Chemical signaling is essential to information processing in the brain. The key events of this process in the mammalian brain are the exocytotic release of signaling molecules by one cell and its binding to a receptor molecule on the recipient cell, which then transduces the chemical message. Here we ask: if we take the receptors away, can there still be signaling?
We find that the lipid bilayer responds to some signaling molecules, such as serotonin, and changes its order and mechanical properties. We then combine non-epifluorescence multiphoton UV with spectral confocal microscopy and show that these changes modulate membrane-mediated processes such as exo- and endocytosis. It also affects the functioning of non-cognate receptor proteins in the membrane. Nature appears to have optimized the membrane compositions of different organelles to tune them for neurotransmitter interactions. In summary, the lipid bilayer membrane can itself be a receptor for many signaling molecules. Potentially, our findings pave the way for a major new class of membrane-active but receptor-silent pharmacological agents that can affect biological function.
Oligomers of disease-causing amyloid proteins (such as Alzheimer’s Amyloid beta or ’Aβ’) are generally amphiphilic and their interactions with lipid membranes are possibly the origin of their toxicity. However, how oligomers of different stoichiometries or different mutants differ in their interaction with the membrane, and how these differences correlate with their toxicity, has largely remained beyond the reach of existing experimental techniques. Here we use Q-SLIP, a single-molecule tool that can resolve the surface exposure of different parts of individual oligomers, and different radical-labeled lipids that act as quenchers, to address these questions.
Small oligomers are widely accepted to be the major toxic agents in amyloid diseases. However, it is difficult to decipher their structure or conformation. Small oligomers of different sizes are in dynamic equilibrium with each other, and are therefore not amenable to separation techniques. Single molecule approaches can study individual members of an ensemble without separating them. Here we apply a single molecule photobleaching technique, called Q-SLIP (quencher induced step length increase in photobleaching), which allows us to probe the arrangement of monomers in human Islet Amyloid Poly Peptide (hIAPP, or simply IAPP) oligomers. IAPP oligomers are the toxic species associated with Type II diabetes, and understanding the monomer-wise arrangement of membraneattached IAPP oligomers is crucial for understanding the toxic mechanism. QSLIP probes the accessibility of fluorophore labels in each monomer of individual membrane-attached IAPP oligomers. We show that the arrangement is far from uniform. When two monomers form a dimer, the Nterminus of the second monomer gets buried near the middle of the bilayer (which has a doxyl quencher at the 16’ position of the lipid chain). We study oligomers until the tetramer, and our results show that the sequential growth of the oligomers produces a structure very different from a tetrameric subunit of a mature fibril. This may explain the difference in toxicity between the oligomeric species and the fibrils.
Deconvolution, pixel reassignment or adaptive optics-based strategies utilize information about the detection profile in improving the resolution of optical microscopy. Here, we show a novel method which allows us to obtain the single-photon detection volume of a laser scanning confocal microscope at any desired location of the object. It can create a stationary, virtual ‘guide star’ at the chosen location while the excitation beam is scanning the sample, by using an optical fiber placed in the non-descanned path of the microscope. Our experimental results are verified by diffraction theory-based calculations. The major advantages of our method are that it is alignment free, affordable, sensitive and applicable to many different modes of confocal imaging.
While dozens of human ailments are now identified as "protein aggregation diseases", aggregation by itself does not
seem to be a clear determinant of the toxicity. The structural transformation that accompanies the initial steps of
aggregation may be an even more important aspect controlling the biological effects of these protein particles. For this,
the key is to develop appropriate fluorescent biomarkers which can probe both aggregation and conformation at low
physiological concentrations. Using Alzheimer's amyloid beta (Aβ) as a model system, we have developed probes
suitable for the application of Fluorescence Correlation Spectroscopy (FCS, which reports aggregation) and Förster
Resonance Energy Transfer (FRET, which reports conformational changes) techniques. To diagnose these changes in
the cerebrospinal fluid of Alzheimer's patients, we are now designing better single molecule detection devices. Here we
report a confocal device with a 4π collection geometry, which detects more than 0.5 million photons per second from a
single rhodamine B molecule in aqueous solution, which to our knowledge is the highest sensitivity achieved so far
with such devices. This allows us to perform quick and sensitive antibunching measurements which report the
aggregate mass and fluorophore lifetime of Aβ oligomers.
