A maximum likelihood estimator to distinguish single molecules by their fluorescence decays

We have developed a maximum likelihood estimator to distinguish between similar molecules at the single molecule level based upon fluorescence decay measurements. Time resolved fluorescence measurements for single Rhodamine 6G and tetramethylrhodamine isothiocyanate molecules in fluid flow are derived from time-correlated single photon counting. A maximum likelihood estimator is developed and applied to data from a mixture of molecules. Single molecules are identified and distinguished by their fluorescence time decays. Comparison is made between identification error rates and theoretical predictions. To our knowledge, this is the first reported example of single molecule identification by fluorescence decay in a mixture.     

Differential stable isotope labeling of peptides for quantitation and de novo sequence derivation.

We have demonstrated the use of per-methyl esterification of peptides for relative quantification of proteins between two mixtures of proteins and automated de novo sequence derivation on the same dataset. Protein mixtures for comparison were digested to peptides and resultant peptides methylated using either d0- or d3-methanol. Methyl esterification of peptides converted carboxylic acids, such as are present on the side chains of aspartic and glutamic acid as well as the carboxyl terminus, to their corresponding methyl esters. The separate d0- and d3-methylated peptide mixtures were combined and the mixture subjected to microcapillary high performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). Parent proteins of methylated peptides were identified by correlative database searching of peptide tandem mass spectra. Ratios of proteins in the two original mixtures could be calculated by normalization of the area under the curve for identical charge states of d0- to d3-methylated peptides. An algorithm was developed that derived, without intervention, peptide sequence de novo by comparison of tandem mass spectra of d0- and d3-peptide methyl esters.     

Continuous countercurrent extraction of hemicellulose from pretreated wood residues

Two-stage dilute acid pretreatment followed by enzymatic cellulose hydrolysisis an effectivemethod for obtaining high sugar yields from wood residuessuchassoftwood forest thinnings. In the first-stage hydrolysis step, most of the hemicellulose is solubilized using relatively mild conditions. The soluble hemicellu losic sugars are recovered from the hydrolysateslurry by washing with water. The washed solids are then subjected tomoresevere hydrolysis conditions to hydrolyze approx 50% of the cellulose to glucose. The remaining cellulose can further be hydrolyzed with cellulase enzyme. Our process simulation indicates that the amount of water used in the hemicellulose recovery step has a significan tim pact on the cost of ethanol production. It is important to keep water usage as low as possible while mainta ining relatively high recovery of solublesugars. To achieve this objective, a prototype pilot-scale continuous countercurrent screw extractor was evaluated for the recovery of hemicellulose from pretreated forest thinnings. Using the 274-cm (9-ft) long extractor, solubles recoveries of 98, 91, and 77% were obtained with liquid-to-insoluble solids (L/1S) ratios of 5.6, 3.4, and 2.1, respectively. An empirical equation was developed to predict the performance of the screwextractor. This equation predicts that soluble sugar recovery above 95% can be obtained with an L/IS ratio as low as 3.0.     

Biotransformation-mediated synthesis of (1S)-1-(2,6-dichloro-3-fluorophenyl)ethanol in enantiomerically pure form

An efficient four-step biotransformation-mediated synthesis of (1S)-1-(2,6-dichloro-3-fluorophenyl)ethanol in enantiomerically pure form is described. This compound is a key intermediate required for the preparation of PF-2341066, a potent inhibitor of c-Met/ALK that is currently in clinical development. The described synthesis was used to manufacture 6 kg of the title compound and can also be employed to produce the corresponding (1R)-enantiomer.     

240-fs pulses with 22-W average power from a mode-locked thin-disk Yb:KY(WO(4))(2) laser

We demonstrate what is to our knowledge the first passively mode-locked thin-disk Yb:KY(WO(4))(2) laser. The laser produces pulses of 240-fs duration with an average power of 22 W at a center wavelength of 1028 nm. At a pulse repetition rate of 25 MHz, the pulse energy is 0.9microJ , and the peak power is as high as 3.3 MW. The beam quality is very close to the diffraction limit, with M(2)=1.1 .     

Diode-pumped passively mode-locked Nd:YAG laser with 10-W average power in a diffraction-limited beam

We present a passively mode-locked Nd:YAG laser with 10.7-W average output power in a diffraction-limited beam. Stable self-starting mode locking with a pulse duration of 16 ps and a pulse energy of 120 nJ is obtained with a semiconductor saturable-absorber mirror. The laser is directly side pumped with two 20-W diode bars. Single-pass frequency doubling in an external 5-mm-thick KTP crystal yields 3.2-W average power at 532 nm.     

Directly diode-pumped millijoule subpicosecond Yb:glass regenerative amplifier

An Yb:glass regenerative amplifier directly side pumped by four 20-W diodes is demonstrated. By use of a novel pumping scheme and introduction of cylindrical optics into the cavity, a free-running average output power as great as 4 W with a TEM(00) -like mode was achieved from the bare cavity, with a 0.56 pump duty cycle. When the regenerative amplifier injected, 1-mJ 200-fs FWHM pulses were obtained following compression by use of 2-ms pump pulses and up to a 150-Hz repetition rate.     

A pair of stream functions for three-dimensional vortex flows

Introducing a vector potential, that is based on a pair of stream functions, and a velocity potential, antisymmetric equations for the stream functions are derived with the help of a variational principle. It is found that the equations are in a suitable form to investigate flows with helical symmetry, and, for example, to connect upstream axisymmetric flows with downstream helical flows. The special case of a transition from an upstream solid-body vortex to a downstream helical flow is investigated in detail. Furthermore, the stream-function equations are particularly useful to investigate general small-amplitude inertia waves on vortex flows. Time-dependent helical flows that are time-independent in a suitably rotating frame of reference can also be discussed with the proposed method.