plate using toner transfer technique followed by ferric chloride etching. Paper also discusses, the issues involved in PCB based master fabrication and their viable solutions. Glass is used as substrate material and the channel is made of Sylgard 184 Polydimethylsiloxane (PDMS). Channel is interfaced with a syringe pump to observe the fluid flow. To predict the behavior of the channel, FEM simulation is performed using COMSOL Multiphysics 5.2a. There is a good match between the theoretical, simulation and test results. Finally, to test the biocompatibility of the channel, genomic DNA is passed through the channel and gel electrophoresis analysis is performed.

Detection of mitochondrial dysfunction in vitro by laser-induced autofluorescence.

Mitochondrion plays a significant role in a variety of biological functions. Because of their diverse character and location in the cellular systems, mitochondria commonly get exposed to various extrinsic and intrinsic cellular stresses. The present study reports a novel approach to detection of mitochondrial dysfunction based on tryptophan autofluorescence of its proteins in mouse liver, using laser-induced fluorescence (LIF) as a tool. Mitochondria, isolated from the mouse liver, were initially tested for purity and integrity using lactate dehydrogenase and succinate dehydrogenase (SDH) assays. Mitochondrial stress was induced by treating the isolated mitochondria with heavy metals at 10 and 0.01 mM for sodium arsenite and mercuric chloride, respectively. Upon treatment with the heavy metal, tryptophan autofluorescence quenching was recorded at 281 nm excitation. The functional integrity of the mitochondria treated with heavy metals was evaluated by measuring SDH and cytochrome c oxidase activities at various concentrations of mitochondria, which showed impaired activity as compared to control upto a concentration of 6.25 µg. A significant shift was also observed in the autofluorescence of proteins upto the level below 1 µg, suggesting their conformational change and hence altered structural integrity of mitochondria. Circular dichroism spectroscopy data of the mitochondrial proteins treated with heavy metals further validates their conformational change as compared to untreated control. The present study clearly shows that the LIF can be a novel detection tool to detect altered structural integrity of cellular mitochondria upon stress, and it also possesses the potentiality to combine with other interdisciplinary modalities.

Probing endogenous collagen by laser-induced autofluorescence in burn wound biopsies: A pilot study.

The focus of the current study was to interrogate the predictive potential of laser-induced autofluorescence (LIAF) by objectively assessing collagen synthesis in burn wound granulation tissues ex vivo. Prior grafting, granulation tissues (20 samples) following burn injury were collected from 17 subjects of age range 18 to 60 years with patient/donor consent and the corresponding autofluorescence spectra were recorded at 325 nm He-Cd laser (≈2 mW) excitations. The resulting endogenous collagen intensity from the above tissue samples was computed by normalizing the nicotinamide adenine dinucleotide levels. In addition, the hydroxyproline content was also estimated biochemically from the same granulation tissues. A comparative assessment of both LIAF and biochemical estimations for endogenous collagen by hydroxyproline resulted in strong positive correlation among them. The above relevant observations suggest that LIAF is equally informative as that of biochemical estimations, in evaluating endogenous collagen content in wound granulation tissues. Thus, it can be concluded that LIAF has the predictive potential, as a noninvasive objective tool to measure the endogenous collagen levels in wound biopsy tissues and provide complementary data conducive for making clinical decisions.

Interrogation of an autofluorescence-based method for protein fingerprinting.

In the present study, we have designed a laser-induced fluorescence (LIF) based instrumentation and developed a sensitive methodology for the effective separation, visualization, identification and analysis of proteins on a single platform. In this method, intrinsic fluorescence spectra of proteins were detected after separation on 1 or 2 dimensional Sodium Dodecyl Sulfate-Tris(2-carboxyethyl)phosphine (SDS-TCEP) polyacrylamide gel electrophoresis (PAGE) and the data were analyzed. The MATLAB assisted software was designed for the development of PAGE fingerprint for the visualization of protein after 1- and 2-dimensional protein separation. These provided objective parameters of intrinsic fluorescence intensity, emission peak, molecular weight and isoelectric point using a single platform. Further, the current architecture could differentiate the overlapping proteins in the PAGE gels which otherwise were not identifiable by conventional staining, imaging and tagging methods. Categorization of the proteins based on the presence or absence of tyrosine or tryptophan residues and assigning the corresponding emission peaks (309-356 nm) with pseudo colors allowed the detection of proportion of proteins within the given spectrum. The present methodology doesn't use stains or tags, hence amenable to couple with mass spectroscopic measurements. This technique may have relevance in the field of proteomics that is used for innumerable applications.

