Two-photon imaging using adaptive phase compensated ultrashort laser pulses (original) (raw)
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Nanomedicine: Nanotechnology, Biology and Medicine, 2006
Two-photon excitation using ultrashort laser pulses can selectively activate nanoparticles or excite fluorophores within thick biological samples. We show how the use of methods such as multiphoton intrapulse interference phase scan (MIIPS) to compensate phase distortions caused by microscope objectives with a high numerical aperture increases signal intensity and reproducibility in twophoton imaging. Using phase shaping of our compensated pulses, we demonstrate selective excitation of fluorophores within a mouse kidney sample, increasing the contrast between different subcellular structures compared to unshaped pulses.
Advantages of Two-photon Microscopy with Ultrashort Pulses
Springer Series in Chemical Physics, 2009
Ultrashort pulses are expected to be beneficial for multiphoton microscopy. To utilize their advantages, however, chromatic dispersion must be compensated. We use multiphoton intrapulse interference phase scan (MIIPS) to measure and then eliminate phase distortions of pulses with a FWHM spectral bandwidth greater than 100 nm. Once compensated, the transform limited pulses (<15 fs) deliver higher twophoton excited fluorescence signal intensity, which translates into deeper optical penetration depth.
Greater signal and contrast in two-photon microscopy with ultrashort pulses
Progress in Biomedical Optics and Imaging - Proceedings of SPIE, 2008
Ultrashort <15 fs pulses are shown to provide higher fluorescence intensity, deeper sample penetration, and single laser selective excitation. To realize these advantages chromatic dispersion effects must be compensated. We use multiphoton intrapulse interference phase scan (MIIPS) to measure and then eliminate high-order distortions on pulses with a bandwidth greater than 100nm FWHM. Once compensated, the transform limited pulses deliver higher signal intensity, and this translates into deeper optical penetration depth with a high signal-to-noise ratio. By using a pulse shaper and taking advantage of the broad spectrum of the ultrafast laser, selective excitation of different cell organelles is observed due to the difference in nonlinear optical susceptibility of different chromophores without the use of an emission filter wheel.
Multiplexed two-photon microscopy of dynamic biological samples with shaped broadband pulses
Optics …, 2009
Coherent control can be used to selectively enhance or cancel concurrent multiphoton processes, and has been suggested as a means to achieve nonlinear microscopy of multiple signals. Here we report multiplexed two-photon imaging in vivo with fast pixel rates and micrometer resolution. We control broadband laser pulses with a shaping scheme combining diffraction on an optically-addressed spatial light modulator and a scanning mirror allowing to switch between programmable shapes at kiloHertz rates. Using coherent control of the two-photon excited fluorescence, it was possible to perform selective microscopy of GFP and endogenous fluorescence in developing Drosophila embryos. This study establishes that broadband pulse shaping is a viable means for achieving multiplexed nonlinear imaging of biological tissues.
Greater signal, increased depth, and less photobleaching in two-photon microscopy with 10fs pulses
Optics Communications, 2008
The fundamental advantages to using ultrafast ((100 fs) laser pulses in two-photon microscopy for biomedical imaging are seldom realized due to chromatic dispersion introduced by the required high numerical aperture microscope objective. Dispersion is eliminated here by using the multiphoton intrapulse interference phase scan (MIIPS) method on pulses with a bandwidth greater than 100 nm full width at half maximum. Higher fluorescence intensity, deeper sample penetration, and improved signal-to-noise ratio are demonstrated quantitatively and qualitatively. Due to the higher signal intensity obtained after MIIPS compensation, lower laser power is required, which decreases photobleaching. The observed advantages are not realized if group delay dispersion is compensated for while higherorder dispersion is not. By using a pulse shaper and taking advantage of the broad spectrum of the ultrafast laser, selective excitation of different cell organelles is achieved due to the difference in nonlinear optical susceptibility of different chromophores without requiring an emission filter wheel. Experiments on biological specimens, such as HeLa cells and mouse kidney tissue samples, illustrate the advantages to using sub-10 fs pulses with MIIPS compensation in the field of two-photon microscopy for biomedical imaging.
Applications of ultrashort shaped pulses in microscopy and for controlling chemical reactions
Chemical Physics, 2008
The fundamental advantages to using ultrafast ((100 fs) laser pulses in two-photon microscopy for biomedical imaging are seldom realized due to chromatic dispersion introduced by the required high numerical aperture microscope objective. Dispersion is eliminated here by using the multiphoton intrapulse interference phase scan (MIIPS) method on pulses with a bandwidth greater than 100 nm full width at half maximum. Higher fluorescence intensity, deeper sample penetration, and improved signal-to-noise ratio are demonstrated quantitatively and qualitatively. Due to the higher signal intensity obtained after MIIPS compensation, lower laser power is required, which decreases photobleaching. The observed advantages are not realized if group delay dispersion is compensated for while higherorder dispersion is not. By using a pulse shaper and taking advantage of the broad spectrum of the ultrafast laser, selective excitation of different cell organelles is achieved due to the difference in nonlinear optical susceptibility of different chromophores without requiring an emission filter wheel. Experiments on biological specimens, such as HeLa cells and mouse kidney tissue samples, illustrate the advantages to using sub-10 fs pulses with MIIPS compensation in the field of two-photon microscopy for biomedical imaging.
Commercial and Biomedical Applications of Ultrafast Lasers VI, 2006
A number of nonlinear imaging modalities, such as two-photon excitation and second harmonic generation, have gained popularity during the last decade. These, and related methods, have in common the use of a femtosecond laser in the near infrared, with the short pulse duration making the nonlinear excitation highly efficient. Efforts toward the use of pulses with pulse duration at or below 10 fs, however, have been a great challenge, in part due to the fact that shorter pulses have been found to cause greater sample damage. Here we provide a brief review of the MIIPS method for correction of phase distortions introduced by high numerical aperture objectives and the introduction of simple phase functions capable of preventing three-photon induced damage, reducing autofluorescence, and providing selective probe excitation.
Ultrafast multiphoton microscopy with high-order spectral phase distortion compensation
Multiphoton Microscopy in the Biomedical Sciences IX, 2009
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 laserscanning 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.
Two-photon laser scanning microscopy with ultrabroad bandwidth 110 nm FWHM femtosecond pulses
Progress in Biomedical Optics and Imaging - Proceedings of SPIE, 2008
Shorter pulses, in theory, should be favorable in nonlinear microscopy and yield stronger signals. However, shorter pulses are much more prone to chromatic dispersion when passing through the microscope objective, which significantly broadens its pulse duration and cancels the expected signal gain. In this paper, multiphoton intrapulse interference phase scan (MIIPS) was used to compensate chromatic dispersion introduced by the 1.45 NA objective. The results show that with MIIPS compensation, the increased signal is realized. We also find that third and higher order dispersion compensation, which cannot be corrected by prism pairs, is responsible for an additional factor of 4.7 signal gain.
Optics Communications, 2018
A variable prism-pair-based pulse compressor with wavefront aberration sensing was used to enhance multiphoton imaging in biomedical samples. This was incorporated into a custom-made microscope to reduce pulse temporal length, improve the quality of images of different layers of thick tissues and increase penetration depth. The laser beam aberrations were found to hardly change with the different experimental configurations of the pulse compressor. The optimum pulse compression state was maintained with depth within the tissue, independently of its thickness. This suggests that for each sample, a single experimental configuration is able to provide the best possible image at any depth location; although this needs to be experimentally obtained. Furthermore, a simple method based on laser average power reduction is presented to minimize the risk of photo-damage in biological samples. The use of pulse compression in multiphoton microscopy might have a potential for accurate and improv...