The dual nature of photons is well known but in spite of several years of work its foundation is not well understood. In
the present paper, a fresh approach is proposed according to which fields and particles are transformed into each other
and propagate together. This is explained by considering the field quantization process with the help of annihilation and
creation operators. Thus, at every point in space and time the sum of the energies associated with electric, magnetic
fields and photons is conserved. The present investigation supports the recently observed experimental data related with
sub wavelength interference and its regain by detecting photons with the help of the photon-echo process.
A Raman spectroscopic investigation of a genetically altered Human Embryonic Kidney Cell (HEK293) along with a
pathologically normal cell has been carried out by a conventional method. The genetic alteration was carried out with a
standard protocol by using a Green Fluorescence Protein (GFP). Raman spectra show that there are dramatic differences
between the spectrum obtained from a genetically altered cell and that obtained from a pathologically normal cell. The
former shows three broad bands; meanwhile the latter shows several sharp peaks corresponding to the ring vibrational
modes of Phen, GFP and DNA. The present analysis provides an indication that the force field near Phen located at 64,
65 and 66 was altered during the genetic transformation. The Raman spectrum could be a direct experimental evidence
for substantial modifications triggered due to the expression of specific genes.
Chondrocytes, obtained from testosterone treated human articular cartilage, were examined by a recently developed
Multiple Beam Interference Microscopy (MBIM) attached to a confocal set up, Video-enhanced differential interference
microphotography and also by cinematography. In the MBIM, the intensity of the transmitted pattern is given by the
Airy function which increases the contrast dramatically as the coefficient of the reflectance of the parallel plates
increases. Moreover, in this configuration, the beam passes several times through a specific organelle and increases its
optical path difference both because of the increase in the trajectory and refractive index (high density) of the organelle.
The improved contrast enhances the resolving power of the system and makes visible several structural details of sub
micron dimensions like nucleolus, retraction fibers, podia, etc. which are not possible to reveal with such a clarity by
conventional techniques such as bright field, phase contrast or DIC. This technique permits to detect the oscillatory and
rotational motions of unstained cilia for the first time. The frequency of oscillations was found to be 0.8 Hz.
Raman spectroscopy is a powerful technique as it provides fundamental information about vibrational modes of a system. Eigenvalues of these modes are very sensitive to the strength of the chemical bonds and perturbations caused by the environment, particularly charge distribution and alterations in the dipole strength of the system. All these parameters are profoundly modified during the tumor formation process nad hence Raman technique could be a unique and also a direct approach for evaluating tumor genesis at early stages. for this pupose the present investigation was carried out.
We used cultured wild type and c-ras transformed NIH 3T3 fibroblast. The samples were treated with methyl alcohol solution ina conventional manner and then Raman spectra nad images were obtained by a specially developed confocal Raman microscope. The present results reveal differences between both cell types in the spectral details as well as in the morphology. Raman images are able to detect the exact site where cancer-related changes have taken place.
These results clearly indicate the superiority of the present technique over conventional methods such as images obtained by X-rays or Nuclear Magnetic Resonance (NMR). Moreover, unlike other approaches, Raman images detect alterations at the submicron level rather than in the centimeter or millimeter range. Being an optical method it can be applied in vivo as a non-invasive technique potentially useful to early detection of cancer (particularly easy accessible cancers such as those of the skin and the digestive tract). The obtained resulsts suggest the great potential of Raman imaging in premature clinical diagnostic approaches.
A recently developed multiple beam interference confocal microscopy and conventional confocal microscopic techniques were employed to obtain 3-D images and morphological details of chondrocytes obtained from a postmortem of 1 day old baby for the first time. Unprecedented images clearly showed that they were circular and internal structures were noticeably visible. The nucleus shows two or three nucleoids. Peripheral nuclear organelles were also perceptible. These techniques permit to examine with precision the thickness of the organelles.
Dictyosphaerium puichellum Wood, a typical a fresh water alga, is known for photosynthesis and expected to have photoluminescence also. We have, therefore, carried out a confocal fluorescence microscopic and spectroscopic study of this species. 3-D image constructed from 50 optical sections clearly reveals that fluorescence is originated from only the outer layer and not from the central core. Spectral investigation shows that a strong and sharp peak is located at 684 nm. At this peak value, the power measurement was carried out and the number of photons emitted by alga in the solid angle 48.59 f was calculated and found to be 3.4*1010/ sec. The other significant feature of the responsible fluorophore is that the present photoluminescence has a very low quenching rate and hence the emitted radiation is observed for a very long time (5 12 sec.). This directly suggests that this fluorophore could have direct applications in dye related industries.
Reliable and economical Human Immnodeficiency Virus (HIV) testing was achieved by optical absorption spectroscopy of the core of the hair in the range of 400 nm to 800 nm. In HIV+ patients, extra optical active material is deposited in the core and optical absorption spectra, recorded in polarized radiation reveal special features, which can be used for guidance, detection, monitoring and control.