Responses of He-Ne laser irradiation on agronomical characters and chlorogenic acid content of brinjal (Solanum melongena L.) var. Mattu Gulla.

Exposure to laser irradiation on seeds brings about the changes in agronomical characteristics of the plants. Solanum melongena L. var. Mattu Gulla, a variety of brinjal is of high economic value due to its unique colour and flavour. The aim of the study was to understand the influence of Helium-Neon (He-Ne) laser irradiation on agronomical characters of Solanum melongena L. var. Mattu Gulla in the field conditions. Various growth characteristics including seed germination percentage, survival rate, plant height, number of branches, and flowers and fruits were estimated during different developmental stages of the brinjal. In addition, the chlorogenic acid content of fruits obtained from the laser irradiated seeds were quantified using reversed-phase high-performance liquid chromatography (RP- HPLC). The plants from the seeds irradiated with different doses (20, 25 and 30J/cm2) of He-Ne laser showed significant enhancement on the growth characteristics when compared to the non-irradiated control groups. He-Ne laser irradiation also improved the yield characteristics of the plants significantly in in vivo conditions in comparison with control group. 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed using methanolic extract of matured fruit of Mattu Gulla on HepG2 and fibroblast cell lines. The IC50 values of fruit extract from laser irradiated groups were found to be similar to non-irradiated control groups. Chlorogenic acid content was found to be higher in 20J/cm2 and lower in 30J/cm2 treated fruit tissue. The current study thus elucidates the role of He-Ne laser as a biostimulator on brinjal var. Mattu Gulla not only in the in vitro conditions but also in the in vivo field conditions.

Objective assessment of endogenous collagen in vivo during tissue repair by laser induced fluorescence.

Collagen, a triple helical protein with the primary role of mechanical function, provides tensile strength to the skin, and plays a pivotal task in tissue repair. During tissue regeneration, collagen level increases gradually and therefore, monitoring of such changes in vivo by laser induced fluorescence was the main objective behind the present study. In order to accomplish this, 15 mm diameter excisional wounds were created on six to eight week old Swiss albino mice. The collagen deposition accelerated upon irradiation of single exposure of 2 J/cm2 He-Ne laser dose immediately after wounding was recorded by laser induced autofluorescence in vivo along with un-illuminated and un-wounded controls. Autofluorescence spectra were recorded for each animal of the experimental groups on 0, 5, 10, 30, 45 and 60 days post-wounding, by exciting the granulation tissue/skin with 325 nm He-Cd laser. The variations in the average collagen intensities from the granulation tissue/skin of mice were inspected as a function of age and gender. Further, the spectral findings of the collagen synthesis in wound granulation tissue/un-wounded skin tissues were validated by Picro-Sirius red- polarized light microscopy in a blinded manner through image analysis of the respective collagen birefringence. The in vivo autofluorescence studies have shown a significant increase in collagen synthesis in laser treated animals as compared to the un-illuminated controls. Image analysis of the collagen birefringence further authenticated the ability of autofluorescence in the objective monitoring of collagen in vivo. Our results clearly demonstrate the potential of laser induced autofluorescence in the monitoring of collegen synthesis during tissue regeneration, which may have clinical implications.

Influence of helium-neon laser irradiation on seed germination in vitro and physico-biochemical characters in seedlings of brinjal (Solanum melongena L.) var. Mattu Gulla.

