We present a multicolor fluorescence microscope system, under a selective plane illumination microscopy (SPIM) configuration, using three continuous wave-lasers and a single-channel-detection camera. The laser intensities are modulated with three time-delayed pulse trains that operate synchronously at one third of the camera frame rate, allowing a sequential excitation and an image acquisition of up to three different biomarkers. The feasibility of this imaging acquisition mode is demonstrated by acquiring single-plane multicolor images of living hyphae of Neurospora crassa. This allows visualizing simultaneously the localization and dynamics of different cellular components involved in apical growth in living hyphae. The configuration presented represents a noncommercial, cost-effective alternative microscopy system for the rapid and simultaneous acquisition of multifluorescent images and can be potentially useful for three-dimensional imaging of large biological samples.
We demonstrate frequency differential CARS (D-CARS) using femtosecond laser pulses linearly chirped by glass
elements of high group-velocity dispersion. By replicating the Pump-Stokes pair into a pulse train at twice the
laser repetition rate, and controlling the instantaneous frequency difference by glass dispersion, we adjust the
Raman frequency probed by each pair in an intrinsically stable way. The resulting CARS intensities are detected
simultaneously by a single photomultiplier as sum and difference using lock-in detection. We demonstrate
imaging of living cells with strongly suppressed non-resonant background. We also show D-CARS using a single
femtosecond laser source.
We have developed a home-built CARS microscope which exploits linearly-chirped ultrafast laser pulses. By using
glass of high group-velocity dispersion, Stokes and Pump pulses of 150 fs duration Fourier-limited are equally
chirped to pulse durations in the 0.5 ps-2.8 ps range. In this way we reduce the spectral width of the instantaneous
frequency difference to the Fourier limit of the chirped pulse duration (spectral focussing). As a proof of
principle, CARS spectroscopy with high spectral resolution is demonstrated on polystyrene beads. We also
show, both theoretically and experimentally, that for chirped pulse durations shorter than or comparable to the
Raman coherence time, maximum CARS signal occurs for a Pump arriving after the Stokes pulse. Furthermore,
we demonstrate the applicability of our CARS microscope to biological sciences by performing CARS microspectroscopy
on different live cells and fixed tissue samples.
A novel signal processing algorithm for quantifying structural disorder in biological tissue using second harmonic generation (SHG) imaging is described. Both the magnitude and the pattern of disorder in collagenous tissues can be determined with this method. Mathematical models are used to determine the range of disordered states over which the algorithm can be used, because highly disordered biological samples do not generate second harmonic signals. The method is validated by measuring disorder in heated fascicles using SHG and showing that results are significantly correlated with morphometric determination. Applicability of the method to tissue pathology is demonstrated by analysis of a mouse model of intervertebral disk injury. Disks were subjected to tensile or compressive forces in vivo for one week. Structural disorder in the annulus fibrosus was measured by SHG scanning and by standard morphometric analysis. Values for disorder obtained by SHG scanning were significantly correlated with values obtained by morphometry (p<0.001). Quantitation of disorder using SHG offers significant advantages over morphometric determination. Data obtained in this study suggest that this method can be used to discriminate between reversible and irreversible tissue damage.
We report the effective excitation of space-charge waves (SCW) in photorefractive ac-biased CdTe:Ge crystal illuminated by a laser beam (λ = 1150 nm) and an auxiliary incoherent light with different wavelength. We show that laser beam scattered from SCW has resonant dependence on the auxiliary light wavelength. The effect of auxiliary illumination on photorefractive two-wave coupling and subharmonic generation is presented.
We study the symmetrical vectorial two-wave mixing in cubic photorefractive crystals when the weak beam is always amplified in spite of its mutual direction in respect of the strong pump beam. We show that this effect takes place both in ac- and dc-biased crystals although the mechanisms underlying this effect are different for local and non-local photorefractive gratings. We present the results of analytical and numerical analyses along with the experimental results obtained with a Bi12TiO20 crystal.