Perception of temporally interleaved ambiguous patterns

BACKGROUND: Continuous viewing of ambiguous patterns is characterized by wavering perception that alternates between two or more equally valid visual solutions. However, when such patterns are viewed intermittently, either by repetitive presentation or by periodic closing of the eyes, perception can become locked or "frozen" in one configuration for several minutes at a time. One aspect of this stabilization is the possible existence of a perceptual memory that persists during periods in which the ambiguous stimulus is absent. Here, we use a novel paradigm of temporally interleaved ambiguous stimuli to explore the nature of this memory, with particular regard to its potential impact on perceptual organization. RESULTS: We found that the persistence of a perceptual configuration was robust to interposed visual patterns, and, further, that at least three ambiguous patterns, when interleaved in time, could undergo parallel, stable time courses. Then, using an interleaved presentation paradigm, we established that the occasional reversal in one pattern could be coupled with that of its interleaved counterpart, and that this coupling was a function of the structural similarity between the patterns. CONCLUSIONS: We postulate that the stabilization observed with repetitive presentation of ambiguous patterns can be at least partially accounted for by processes that retain a recent perceptual interpretation, and we speculate that such memory may be important in natural vision. We further propose that the interleaved paradigm introduced here may be of great value to gauge aspects of stimulus similarity that appeal to particular mechanisms of perceptual organization.     

Structured illumination microscopy with interleaved reconstruction (SIMILR)

Structured illumination microscopy (SIM) is the commonly used super‐resolution (SR) technique for imaging subcellular dynamics. However, due to its need for multiple illumination patterns, the frame rate is just a fraction of that of conventional microscopy and is thus too slow for fast dynamic studies. A new SR image reconstruction method that maximizes the use of each subframe of the acquisition series is proposed for improving the super‐resolved frame rate by N times for N illumination directions. The method requires no changes in raw data and is appropriate for many versions of SIM setup, including those implementing fast illumination pattern generation mechanism based on spatial light modulator or digital micromirror device. The performance of the proposed method is demonstrated through imaging the highly dynamic endoplasmic reticulum where continuous rapid growths or shape changes of tiny structures are observed.      

Pulsed EPR spectroscopy

Photosynthesis Research -     

Pulsed field electrophoresis

Pulsed field electrophoresis, or PFE, provides good separation between large molecules that under constant field electrophoresis are hard to isolate. This is due to the weak dependence of the constant field mobility on the molecular weight for these large molecules. If a spectrum of relaxation times exists that describes the recovery of the mobility to its constant field value after a reversal of the field, then we show that molecules with differing molecular weights are separated into two groups. Those with short relaxation times are unaffected by the cycling of the field and those with long relaxation times exhibit reduced mobilities. If the molecules adopt conformations that decrease their mobility initially, after a field reversal we demonstrate that a minimum develops in the mobility as a junction of the relaxation time. Using the model we demonstrate that effects of varying the switching times as a function of time. We predict that exponential rather than linear dependencies of the switching times on time increase the range of molecular weights over which enhanced separation can occur.     

Pulsed cytochrome c oxidase

The identification of two functionally distinct states, called pulsed and resting, has led to a number of investigations on the conformational variants of the enzyme. However, the catalytic properties of cytochrome oxidase may depend on a number of experimental conditions related to the solvent as well as to the protocol followed to determine the turnover number of the enzyme. This paper reports results which illustrate that the steady-state differences between pulsed and resting oxidase may, or may not, be detected depending on experimental conditions.     

Multi-photon excitation microscopy

Multi-photon excitation (MPE) microscopy plays a growing role among microscopical techniques utilized for studying biological matter. In conjunction with confocal microscopy it can be considered the imaging workhorse of life science laboratories. Its roots can be found in a fundamental work written by Maria Goeppert Mayer more than 70 years ago. Nowadays, 2PE and MPE microscopes are expected to increase their impact in areas such biotechnology, neurobiology, embryology, tissue engineering, materials science where imaging can be coupled to the possibility of using the microscopes in an active way, too. As well, 2PE implementations in noninvasive optical bioscopy or laser-based treatments point out to the relevance in clinical applications. Here we report about some basic aspects related to the phenomenon, implications in three-dimensional imaging microscopy, practical aspects related to design and realization of MPE microscopes, and we only give a list of potential applications and variations on the theme in order to offer a starting point for advancing new applications and developments.     