In the present study, the seeds of brinjal (Solanum melongena L.) var. Mattu Gulla were irradiated with single exposure of He-Ne laser at different doses of 5-40 J cm(-2) and germinated aseptically. Thirty day old seedlings were harvested and the germination, growth, physiological and biochemical parameters were estimated and compared with un-irradiated control seedlings. A significant enhancement in growth characters were noted with respect to length, fresh and dry weight of shoots and roots. In addition, chlorophyll (a and b), carotenoid content, anthocyanin and amylases (α and ß) activities were found to be altered. Significant alterations in percentage of seed germination (P < 0.001) and time to 50% germination (P < 0.001) were observed in the irradiated seeds compared with the un-irradiated controls. In conclusion, the results of the present study demonstrated that low dose (5-30 J cm(-2) ) of He-Ne laser irradiation enhanced the germination process and altered growth, by positively influencing physiological and biochemical parameters of the brinjal seedlings compared with un-irradiated control under in vitro conditions.

Spectroscopic and histological evaluation of wound healing progression following Low Level Laser Therapy (LLLT).

The present study focuses on the evaluation of the effect of He-Ne laser on tissue regeneration by monitoring collagen synthesis in wound granulation tissues in Swiss albino mice using analysis of laser induced fluorescence (LIF) and light microscopy techniques. The spectral analyses of the wound granulation tissues have indicated a dose dependent increase in collagen levels during the post-wounding days. The histological examinations on the other hand have also shown a significant increase in collagen deposition along with the reduced edema, leukocytes, increased granulation tissue, and fibroblast number in the optimal laser dose treated group compared to the non-illuminated controls.

Effect of laser dose and treatment schedule on excision wound healing in diabetic mice.

The present study was undertaken to evaluate a He-Ne laser (632.8 nm; 7 mW; 4.02 mW cm(-2); 15 mm spot size) dose and the treatment schedule on diabetic wound healing in a mouse model. Circular wounds of 15 mm diameter were created on streptozotocin induced diabetic Swiss albino mice, and were uniformly illuminated with the single exposure of various He-Ne laser doses of 1, 2, 3, 4 and 5 J cm(-2) respectively. Further, the treatment schedule was also optimized by exposing the wounds with 3 J cm(-2) at 0, 24 h, 48 h postwounding. Contraction kinetics, mean area under the curve and the mean healing time of the wounds were computed along with the collagen and the glucosamine levels in the wound ground tissues at various postwounding treatment schedules. Results of this study indicated that the single exposure of 3 J cm(-2) laser dose applied immediately after the wounding caused a significant reduction in the mean area under the curve and the mean healing time along with the elevated levels of collagen and glucosamine contents in the tissue compared to the controls. In conclusion, He-Ne laser dose of 3 J cm(-2) applied immediately after the wounding has demonstrated optimum wound healing compared to the other doses and treatment schedules.

Photoacoustic spectroscopy of ovarian normal, benign, and malignant tissues: a pilot study.

Photoacoustic spectra of normal, benign, and malignant ovarian tissues are recorded using 325-nm pulsed laser excitation in vitro. A total of 102 (34 normal, 38 benign, and 30 malignant) spectra are obtained from 22 samples belonging to normal, benign, and malignant subjects. Applying multi-algorithm approach, comprised of methods such as, principal component analysis (PCA) based k-nearest neighbor (k-NN) analysis, artificial neural network (ANN) analysis, and support vector machine (SVM) analysis, classification of the data has been carried out. For PCA, first the calibration set is formed by pooling 45 spectra, 15 belonging to each of pathologically certified normal, benign, and malignant samples. PCA is then performed on the data matrix, comprised of the six spectral features extracted from each of 45 calibration samples, and three principal components (PCs) containing maximum diagnostic information are selected. The scores of the selected PCs are used to train the k-NN, ANN, and SVM classifiers. The ANN used is a classical multilayer feed forward network with back propagation algorithm for its training. For k-NN, the Euclidean distance based algorithm is used and for SVM, one-versus-rest multiclass kernel-radial basis function is used. The performance evaluation of the classification results are obtained by calculating statistical parameters like specificity and sensitivity. ANN and k-NN techniques showed identical performance with specificity and sensitivity values of 100 and 86.76%, whereas SVM had these values at 100 and 80.18%, respectively. In order to determine the relative diagnostic performance of the techniques, receiver operating characteristics analysis is also performed.

