Abstract
.Significance: Stimulated Raman scattering (SRS) and pump-probe microscopy are implementations of multiphoton microscopy that acquire high-resolution, label-free images of live samples encoded with molecular contrast. Most commercial multiphoton microscopes cannot access these techniques since they require sample illumination by two temporally synchronized ultrafast pulse trains. We present a compact and robust way of synchronizing an additional Ti:sapphire laser with a conventional single-beam multiphoton microscope to realize an instrument that can acquire images with enhanced molecular specificity.Aim: A passive optical synchronization scheme for a pair of commercially available, unmodified modelocked Ti:sapphire lasers was developed. The suitability of this synchronization scheme for advanced biomedical microscopy was investigated.Approach: A pair of modelocked Ti:sapphire lasers were aligned in master–slave configuration. Five percent of the master laser output was used to seed the modelocking in the slave laser cavity. The timing jitter of the master and slave pulse trains was characterized using an optical autocorrelator. The synchronized output of both lasers was coupled into a laser scanning microscope and used to acquire spectral focusing SRS and pump-probe microscopy images from biological and nonbiological samples.Results: A timing jitter between the modelocked pulse trains of 0.74 fs was recorded. Spectral focusing SRS allowed spectral discrimination of polystyrene and polymethyl methacrylate beads. Pump-probe microscopy was used to record excited state lifetime curves from hemoglobin in intact red blood cells.Conclusion: Our work demonstrates a simple and robust method of upgrading single-beam multiphoton microscopes with an additional ultrafast laser. The resulting dual-beam instrument can be used to acquire label-free images of sample structure and composition with high biochemical specificity.
Highlights
Our work demonstrates a simple and robust method of upgrading single-beam multiphoton microscopes with an additional ultrafast laser
With the autocorrelator delay line not scanning, the two-photon absorption (TPA) signal was recorded under two conditions: the master and slave pulse trains were temporally offset by half a pulse width, and the temporal offset was larger than the pulse width
To determine the timing jitter, we calculated the standard deviation of the TPA signal for the two conditions and converted these values from intensity to timing fluctuations using the linear slope of the cross-correlation signal
Summary
Since its development in 1990, multiphoton microscopy[1] has become a key enabling technology in the biomedical sciences.[2,3,4] Multiphoton microscopy uses the near-infrared, pulsed output of an ultrafast laser system to excite and image nonlinear optical processes in biological samples.Journal of Biomedical OpticsDownloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Biomedical-Optics on 08 Nov 2021 Terms of Use: https://www.spiedigitallibrary.org/terms-of-useJune 2020 Vol 25(6)Sherlock et al.: Ultra-low timing jitter, Ti:Al2O3 synchronization. . .Modern multiphoton microscopes routinely perform live cell imaging at depths >1 mm below the sample surface, with minimal phototoxicity and photodamage.The majority of commercial multiphoton microscopes use a single ultrafast laser to excite processes such as two photon excited fluorescence or second harmonic generation (SHG) to image sample structure and to a limited degree, composition. Since its development in 1990, multiphoton microscopy[1] has become a key enabling technology in the biomedical sciences.[2,3,4] Multiphoton microscopy uses the near-infrared, pulsed output of an ultrafast laser system to excite and image nonlinear optical processes in biological samples. Alternative nonlinear optical processes that require two spatially and temporally overlapped ultrafast pulse trains can be used to provide biochemically specific contrast in nonfluorescent samples. The chemical specificity of SRS can be further enhanced using spectral focusing, where the relative time delay between two chirped pulse trains is scanned, allowing the acquisition of spectroscopic images.[7] Pump-probe microscopy records the transient evolution of ground and first electronic excited state populations to provide distinctive signatures from molecules with similar linear absorption spectra
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