Straightforward FRAP for quantitative diffusion measurements with a laser scanning microscope.

Confocal or multi-photon laser scanning microscopes are convenient tools to perform FRAP diffusion measurements. Despite its popularity, accurate FRAP remains often challenging since current methods are either limited to relatively large bleach regions or can be complicated for non-specialists. In order to bring reliable quantitative FRAP measurements to the broad community of laser scanning microscopy users, here we have revised FRAP theory and present a new pixel based FRAP method relying on the photo bleaching of rectangular regions of any size and aspect ratio. The method allows for fast and straightforward quantitative diffusion measurements due to a closed-form expression for the recovery process utilizing all available spatial and temporal data. After a detailed validation, its versatility is demonstrated by diffusion studies in heterogeneous biopolymer mixtures.

Structural typing of systemic amyloidoses by luminescent-conjugated polymer spectroscopy.

Most systemic amyloidoses are progressive and lethal, and their therapy depends on the identification of the offending proteins. Here we report that luminescent-conjugated thiophene polymers (LCP) sensitively detect amyloid deposits. The heterodisperse polythiophene acetic acid derivatives, polythiophene acetic acid (PTAA) and trimeric PTAA, emitted yellow-red fluorescence on binding to amyloid deposits, whereas chemically homogeneous pentameric formic thiophene acetic acid emitted green-yellow fluorescence. The geometry of LCPs modulates the spectral composition of the emitted light, thereby reporting ligand-induced steric changes. Accordingly, a screen of PTAA-stained amyloid deposits in histological tissue arrays revealed striking spectral differences between specimens. Blinded cluster assignments of spectral profiles of tissue samples from 108 tissue samples derived from 96 patients identified three nonoverlapping classes, which were found to match AA, AL, and ATTR immunotyping. We conclude that LCP spectroscopy is a sensitive and powerful tool for identifying and characterizing amyloid deposits.

Effect of confinement and kinetics on the morphology of phase separating gelatin-maltodextrin droplets.

The effect of confinement on the structure evolution and final morphology during phase separation and gelation of gelatin and maltodextrin was investigated and compared to the structures seen in bulk phase. Emulsion droplets with diameters from 4 to 300 mum were analyzed using confocal laser scanning microscopy and image analysis. With the confocal laser scanning microscope it was possible to follow the entire phase separating process inside the droplets in real-time. The samples were either quenched directly from 70 degrees C down to 20 degrees C or exposed to holding times at 40 degrees C. Different cooling procedures were studied to examine the structure evolution both before and after gelation in the restricted geometries. The concentration of the biopolymer mixture was kept constant at 4 w/w% gelatin and 6 w/w% maltodextrin. The results revealed that the size of the confinement had a great effect on both the initiation of phase separation and the final morphology of the microstructure inside the emulsion droplets. The phase separation in small droplets was observed to occur at a temperature above the phase separating temperature for bulk. Small droplets had either a microstructure with a shell of maltodextrin and core of gelatin or a microstructure where the two biopolymers had formed two separate bicontinuous halves. The initiation of phase separation in large droplets was similar to what was seen in bulk. The microstructure in large droplets was discontinuous, resembling the morphology in bulk phase. The kinetics had an effect on the character of the maltodextrin inclusions, as the cooling procedure of a direct quench gave spherical inclusions with an even size distribution, while a holding time at 40 degrees C resulted in asymmetrical and elongated inclusions.

Protein biochips patterned by microcontact printing or by adsorption-soft lithography in two modes.

Patterning of proteins is critical to protein biochips. Printing of layers of proteins is well established, as is adsorption of proteins to surfaces properly modified with surface chemical functionalities. The authors show that simple methods based on soft lithography stamps can be used to prepare functional antibody chips through both these routes. Both methods incorporate transfer of the stamp material poly(dimethylsiloxane) (PDMS) to the biochip, whether intended or not intended. The results indicate that microcontact printing of proteins always includes PDMS transfer, thereby creating a possibility of unspecific adsorption to a hydrophobic domain.