Development and evaluation of fiber optic probe-based helium-neon low-level laser therapy system for tissue regeneration--an in vivo experimental study.

We report the design and development of an optical fiber probe-based Helium-Neon (He-Ne) low-level laser therapy system for tissue regeneration. Full thickness excision wounds on Swiss albino mice of diameter 15 mm were exposed to various laser doses of 1, 2, 3, 4, 6, 8 and 10 J cm(-2) of the system with appropriate controls, and 2 J cm(-2) showing optimum healing was selected. The treatment schedule for applying the selected laser dose was also standardized by irradiating the wounds at different postwounding times (0, 24 and 48 h). The tissue regeneration potential was evaluated by monitoring the progression of wound contraction and mean wound healing time along with the hydroxyproline and glucosamine estimation on wound ground tissues. The wounds exposed to 2 J cm(-2) immediately after wounding showed considerable contraction on days 5, 9, 12, 14, 16 and 19 of postirradiation compared with the controls and other treatment schedules, showing significant (P < 0.001) decrease in the healing time. A significant increase in hydroxyproline and glucosamine levels was observed for the 2 J cm(-2) irradiation group compared with the controls and other treatment groups. In conclusion, the wounds treated with 2 J cm(-2) immediately after the wounding show better healing compared with the controls.

Principal component analysis (PCA)-based k-nearest neighbor (k-NN) analysis of colonic mucosal tissue fluorescence spectra.

OBJECTIVE: The objective of this study was to verify the suitability of principal component analysis (PCA)-based k-nearest neighbor (k-NN) analysis for discriminating normal and malignant autofluorescence spectra of colonic mucosal tissues. BACKGROUND DATA: Autofluorescence spectroscopy, a noninvasive technique, has high specificity and sensitivity for discrimination of diseased and nondiseased colonic tissues. Previously, we assessed the efficacy of the technique on colonic data using PCA Match/No match and Artificial Neural Networks (ANNs) analyses. To improve the classification reliability, the present work was conducted using PCA-based k-NN analysis and was compared with previously obtained results. METHODS: A total of 115 fluorescence spectra (69 normal and 46 malignant) were recorded from 13 normal and 10 malignant colonic tissues with 325 nm pulsed laser excitation in the spectral region 350-600 nm in vitro. We applied PCA to extract the relevant information from the spectra and used a nonparametric k-NN analysis for classification. RESULTS: The normal and malignant spectra showed large variations in shape and intensity. Statistically significant differences were found between normal and malignant classes. The performance of the analysis was evaluated by calculating the statistical parameters specificity and sensitivity, which were found to be 100% and 91.3%, respectively. CONCLUSION: The results obtained in this study showed good discrimination between normal and malignant conditions using PCA-based k-NN analysis.

Serum protein profile study of normal and cervical cancer subjects by high performance liquid chromatography with laser-induced fluorescence.

High performance liquid chromatography with high sensitivity laser-induced fluorescence detection is used to study the protein profiles of serum samples from healthy volunteers and cervical cancer subjects. The protein profiles are subjected to principal component analysis (PCA). PCA shows that the large number of chromatograms of a given class of serum samples--say normal/malignant--can be expressed in terms of a small number of factors (principal components). Three parameters--scores of the factors, squared residuals, and Mahalanobis distance--are derived from PCA. The parameters are observed to have a narrow range for protein profiles of standard calibration sets formed from groups of clinically confirmed normal/malignant classes. Limit tests using match/no match of the parameters of any test sample with parameters derived for the standard calibration sets give very good discrimination between malignant and normal samples with high sensitivity (approximately 100%) aand specificity (approximately 94%).