Instruments with single-molecule level detection capabilities can potentially benefit a wide variety of fields, including medical diagnostics. However, the size, cost, and complexity of such devices have prevented their widespread use outside sophisticated research laboratories. Fiber-only devices have recently been suggested as smaller and simpler alternatives, but thus far, they have lacked the resolution and sensitivity of a full-fledged system, and accurate alignment remains a critical requirement. Here we show that through-space reciprocal optical coupling between a fiber and a microscope objective, combined with wavelength division multiplexing in optical fibers, allows a drastic reduction of the size and complexity of such an instrument while retaining its resolution. We demonstrate a 4×4×18 cm3 sized fluorescence correlation spectrometer, which requires no alignment, can analyze kinetics at the single-molecule level, and has an optical resolution similar to that of much larger microscope based devices. The sensitivity can also be similar in principle, though in practice it is limited by the large background fluorescence of the commonly available optical fibers. We propose this as a portable and field deployable single molecule device with practical diagnostic applications.
Coupling three-photon microscopy with automated stage movement can now produce a live high resolution
map of the neurotransmitter serotonin in a single cross section of the whole rat brain. Accurate quantification of these
serotonin images demands appropriate spectral filtering. This requires one to consider that the spectral characteristics of
serotonin show a remarkable variation as it non-covalently associates with different molecules, as we discuss here. Also
it is known that serotonin emission changes when it forms a covalent adduct with para-formaldehyde. This provides a
potential route for producing a whole brain serotonin map using multiphoton microscopy in a fixed rat brain. Here we
take the initial step showing that multiphoton microscopy of this adduct can quantitatively image chemically induced
changes in serotonin distribution.
Two-photon excitation can excite physiological ultraviolet (UV) chromophores at visible wavelengths and holds significance for applications such as non-linear microscopy, micro-pharmacology, localized heating and tissue ablation. However, little quantitative data is available on the absolute two-photon absorption cross sections ((sigma) 2) of these molecules. Their low two-photon absorbance and limited solubility implies that the sensitivity required for such measurements is much higher than that available from standard techniques. We employ the recently developed generalized z-scan technique to measure the absolute value of the (sigma) 2 of tryptophan, the most ubiquitous of the physiological near-UV chromophores, and obtain the value of 32.0 +/- 1.2 mGM (1 GM equals 10-50 cm4sec/photon/molecule) at 532 nm. This is the first determination of the absolute (sigma) 2 of any biological UV chromophore, and can be used to calibrate the previously reported relative two-photon excitation spectra of a number of such molecules.
We derive an exact steady state solution for a diffusion-coupled reaction localized to a small but open spherical sub-volume in a fluid reaction medium. We show that this leads to a fluorescence-based method for measuring the diffusion constant and the photochemical properties of fluorophores that is simpler and more robust than existing techniques such as fluorescence correlation spectroscopy (FCS). We use this method to study the photobleaching of rhodamine-B labeled protein molecules under different illumination intensities. Together with complementary data provided by FCS, this determines the average number of photons emitted by the fluorophores before photobleaching (~5x104). We demonstrate that this technique can be easily implemented on any confocal or multiphoton microscope or spectrometer and thus it should be adaptable to a variety of biological and chemical problems.
Vibration reduction possibilities in laminated composite with one of the plies as actuation layer, have been investigated. Magnetostrictive and piezoelectric actuation have been considered. A comparative assessment of the vibration suppression performance of piezoelectric and magnetostrictive actuation in laminated composite beam has been presented. Variation of closed loop frequencies and damping coefficients with respect to the changes in ply lay- up, active layer position and controller gain have been studied. A smart composite finite element for laminated beam analysis has been developed. Numerical results have been presented in typical cases, to indicate performance of the finite elements developed and also to indicate the variation of vibration reduction times with various geometric and control parameters.
Recent infrared spectroscopic studies of the ultrafast responses of optically triggered changes in proteins are discussed. Examples from research on bacteriorhodospin and reaction centers illustrate the potential of time resolved infrared spectroscopy in the field of protein dynamics.
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