High-speed volumetric imaging in vivo based on structured illumination microscopy with interleaved reconstruction

Wide-field fluorescence microscopy (WFFM) is widely adopted in biomedical studies, due to its high imaging speed over large field-of-views. However, WFFM is susceptible to out-of-focus background. To overcome this problem, structured illumination microscopy (SIM) was proposed as a wide-field, optical-sectioning technique, which needs multiple raw images for image reconstruction and thus has a lower imaging speed. Here we propose SIM with interleaved reconstruction, to make SIM of lossless speed. We apply this method in volumetric imaging of neural network dynamics in brains of zebrafish larva in vivo.     

Pulsed Interleaved Excitation Fluctuation Imaging

Fluorescence fluctuation imaging is a powerful means to investigate dynamics, interactions, and stoichiometry of proteins inside living cells. Pulsed interleaved excitation (PIE) is the method of nanosecond alternating excitation with time-resolved detection and allows accurate, independent, and quasi-simultaneous determination of fluorescence intensities and lifetimes of different fluorophores. In this work, we combine pulsed interleaved excitation with fluctuation imaging methods (PIE-FI) such as raster image correlation spectroscopy (RICS) or number and brightness analysis (N&B). More specifically, we show that quantitative measurements of diffusion and molecular brightness of Venus fluorescent protein (FP) can be performed in solution with PIE-RICS and compare PIE-RICS with single-point PIE-FCS measurements. We discuss the advantages of cross-talk free dual-color PIE-RICS and illustrate its proficiency by quantitatively comparing two commonly used FP pairs for dual-color microscopy, eGFP/mCherry and mVenus/mCherry. For N&B analysis, we implement dead-time correction to the PIE-FI data analysis to allow accurate molecular brightness determination with PIE-NB. We then use PIE-NB to investigate the effect of eGFP tandem oligomerization on the intracellular maturation efficiency of the fluorophore. Finally, we explore the possibilities of using the available fluorescence lifetime information in PIE-FI experiments. We perform lifetime-based weighting of confocal images, allowing us to quantitatively determine molecular concentrations from 100 nM down to <30 pM with PIE-raster lifetime image correlation spectroscopy (RLICS). We use the fluorescence lifetime information to perform a robust dual-color lifetime-based FRET analysis of tandem fluorescent protein dimers. Lastly, we investigate the use of dual-color RLICS to resolve codiffusing FRET species from non-FRET species in cells. The enhanced capabilities and quantitative results provided by PIE-FI make it a powerful method that is broadly applicable to a large number of interesting biophysical studies.     

Pulsed optoacoustic spectroscopy of NADH

Optoacoustic spectroscopy is a potentially powerful tool for the determination of trace and ultratrace components of biochemical interest. We derive here an abbreviated theory of pulsed optoacoustic spectroscopy, describe the experimental apparatus, and report on the performance in the determination of NADH. Results are consistent with theory over the concentration range 1 × 10^?5 to 5 × 10^?7, m. A projected lower limit for detection is 1 × 10^?8, m. Possible applications to the study of nucleotide interactions are discussed.     

Pulsed Interleaved Excitation Fluctuation Imaging

Fluorescence fluctuation imaging is a powerful means to investigate dynamics, interactions, and stoichiometry of proteins inside living cells. Pulsed interleaved excitation (PIE) is the method of nanosecond alternating excitation with time-resolved detection and allows accurate, independent, and quasi-simultaneous determination of fluorescence intensities and lifetimes of different fluorophores. In this work, we combine pulsed interleaved excitation with fluctuation imaging methods (PIE-FI) such as raster image correlation spectroscopy (RICS) or number and brightness analysis (N&B). More specifically, we show that quantitative measurements of diffusion and molecular brightness of Venus fluorescent protein (FP) can be performed in solution with PIE-RICS and compare PIE-RICS with single-point PIE-FCS measurements. We discuss the advantages of cross-talk free dual-color PIE-RICS and illustrate its proficiency by quantitatively comparing two commonly used FP pairs for dual-color microscopy, eGFP/mCherry and mVenus/mCherry. For N&B analysis, we implement dead-time correction to the PIE-FI data analysis to allow accurate molecular brightness determination with PIE-NB. We then use PIE-NB to investigate the effect of eGFP tandem oligomerization on the intracellular maturation efficiency of the fluorophore. Finally, we explore the possibilities of using the available fluorescence lifetime information in PIE-FI experiments. We perform lifetime-based weighting of confocal images, allowing us to quantitatively determine molecular concentrations from 100 nM down to <30 pM with PIE-raster lifetime image correlation spectroscopy (RLICS). We use the fluorescence lifetime information to perform a robust dual-color lifetime-based FRET analysis of tandem fluorescent protein dimers. Lastly, we investigate the use of dual-color RLICS to resolve codiffusing FRET species from non-FRET species in cells. The enhanced capabilities and quantitative results provided by PIE-FI make it a powerful method that is broadly applicable to a large number of interesting biophysical studies.     

