Multiphoton microscopy has enabled unprecedented dynamic exploration in living organisms. A significant challenge in biological research is the dynamic imaging of features deep within living organisms, which permits the real-time analysis of cellular structure and function. To make progress in our understanding of biological machinery, optical microscopes must be capable of rapid, targeted access deep within samples at high resolution. In this Review, we discuss the basic architecture of a multiphoton microscope capable of such analysis and summarize the state-of-the-art technologies for the quantitative imaging of biological phenomena.

Despite all the advances in nonlinear microscopy, all existing instruments are constrained to obtain images of one focal plane at a time. In this Letter we demonstrate a two-photon absorption fluorescence scanning microscope capable of imaging two focal planes simultaneously. This is accomplished by temporally demultiplexing the signal coming from two focal volumes at different sample depths. The scheme can be extended to three or more focal planes.

The measurement of cell elastic parameters using optical forces has great potential as a reagent-free method for cell classification, identification of phenotype, and detection of disease; however, the low throughput associated with the sequential isolation and probing of individual cells has significantly limited its utility and application. We demonstrate a single-beam, high-throughput method where optical forces are applied anisotropically to stretch swollen erythrocytes in microfluidic flow. We also present numerical simulations of model spherical elastic cells subjected to optical forces and show that dual, opposing optical traps are not required and that even a single linear trap can induce cell stretching, greatly simplifying experimental implementation. Last, we demonstrate how the elastic modulus of the cell can be determined from experimental measurements of the equilibrium deformation. This new optical approach has the potential to be readily integrated with other cytometric technologies and, with the capability of measuring cell populations, enabling true mechanical-property-based cell cytometry.

We demonstrate, for the first time, OCT imaging capabilities of a novel, akinetic (without any form of movement in the tuning mechanism), all-semiconductor, all-electronic tunable, compact and flexible swept source laser technology at 1550 nm and 1310 nm. To investigate its OCT performance, 2D and 3D ex vivo and in vivo OCT imaging was performed at different sweep rates, from 20 kHz up to 200 kHz, with different axial resolutions, about 10 µm to 20 µm, and at different coherence gate displacements, from zero delay to >17 cm. Laser source phase linearity and phase repeatability standard deviation of <2 mrad (<160 pm) were observed without external phase referencing, indicating that the laser operated close to the shot noise limit (~2 × factor); constant percentile wavelengths variations of sliding RIN and ortho RIN <0.2% could be demonstrated, ~5 times better as compared to other swept laser technologies.

We demonstrate a novel multifocal, multiphoton microscope that is capable of simultaneous dynamic imaging of multiple focal planes. We show for the first time that multimodal, multiphoton images excited with orthogonal polarizations can be acquired simultaneously in both the transmission and epi directions.