An IR spectroscopic study was carried out at room temperature for Human Serum albumin (HSA) glycated with fructose and glucose and inhibited with acetylsalicylic acid. The glycation process was carried out in our laboratory by a conventional method to confirm earlier reported observation of the effect of glycation on the intensity variation of the IR spectra, particularly, in the range 1500 cm-1 to 1700 cm-1 and around 3300 cm-1. IR spectra reveal that the effects of glycation of HSA by fructose are more intense than with glucose, which is the expected. Bovine serum albumin was also glycated using Glucose-6-phosphate disodium salt, and gamma-globulin was glycate with glucose, As expected, the glycation process was more intense with glucose-t-phosphate disodium salt. Acetyl salicylic acid was also used and its inhibitor effects could be observed in both cases, with glucose and with glucose-6-phosphate disodium salt even though, to a smaller extent with the latter. This is consistent with the earlier data and is explained on the basis of the attachment of macromolecules to (epsilon) -NH2 groups of lysines. The experimental results confirm that acetylsalicylic acid, indeed, acts as an inhibitor by acetylation of the (epsilon) -NG2 group where the sugars are supposed to be attached.
Multiple beam interference system is used in conjunction with a conventional scanning confocal microscope to examine the morphology and construction of 3D images of Histolytic Ameba and parasite Candida Albicans. The present combination permits to adjoin advantages of both systems, namely the vertical high contrast and optical sectioning. The interference pattern obtained from a multiple internal reflection of a simple, sandwiched between the glass plate and the cover plate, was focussed on an objective of a scanning confocal microscope. According to optical path differences, morphological details were revealed. The combined features, namely improved resolution in z axis, originated from the interference pattern and the optical sectioning of the confocal scanning system, enhance the resolution and contrast dramatically. These features permitted to obtain unprecedented images of Histolytic Ameba and parasite Candida Albicans. Because of the improved contrast, several details like double wall structure of candida, internal structure of ameba are clearly visible.
KEYWORDS: Atomic force microscope, Atomic force microscopy, Proteins, Nanoimaging, 3D image processing, Electron microscopy, Tissues, Blood, Imaging systems, 3D vision
Nan-imaging of Entamoeba histolytica was carried out by using Atomic Force Microscope (AFM). The structure of the nucleus, endoplasm and ectoplasm were studied separately. The diameter of the nucleus in living E. histolytica was found to be of the order of 10 micrometers which is slightly higher than the earlier reported value. The presence of karysome was detected in the nucleus. Well-organized patterns of chromatoid bodies located within the endoplasm, were detected and their repetitive patterns were examined. The organized structure was also extended within the ectoplasm. The dimensions and form of the organization suggest that chromatic bodies are constituted with ribosomes ordered in the form of folded sheet. Such structures were found to be absent in non-living E. histolytica. AFM images were also captured just in the act when ameba was extending its pseudopods. Alteration in the ultrastructure caused during the process of extension was viewed. Well marked canals of width 694.05 nm. And height 211.05 nm are clearly perceptible towards the direction of the pseudopods. 3D images are presented to appreciate the height variation, which can not be achieved by conventional well-established techniques such as electron microscopy.
Glucose is known to be an optically active material and therefore in cross polarized light, a bright color will be visualized against a dark background. In non enzymatic glycation glucose is attached to (epsilon) -NH2 group of Lysine residues of proteins and therefore an irreversible alteration takes place. Glucose molecules along with proteins are deposited in the core of the hair in the growth process and when it is examined under cross polarized radiation bright colored patches, depending upon the thickness of glucose conglomerate, are expected. With this view, we have examined 25 diabetic patients and we have observed the presence of such patches. In controlled cases, these patches were absent provided that the hairs had not received any chemical or heat treatment. If properly exploited, this could be a very useful non-invasive diagnostic tool or guidance for diabetes related phenomena. Moreover, the proteins deposited in the hair core are not altered and are preserved for a long period and hence, it could reveal the history of the patient in relation with his glucose metabolism.
Nonenzymatic glycation, also known as Maillard reaction, plays an important role in the secondary complications of the diabetic pathology and aging, therefore, human serum albumin (HSA) and bovine serum albumin (BSA) were glycated by a conventional method in our laboratory using glucose as the glycating agent. Fluorescence lifetime measurements were carried out with a laser strobe fluorometer equipped with a nitrogen/dye laser and a frequency doubler as a pulsed excitation source. The samples were excited at 295 nm and the emission spectra were recorded at 345 nm. The obtained decay curves were tried for double and triple exponential functions. It has been found that the shorter lifetime increases for glycated proteins as compared with that of the native ones. For example, in the case of glycated BSA the lifetime increased from 1.36 ns to 2.30 ns. Similarly, for HSA, the lifetime increases from 1.58 ns to 2.26 ns. Meanwhile, the longer lifetime changed very slightly for both proteins (from 6.52 ns to 6.72 ns). The increase in the lifetime can be associated with the environmental effect; originated from the attachment of glucose to some lysine residues. A good example is Trp 214 which is in the cage of Lys 225, Lys 212, Lys 233, Lys 205, Lys 500, Lys 199 and Lys 195. If fluorescence lifetime technique is calibrated and properly used it could be employed for assessing glycation of proteins.