Pulsed feedback defers cellular differentiation

Environmental signals induce diverse cellular differentiation programs. In certain systems, cells defer differentiation for extended time periods after the signal appears, proliferating through multiple rounds of cell division before committing to a new fate. How can cells set a deferral time much longer than the cell cycle? Here we study Bacillus subtilis cells that respond to sudden nutrient limitation with multiple rounds of growth and division before differentiating into spores. A well-characterized genetic circuit controls the concentration and phosphorylation of the master regulator Spo0A, which rises to a critical concentration to initiate sporulation. However, it remains unclear how this circuit enables cells to defer sporulation for multiple cell cycles. Using quantitative time-lapse fluorescence microscopy of Spo0A dynamics in individual cells, we observed pulses of Spo0A phosphorylation at a characteristic cell cycle phase. Pulse amplitudes grew systematically and cell-autonomously over multiple cell cycles leading up to sporulation. This pulse growth required a key positive feedback loop involving the sporulation kinases, without which the deferral of sporulation became ultrasensitive to kinase expression. Thus, deferral is controlled by a pulsed positive feedback loop in which kinase expression is activated by pulses of Spo0A phosphorylation. This pulsed positive feedback architecture provides a more robust mechanism for setting deferral times than constitutive kinase expression. Finally, using mathematical modeling, we show how pulsing and time delays together enable “polyphasic” positive feedback, in which different parts of a feedback loop are active at different times. Polyphasic feedback can enable more accurate tuning of long deferral times. Together, these results suggest that Bacillus subtilis uses a pulsed positive feedback loop to implement a “timer” that operates over timescales much longer than a cell cycle.     

Note on anodal excitation

A general, necessary and sufficient condition for the occurrence of anodal excitation is given on the basis of Rashevsky's two-factor theory of excitation, expressions for the anodal rheobase and the minimal shock intensity following as simple special cases.     

Biophysics of nerve excitation

Experimental studies testifying to the presence of an interrelation between the physiological functions of the organism and physical and chemical processes in nerves are discussed. Changes in some physical and chemical parameters observed both upon elicited rhythmic excitation of nerves and during the spontaneous rhythmic activity of neurons are analyzed. Upon rhythmic excitation, a complex of physical and chemical processes is triggered, and reversible structural and metabolic rearrangements at the subcellular and molecular levels occur that do not take place during the generation of a single action potential. Thus, only in conditions of rhythmic excitation of a nerve, it is possible to reveal those processes that provide excitation of nerves in the organism. The future possibilities of the investigations combining the biophysical and physiological approaches are substantiated. Characteristic changes in physicochemical parameters are observed in nerves during the generation of a series of action potentials of different frequency and duration (“frequency dependence”) under normal physiological conditions, as well as in extreme situations and in nerve pathology. The structural and metabolic rearrangements are directly related to the mode of rhythmic excitation and proceed both in the course of rhythmic excitation and after its termination. Shown also is participation of the basic components of the nervous trunk (axon, Schwann cell, myelin, subcellular organelles) in the realization of rhythmic excitation. In the coordination of all processes involved in rhythmic excitation, the main role is played by the systems of redistribution and transport of intercellular and intracellular calcium. The idea is put forward that myelin of nerve fibers is not only an insulator, but also an “intercellular depot” of calcium and participates in the redistribution of different ions. Thus, the rhythmic excitation is of great importance in the realization of some physiological functions, the adaptation to changing conditions, the liquidation of consequences of paralogical processes, the formation of mechanisms of “memory,” etc.     

Dual-photon excitation of albumin

Fluorescence emission after two-photon excitation at 580 nm is observed in albumin by means of Nd:YAG laser at room temperature. The two-photon excitation spectral range 550-590 nm was obtained. The experimental results show that albumin fluorescence originates from tryptophan residues.