Laser Doppler spectroscopy as applied to electrophoresis in protein solutions

The application of Laser Doppler spectroscopy (LDS) to the electrophoretic migration of macromolecules in solution by heterodyne light beating technique, previously developed by Ware and Flygare, has been improved by the design of a new microelectrophoresis cell and a high resolution in the frequency power spectrum. A 1024-channel correlator was used in combination with a software-controlled Fast Fourier transformation (FFT).This technique has been applied to single protein solution, bovine serum albumin (BSA), and to multicomponent systems, in particular to human blood serum. In comparison to normal free electrophoresis, LDS is more convenient and reveals more information in a much shorter period of time.     

Laser Doppler fluxmetry

BACKGROUND: Laser Doppler fluxmetry (LDF) is an extraordinary sensitive noninvasive method of examination. It can be used for monitoring changes in the cutaneous peripheral microcirculation(15). It uses a monochromatic low-energy laser beam. This beam penetrates the tissue and, depending on individual tissue penetration, it is reflected, recorded by a sensitive sensor and subsequently analyzed using the Doppler. Laser Doppler fluxmetry detects movement of cells in the peripheral circulation and microcirculation. METHODS: The light from the laser source is delivered via optical fibers to the tissue. In the tissue the light collides with moving blood elements; after a collision, the wave length of the light changes--this phenomenon is called Doppler shift. Our own method of measurement has already been fully developed. We have a group of patients with physiological findings and also groups of patients with vasoneurosis, patients with type 1 diabetes mellitus, vasculitis patients, and other patient groups. We examine the blood flow on the dorsal side of fingers or toes. There are many protocols for laser Doppler examination. For all the patient groups we used a protocol evaluating an algorithm, developed in the training department of the Perimed company, using provocation tests for the assessment of the vascular wall function in addition to records at rest. USE OF LASER DOPPLER: We have developed a method of use of laser Doppler examination in about 8 years. We have examined several groups of patients. First a patient group with physiological findings was examined, and subsequently groups of patients with vasculitis, vasoneurosis, and a group of patients with type l diabetes mellitus. Recently, monitoring has been performed in patients after cardioversion in chronic atrial fibrillation with sinus rhythm restitution, and also data obtained before and after a varicose vein operation on lower limbs have been compared. CONCLUSION: Clear diagnostic criteria for this method do not exist so far. For the time being this is a rather theoretical method that we used even in practice for the above mentioned diseases. The method provides enough data even for more detailed analyses. The information value of the curve arises after statistical data analysis with the t-test.     

Euclidian space and grouping of biological objects

MOTIVATION: Biological objects tend to cluster into discrete groups. Objects within a group typically possess similar properties. It is important to have fast and efficient tools for grouping objects that result in biologically meaningful clusters. Protein sequences reflect biological diversity and offer an extraordinary variety of objects for polishing clustering strategies. Grouping of sequences should reflect their evolutionary history and their functional properties. Visualization of relationships between sequences is of no less importance. Tree-building methods are typically used for such visualization. An alternative concept to visualization is a multidimensional sequence space. In this space, proteins are defined as points and distances between the points reflect the relationships between the proteins. Such a space can also be a basis for model-based clustering strategies that typically produce results correlating better with biological properties of proteins. RESULTS: We developed an approach to classification of biological objects that combines evolutionary measures of their similarity with a model-based clustering procedure. We apply the methodology to amino acid sequences. On the first step, given a multiple sequence alignment, we estimate evolutionary distances between proteins measured in expected numbers of amino acid substitutions per site. These distances are additive and are suitable for evolutionary tree reconstruction. On the second step, we find the best fit approximation of the evolutionary distances by Euclidian distances and thus represent each protein by a point in a multidimensional space. The Euclidian space may be projected in two or three dimensions and the projections can be used to visualize relationships between proteins. On the third step, we find a non-parametric estimate of the probability density of the points and cluster the points that belong to the same local maximum of this density in a group. The number of groups is controlled by a sigma-parameter that determines the shape of the density estimate and the number of maxima in it. The grouping procedure outperforms commonly used methods such as UPGMA and single linkage clustering.     

Volume estimation of biological objects by systematic sections

The absolute volume of biological objects is often estimated stereologically from an exhaustive set of systematic sections. The usual volume estimator is the sum of the section contents times the distance between sections. For systematic sectioning with a random start, it has been recently shown that is unbiased when m, the ratio between projected object length and section distance, is an integer number (Cruz-Orive 1985). As this quantity is no integer in the real world, we have explored the properties of in the general and realistic situation m . The unbiasedness of under appropriate sampling conditions is demonstrated for the arbitrary compact set in 3 dimensions by a rigorous proof. Exploration of further properties of for the general triaxial ellipsoid leads to a new class of non-elementary real functions with common formal structure which we denote as np-functions. The relative mean square error (CE ²) of in ellipsoids is an oscillating differentiable np-function, which reduces to the known result CE ²= 1/(5m ⁴) for integer m. As a biological example the absolute volumes of 10 left cardiac ventricles and their internal cavities were estimated from systematic sections. Monte Carlo simulation of replicated systematic sectioning is shown to be improved by using m instead of m . In agreement with the geometric model of ellipsoids with some added shape irregularities, mean empirical CE was proportional to m ^–1.36 and m^–1.73 in the cardiac ventricle and its cavity. The considerable variance reduction by systematic sectioning is shown to be a geometric realization of the principle of antithetic variates.     

