A fluorescence lifetime imaging microscopy (FLIM) integrated with two-photon excitation technique was developed. A wavelength-tunable femtosecond pulsed laser with nominal pulse repetition rate of 76-MHz was used to acquire FLIM images with a high pixel rate of 3.91 MHz by processing the pulsed two-photon fluorescence signal. Analog mean-delay (AMD) method was adopted to accelerate the lifetime measurement process and to visualize lifetime map in real-time. As a result, rapid tomographic visualization of both structural and chemical properties of the tissues was possible with longer depth penetration and lower photo-damage compared to the conventional single-photon FLIM techniques.

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Multimodal nonlinear microscopy has been widely applied in biology and medicine due to its relatively deep penetration into tissue and its label-free manner. However, current multimodal systems require the use of multiple sources and detectors, leading to bulky, complex, and expensive systems. In this Letter, we present a novel method of using a single light source and detector for nonlinear multimodal imaging of biological samples. Using a photonic crystal fiber, a pulse picker, and multimode fibers, our developed system successfully acquired multimodal images of swine coronary arteries, including two-photon excitation fluorescence, second-harmonic generation, coherent anti-Stokes Raman scattering, and backreflection. The developed system could be a valuable tool for various biomedical applications.

Wavefront sensing using a Shack-Hartmann sensor has been widely used for estimating wavefront errors or distortions. The sensor combines the local slopes, which are estimated from the centroids of each lenslet images, to give the overall wavefront reconstruction. It was previously shown that the pupil-plane irradiance profile effects on the centroid estimation. Furthermore, a previous study reported that the reconstructed wavefront from a planar wavefront with a Gaussian pupil irradiance profile contain large focus and spherical aberration terms when there is a focus error. However, it has not been reported yet how serious the pupil irradiance profiles, which can be occurred in practical applications, effects on the sensing errors. This paper considered two cases when the irradiance profiles are not uniform: 1) when the light source is Gaussian and 2) when there is a partial interference due to a double reflection by a beam splitting element. The images formed by a Shack-Hartmann sensor were simulated through fast Fourier transform and were then supposed to be detected by a noiseless CCD camera. The simulations found that sensing errors, due to the Gaussian irradiance profile and the partial interference, were found to be smaller than λ/50 which can be ignored in most practical cases where the reference and test beams have the same irradiance profiles.

Optical microscopy has been widely used in biomedical research as it provides photophysical and photochemical information of the target in subcellular spatial resolution without requiring physical contact with the specimen. To obtain a deeper understanding of biological phenomena, several efforts have been expended to combine such optical imaging modalities into a single microscope system. However, the use of multiple light sources and detectors through separated beam paths renders previous systems extremely complicated or slow for in vivo imaging. Herein, we propose a novel high-speed multimodal optical microscope system that simultaneously visualizes five different microscopic contrasts, i.e., two-photon excitation, second-harmonic generation, backscattered light, near-infrared fluorescence, and fluorescence lifetime, using a single femtosecond pulsed laser. Our proposed system can visualize five modal images with a frame rate of 3.7 fps in real-time, thereby providing complementary optical information that enhances both structural and functional contrasts. This highly photon-efficient multimodal microscope system enables various properties of biological tissues to be assessed.