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Selective two-photon excitation of fluorescent probe molecules using phase-only modulated ultrashort 15-fs laser pulses is demonstrated. The spectral phase required to achieve the maximum contrast in the excitation of different probe molecules or identical probe molecules in different micro-chemical environments is designed according to the principles of multiphoton intrapulse interference (MII). The MII method modulates the probabilities with which specific spectral components in the excitation pulse contribute to the two-photon absorption process due to the dependence of the absorption on the power spectrum of E2(t) [1-3]. Images obtained from a number of samples using the multiphoton microscope are presented.

Mass spectrometry (MS) is one of the oldest and most trusted analytical methods for chemical identification. Advances in biology, such as metabolic analysis and proteomics, have fueled a growing number of refinements in this method. Unfortunately, isomers, for example, o- and p-xylene, are seldom identifiable by MS because they produce identical spectra. Time-consuming and less sensitive multidimensional methods are subsequently required for structural determination. The sensitivity of MS coupled with shaped femtosecond laser pulses that control molecular fragmentation and ionization results in a new, fast, and reproducible method for molecular identification which is used here to distinguish positional and geometric isomer compounds and quantify their relative concentration in mixtures.

ABSTRACT:In a quality control program, seed vigor evaluation is of fundamental importance for the success of the program. The objective of this project was to evaluate the efficiency of several vigor tests to evaluate the physiological quality of Brachiaria brizantha cv. 'BRS Piatã' seed lots and to preview seedling emergence in the field. Ten grass seed lots of the B. brizantha cv. 'BRS Piatã' were evaluated by the following tests: germination, first count of germination, accelerated aging (43 °C/48 hours), cold test (seeds rolled in paper towel), seed water content before and after accelerated aging, electrical conductivity (50 and 75 mL, 25 °C and readings made 2, 4, 6, 8, and 24 hours after beginning the test), speed of germination index, and seedling emergence in sand substratum in the laboratory (26 ± 3 °C) and daily seedling counts between the 7th and the 21th day after sowing. The treatment replications were distributed in the laboratory according to a completely random design with four replications. Seedling emergence in the field data were analyzed according to a randomized complete block design. Treatment means were compared by the Scott Knott test, at the 5% level of probability. The tests for germination and first germination count, seedling emergence and first emergence count in the sand, and accelerated aging are useful to assess the vigor of seed batches of the piatã grass, and provide similar evidence to the seedling emergence in the field.

Rapid and direct imaging of microscopic tissue morphology and pathology can be achieved by multiphoton imaging of intrinsic tissue fluorophores and second harmonic signals. Engineering parameters for developing this technology for clinical applications include excitation levels and collection efficiencies required to obtain diagnostic quality images from different tissue types and whether these levels are mutagenic. Here we provide data on typical average powers required for high signal-to-noise in vivo tissue imaging and assess the risk potential of these irradiance levels using a mammalian cell gene mutation assay. Exposure times of ~16 milliseconds per cell to 760 nm, ~200 fs raster-scanned laser irradiation delivered through a 0.75 NA objective produced negligible mutagenicity at powers up to about 50 mW.

Phase modulation, through its influence on the probability of two-photon excitation at specific frequencies, is used to probe a molecule's microscopic chemical environment. The spectral phase required is designed according to the principles of multiphoton intrapulse interference. We show experimental results where phase modulation of 20 fs pulses from a titanium sapphire laser oscillator controls the intensity of two-photon induced fluorescence from the pH-sensitive dye 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) in solution. We show that the dependence of fluorescence yield on the phase can be utilized as a means of distinguishing molecules in different environments. Consequently, this method can be used to achieve selective multiphoton microscopy. To illustrate this capability, we show images where phase modulation was used to selectively excite HPTS in acidic or basic microscopic regions.

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