Repetitively synchronized systems

While the studies here reported have not resulted in any improvement of the division synchrony over what we have had for years (see, however, addendum, note 4, p. 149), they have yielded a repetitively synchronized Tetrahymem system that seems superior to its nearest predecessor (system of Padilla and Cameron, 1964) in that cell division is better synchronized, macronuclear DNA synthesis is synchronized, and it shows good free-running synchrony. An expected advantage of the repetitively synchronized system over the one obtained with multiple heat shocks is that all cell parameters should double in each repetitive cycle (see p. 142 and Fig. 21). The weakness, that in this system the environment and cells are both cyclic, can be controlled by parallel analysis of the free-running system, i.e., under conditions when only the biologic cycle remains. 

The technical realization of this synchronized system is shown in Fig. 6.16a. The frequency Vs = /27t of the ultrasonic wave is chosen as an integer multiple v =q c I Id of the mode-locking frequency. A fast photodiode, which detects the mode-locked optical pulses, provides the trigger signal for the RF generator for the ultrasonic wave. This allows the adjustment of the phase of the ultrasonic wave in such a way that the arrival time of the mode-locked pulse in the cavity dumper coincides with its maximum extraction efficiency. During the ultrasonic pulse only one mode-locked pulse is extracted. The extraction repetition frequency Vq = (cjld jk can be chosen between 1 Hz to 4 MHz by selecting the repetition rate of the ultrasonic pulses [669]. 

In all free-running synchronous systems there is quick decay of synchrony. In the case of Tetrahymena this pertains to synchrony obtained with multiple heat shocks, with repetitive shocks, and by collection of normal cells in the division stage. In all cases the rate of decay of synchrony is about the same, which immediately tells us that no cure for the special weaknesses in the system obtained with multiple heat shocks (p. 134) can be expected to improve the synchrony greatly. Full synchronization would always be resisted by the existence of a natural spread in cell generation times in the population... 

The commercially available laser source is a mode-locked argon-ion laser synchronously pumping a cavity-dumped dye laser. This laser system produces tunable light pulses, each pulse with a time duration of about 10 picoseconds, and with pulse repetition rates up to 80 million laser pulses/second. The laser pulses are used to excite the sample under study and the resulting sample fluorescence is spectrally dispersed through a monochromator and detected by a fast photomultiplier tube (or in some cases a streak camera (h.)) ... 

These features are exploited in an experiment which is taking place in our laboratory. A schematic diagram of this experiment is shown in figure 4. The system consists of an all-lines violet mode-locked Kr+ ion laser operating at a repetition rate of about 250 MHz Which synchronously pumps a C102 dye laser. The dye laser typically produces about 300 mW of average power and pulse durations of about 3 psec. This is frequency doubled to 243 nm in a crystal of p-barium borate to produce in excess of 2 mW average power. The output from the second harmonic crystal is then mode-matched into an ultra-violet enhancement cavity. The free... 

Picosecond pulses can be produced in a number of different types of laser systems. As an example, a brief description is first given of a synchronously pumped c.w. dye laser such as can be readily assembled from commercially available units. Generation of repetitive subnanosecond pulses in a c.w. laser by mode-locked synchronous pumping was first described by Harris et al. [12]. The essential features of such a system are shown in Fig. 3. In this system, an acousto-optically mode-locked ion laser is used to pump the dye laser. In order to achieve synchronous pumping, the length of the dye cavity must be adjusted so that the dye laser intermode spacing is an integral multiple of the pump mode-locker frequency. 

A streak camera system capable of operating repetitively at a rate of 140 MHz and with a resolution limit of <5ps has been described by Adams et al. [68]. This system permits streak records from relatively weak luminous events, e.g. fluorescence, to be accumulated in order to increase the signal-to-noise ratio. It also allows the use of lower intensity excitation pulses, thus avoiding non-linear effects in the sample. The system relies on the precise synchronization of the streak camera deflection plates to the repetition rate of a mode-locked CW laser. 

The basis of the experimental femtosecond CARS apparatus developed by Okamoto and Yoshihara (1990) which is reproduced in Fig. 3.6-10 is essentially the same as that of Leonhardt et al. (1987) and Zinth et al. (1988) with the addition of the possibility to change the polarization of the laser radiation. The main parts of the system are two dye lasers with short pulses and high repetition rates, pumped by a cw mode-locked Nd YAG laser (1064 nm, repetition rate 81 MHz). The beam of the first dye-laser which produces light pulses with 75-100 fsec duration is divided into two parts of equal intensities and used as the pump and the probe beam. After fixed (for the pump beam) and variable (for the probe beam) optical delay lines, the radiation is focused onto the sample together with the Stokes radiation produced by the second laser (DL2), which is a standard synchronously pumped dye laser. The anti-Stokes signal generated in the sample is separated from the three input laser beams by an aperture, an interference filter, and a monochromator, and detected by a photomultiplier. For further details we refer to Okamoto and Yoshihara (1990). 

In photocathode electron guns, the timing between the microwaves used for acceleration and the photocurrent-generating laser pulse is of critical importance. Precise synchronization between the laser and electron beams is obtained by using a MHz quartz master oscillator to control the cathode pump laser repetition rate and the microwave amplifier system seed frequency (Fig. 3). 

Transient spectroscopy experiments were performed with a pump-probe spectrometer [7] based on a home-made original femtosecond Ti saphire pulsed oscillator and a regenerative amplifier system operated at 10 Hz repetition rate. The Tirsaphire master oscillator was synchronously pumped with doubled output of feedback controlled mode-locked picosecond pulsed Nd YAG laser. The pulse width and energy of Ti saphire system after the amplifier were ca. 150 fs and 0.5 mJ, respectively, tunable over the spectral range of 760-820 nm. The fundamental output of the Ti saphire system (790 nm output wavelength was set for present study) splitted into two beams in the ratio 1 4. The more intense beam passed through a controlled delay line and was utilized for sample... 

Fig. 5. High repetition rate pulsed excitation systems for picosecond absorption and emission studies, (a) Pulsed e -beam with Cerenkov or laser probe pulses (b) actively mode-locked, synchronously pumped argon ion jet stream dye laser. See text for further details.

Unfortunately, pulse piekers have some severe drawbacks. One is that the minimum rate reduetion faetor is about 10. Repetition rates around 40 or 20 MHz are thus not available. Moreover, there is a leakage in the intermediate pulses of the order of 1%. For the first suppressed pulse the leakage can be even higher. Finally, most pulse piekers generate RF noise, which is synchronous with the pulses and ean severely impair the differential linearity of a TCSPC system. 

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