Selective two-photon microscopy with shaped femtosecond pulses

Michigan State University, Ист-Лансинг, Michigan, United States
Optics Express (Impact Factor: 3.49). 08/2003; 11(14):1695-701. DOI: 10.1364/OE.11.001695
Source: PubMed


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.

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Available from: Vadim V Lozovoy
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    • "With ultrashort transform-limited (TL) pulses to start with, one can use the same shaper to add a pre-programmed amount of dispersion and adjust the pulse duration over a large time range. The added phase distortion can be as simple as a linear chirp, but the same system can be used to implement more advanced pulse shaping schemes, e.g., selective twophoton excitation of fluorophores [13] [14] [15], or even other modalities of nonlinear microscopy such as second-harmonic generation (SHG) imaging and single-beam coherent anti- Stokes Raman scattering microscopy [16]. The actual types and mechanisms of damage that are inflicted by the laser pulse illumination are not only sample dependent but also problem specific. "
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    ABSTRACT: We examine the effects of pulse duration tuning on the photodamage inflicted by laser light illumination on the imaged sample and, thereby, explore the optimization of optical pulse parameters for multiphoton microscopy imaging under variable conditions. We discuss the dependence of the nonlinear excitation efficiency and associated photodamage rates on pulse energy and duration, and use the controlled amount of second-order dispersion (linear chirp), introduced by a pulse shaper, to adjust the pulse duration at the imaging plane of the microscope. The pulse energy is varied to maintain a constant two-photon excitation efficiency when switching between short (∼14 fs) and long (∼280 fs) pulses, and the damage is assessed by monitoring the photobleaching rates and sample morphology. We have found that in addition to the well-known photobleaching effects, significant enhancement of the two-photon excited autofluorescence intensity can be observed. Photobleaching rates at the onset of the laser light exposure are shown to be independent of the pulse shape under our experimental conditions, which indicates that the primary damage (bleaching) mechanism stems from the two-photon excitation process. The photoenhancement, however, is found to occur more readily with longer pulses, having higher energies per pulse. Experiments are carried out on human melanoma tissue and on rabbit red blood cells.
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    • "Five statistically significant features were able to differentiate between the normal and the precancerous or cancerous images, thus showing good potential for future clinical use [82]. Pulse shaping and phase modulation can be used for selective excitation of fluorescent probe molecules, as well as to compensate for unwanted phase distortions at the sample [83]. Multiphoton microscopy already extends past the maximum imaging depth of confocal microscopy, but further depths are always desirable. "
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    • "From the peak position of the SHG spectrum, the algorithm retrieves the second derivative of the unknown phase and constructs the compensation phase mask. Several consecutive iterations, made by MIIPS software, insure an accurate correction of the phase distortion; for details, see [7] [12] [13] [14] [15] "
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    ABSTRACT: High-order dispersion of ultrashort laser pulses (with ~100 nm bandwidth) is shown to account for significant reduction of two-photon excitation fluorescence and second harmonic generation signal produced at the focal plane of a laser-scanning two-photon microscope. The second- and third-order corrections recover 20-40% of the signal intensity expected for a transform-limited laser pulse, while the rest depends on the proper compensation of higher-order terms. It can be accomplished through the use of a pulse shaper by measuring and correcting all nonlinear spectral phase distortions.
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