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.