Computer based colour intensity measurement of plant anatomical sections for proximate chemical analysis.

Abstract

The aim and objectives of this study work is to evolve and develop a low cost method for chemical analysis. This method will be highly useful for the chemist, biochemist and botanist for proximal chemical analysis. Microscope is one of the most common techniques used for the investigation of histological and biological material and plant sectioning and staining it is an old method of research in diagnostic field. Anatomical sections provide us more information about the tissues or cells but differentiations and quantification of different compounds are very difficult or one can say that it is impossible but now a day with the help of Microscope, Camera and Computer software one can see 100 times larger image and also observe or quantify them easily. But Computational biology is utterly incomplete without microscopic section staining because computer software differentiates or quantifies to the specific compound on the basis of their colour, so for this purpose different dyes/stains were used for specific area or molecule. In the present research work, first of all proteins were determined from three plants by different reported methods, after the confirmation of protein concentration, same plant’s section were stained with two protein dyes and then were analyzed by own developed computer software that is ABAS. Stains or dyes are Coomassie Brilliant blue (CBB dye) and Lawsone dye (Ls dye). Coomassie stain is well known protein dye and Lawsone dye is also protein dye because it is commonly used for dying hairs, nails and skin proteins internationally. In this research concentration of Lawsone dye was observed in whole and powdered leaves of Henna (Lawsonia inermis) extractions at different time intervals i.e 1hr, 2hrs, 3hrs, 4hrs, 5hrs, 24hrs, 48hrs and temperatures i.e 10oC, 20oC, 30oC and 40oC at different concentration (at 452nm) for staining purpose and pH. In this work one hour of powdered leaves extraction was used for the staining of anatomical sections of three plants. Siris (Albizia lebbbeck) is widely available on the campus of Sindh University, so first of all experimental work was started with the above plants followed by two other plants i.e Pea (Pisum sativum ) and Gram (Cicer arietinum) which were grown at the research plot, Institute of Plant Sciences, University of Sindh. Protein was analyzed from rachis of Siris by Lowry’s and Bradford’s quantitative method, molecular weight of protein was determined by Electrophoresis and staining pH of Lawsone dye assisted by paper chromatography. Sections of rachis of Siris were 3stained in CBB dye for different time intervals at room temperatures and then same plant sections were stained with 2% pure Lawsone dye (Ls dye) and 2% Henna powdered leaves extraction for one hour (Hple1) at different time of intervals i.e 30min, 1hr, 2hrs, 3hrs & 24hrs and temperatures i.e 40oC, 50oC, 60oC, 70oC, and 80oC. All of these sections were observed under microscope which was connected to computer through USB cable and saved all section’s photos (2D images) in computer memory for the assessment of stained image colour intensity by ABAS computer software, after confirmation of protein by computer software, protein concentration were observed at three stages of growth i.e 1 st , 2 nd and 3 rd of the stem portion of both plants i.e Pea & Gram. Protein concentration in both plants were also analyzed by Lowry and Bradford quantitative methods and Electrophoresis and paper chromatography were also done, after that both plants stem sections were stained with CBB dye, Ls dye and Hple1 at different time intervals followed by analysis by ABAS computer software. With the help of these staining methods we can easily quantify even a small quantity of protein and in future we may identify the type of protein at different growth stages of plants, this method can also be applied on plants for quantifying other compounds e.g Alkaloids but for that purpose we need specific dye and desired software. For full and semi automation of microscope, we need different hardwares (Motors) and computer software. Semi automated microscope performed more than one task by computer and others were performed by human. In this research work, Vertical and Horizontal movement of stage of microscope (were observed by putting slide of section/image) controlled with the help of Stepper motor and computer programming in Visual Basic6 (VB6). For the 2D image analysis, ABAS image processing software was developed in Visual Basic. Net frame work (C#.net; pronounced C sharp dot net and Visual Basic.net; pronounced VB.net) computer language. Stained and unstained 2D images of three plants section determined by histograms (8-bit) and on the basis of their changes in staining colour, intensities of colour of unstained & stained plant section (2 images) was determine then protein content was analyzed with the help of formula and compared with each other.