Ultrafast spectroscopy of biological photoreceptors

We review recent new insights on reaction dynamics of photoreceptors proteins gained from ultrafast spectroscopy. In Blue Light sensing Using FAD (BLUF) domains, a hydrogen-bond rearrangement around the flavin chromophore proceeds through a radical-pair mechanism, by which light-induced electron and proton transfer from the protein to flavin result in rotation of a conserved glutamine that switches the hydrogen bond network. Femtosecond infrared spectroscopy has shown that in photoactive yellow protein (PYP), breaking of a hydrogen bond that connects the p-coumaric acid chromophore to the backbone is crucial for trans-cis isomerization and successful entry into the photocycle. Furthermore, isomerization reactions of phycocyanobilin in phytochrome and retinal in the rhodopsins have been revealed in detail through application of femtosecond infrared and femtosecond-stimulated Raman spectroscopy.     

Laser Doppler imaging and cryoglobulinemia]

Continuous microcirculation monitoring of the right foot was carried out for the first time in a 62-year-old patient with cryoglobulinaemia prior to, during and after manual needle insertion at Jie Xi(St.41) acupuncture point using a new method of laser Doppler perfusion imaging (PIMII, Lisca AB, Link?ping, Sweden). In addition to visual inspection, changes in mean perfusion were also used as evaluation parameters. Our results suggest that the new biomedical technique of laser Doppler imaging is a useful method for monitoring the effects of acupuncture on the peripheral microcirculation.     

On a possible biological spectroscopy

A new type of physical transition, denotedS→S ^*, has been detected in irradiated organic molecules (λ=546 nm) through their interaction with specific biological macromolecules. In a specific enzyme-substrate interaction, a clear enhancement of the reaction rate is observed, when the substrate is irradiated with sharply well defined times. These “efficient irradiation times” are always of the 5k sec type (k=1, 2, 3, …). They have been consistently revealed in a great number of specific biological interactions. The present note demonstrates an important property, i.e. that forevery irradiation time aS→S ^* transition is induced in organic molecules. It is shown that for any irradiation times different from the 5k sec type (k=1, 2, 3, …) states of theS ^* type may occur, but the biological macromolecules may “detect” only theS ^* states induced by irradiations of the 5k sec type.     

Interactive visualization and exploration of relationships between biological objects

Genome sequencing and microarray technology produce ever-increasing amounts of complex data that need analysis. Visualization is an effective analytical technique that exploits the ability of the human brain to process large amounts of data. Here, we review traditional visualization methods based on clustering and tree representation, and also describe an alternative approach that involves projecting objects onto a Euclidean space in a way that reflects their structural or functional distances. Data are visualized without preclustering and can be dynamically explored by the user using ‘virtual-reality’. We illustrate this approach with two case studies from protein topology and gene expression.     

Device and programs for investigation of biological objects autofluorescence

A method of fluorescent sub-diffractional microscopy is proposed based on using object dyeing by special dye, which is able to bind to some object structures and initially not absorb light used for excitation of fluorescence. As a result of some processes small part of molecules is activated, that is converted to molecules able to absorb light and fluoresce sufficiently long to be registered by a sensitive video camera as separate spots covering tens of pixels of camera matrix. Then registered molecules must be photobleached or transformed to nonfluorescing state by other influence and new portion of molecules is activated. This circle of «activation» — «fluorescence excitation and registration» — «photobleaching» may be repeated thousands times. The accuracy of center spots determination may be up to 2 nm. A collection of all calculated centers of spots in all registered frames may be used for reconstruction of object image with resolution not limited by diffraction. A method of improving visibility of spots produced by single molecules in object having autofluorescence is described.     

Laser Raman spectroscopy of lyophilized bacterial spores

Laser-excited Raman spectra were examined in lyophilized spores of Bacillus cereus. In a comparison of the spectrum of the dormant spore with that of the germinated spore, we found several Raman bands which occurred in the former but not in the latter. Among these Raman bands, the 1,573, 1,395, 1,017, 822, and 662 cm-1 bands were assigned to the vibrational frequencies of calcium dipicolinate (CaDPA). No Raman bands and peaks due to dipicolinic acid (H2DPA) were observed. This Raman evidence indicates that CaDPA is the predominant DPA species in this spore. We also proposed a tentative assignment for other vibrational frequencies due to several components of the spore.