Detection of nonenzymatic glycated proteins is a very significant feature in diabetes, aging and related diseases, therefore we have carried out an FTIR spectroscopic study for glycated and native proteins such as (gamma) -globulin, human serum albumin. For this purpose, commercially available proteins were glycated by a usual procedure and their FTIR spectra were recorded together with that of the native ones. In order to follow the changes in time, (gamma) -globulin was glycated during 1, 2, 3, 5 and 8 weeks and their spectra were recorded. Direct verification was obtained by examining a model unit where the -NH2 group was attached to glucose. The spectrum shows a strong peak at 3500 cm-1 confirming the observed variation in time dependent spectra. The general features of the spectra are very similar and there was no additional structure or change in the peaks. This is understandable as not all the lysine residues are glycated, only a small fraction. Glucose is attached to the (epsilon) -amino group of lysine to form Amadori products, and therefore, the vibrational modes corresponding to the (epsilon) -NH2 unit of lysine are expected to be altered. This region exactly lies in the Amide I region of protein structure. Careful investigation of this part, indeed, shows a complex structure originated from alternations of -NH2 group. Thus, the present investigation indicates that an optical approach could be a rapid and effective method to identify the nonenzymatic glycation process.
The Chediak-Higashi syndrome (CHS) is a rare disease and is very often observed in a single family. In Venezuelan Los Andes area, its appearance is not uncommon. One of the peculiar aspects of CHS patients is the color, texture and structure of the hairs. We have, therefore, investigated structure, morphology and composition of hairs and compared those properties with that of normal human subject grown in the same geographical region. A few hairs were cut along its length and their fluorescence was investigated. For CHS patients, dark patches were observed. Investigations of chemical compositions of these regions were carried out of using Energy Dispersive Analysis and it was found the presence of Na, K and Ca ions. Fluorescence spectral studies were carried out at room temperature. Observed spectra reveal two peaks, however, those were not useful for positive identification of CHS.
Albumin is an important protein and its glycated form increases in diabetes mellitus and is partly responsible for the age related phenomena. We have, therefore, investigated the optical absorption and fluorescence spectra of glycated albumin along with a kinetic study by means of a proteolytic reaction. For this purpose, albumin was glycated by a conventional method and the optical absorption spectra were recorded for both the glycated and the native forms, from 240 nm to 400 nm and found that they were dominated by a strong peak at 280 nm which was followed by a long tail up to 300 nm for the native and 400 nm for the glycated protein. Non tryptophane fluorescence spectra of both glycated and native albumins in solid form were investigated and it was found that the general features of both spectra are similar. The spectra are dominated by a weak peak located at 450 nm and an intense broad peak at 550 nm. This peak is followed by a shoulder at 625 nm and then a long tail up to 800 nm. One of the major effects of glycation in this investigation is in their physical forms. Native albumin has a crystalline form while the glycated one has a conglomerate form. This effect can be easily visualized when the sample is examined with the help of a fluorescence microscope. This result is in accordance with the data obtained from electron microscopy. Thus, this quick and direct approach could identify the presence of the glycated protein and could provide information on the pathology of diabetes art an early stage.
Spectral investigations of absorbance in deep ultra-violet region (from 200 nm to 350 nm) of (STM) was carried out for different pH values. On the high energy side the peak is located at 195 nm which is generally attributed to peptide bonds. This peak, as expected, does not show any shift with pH value (4.3 to 10.8). A rather broad peak is spread in the region from 200 nm to 240 nm which could be the superposition of the peaks corresponding to the absorption due to (alpha) helix and (beta) structure. This peak shows a red shift as pH value increases. The same hormone was glycated by a conventional method and the process was estimated with the absorption spectra. The results are discussed in the light of nonenzymatic glycation. It was found that glycation mucus somatotropin resistant towards the denaturation process.
A conventional collinear laser induced fluorescence detection system for capillary electrophoresis was modified by replacing a GaAs photomultiplier by a Charge coupled Device which was operated at liquid nitrogen temperature. This permits longer exposure time to collect the weak radiation and hence extends the limit of detection and spectral analysis of lower concentration of biological active molecules. Using the above technique, and passing the solution through a capillary of 20 micrometers inside diameter, we have recorded fluorescence spectra of biological important molecules such as Arginine, Glutamate and Glutamine; all of them were derivatized with Fluorescein Isothiocyanate (FITC) isomer I. Spectra of FITC as a function of the pH were examined to lower the detection limit. The concentration of the molecules was as low as 10-14 M. All the spectra recorded lied in the expected region (510 nm to 550 nm) and showed considerable similarity. The spectrum of low concentration of molecules provides significant information as the intensity of intra-molecular interaction is reduced. The recorded spectra gave information about the distribution of vibrational states near S0 and S1 levels.
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