Differential Multiphoton Laser Scanning Microscopy.

Multifocal multiphoton microscopy (MMM) in the biological and medical sciences has become an important tool for obtaining high resolution images at video rates. While current implementations of MMM achieve very high frame rates, they are limited in their applicability to essentially those biological samples that exhibit little or no scattering. In this paper, we report on a method for MMM in which imaging detection is not necessary (single element point detection is implemented), and is therefore fully compatible for use in imaging through scattering media. Further, we demonstrate that this method leads to a new type of MMM wherein it is possible to simultaneously obtain multiple images and view differences in excitation parameters in a single shot.

Optimizing the fluorescent yield in two-photon laser scanning microscopy with dispersion compensation.

A challenge for nonlinear imaging in living tissue is to maximize the total fluorescent yield from each fluorophore. We investigated the emission rates of three fluorophores-rhodamine B, a red fluorescent protein, and CdSe quantum dots-while manipulating the phase of the laser excitation pulse at the focus. In all cases a transform-limited pulse maximized the total yield to insure the highest signal-to-noise ratio. Further, we find evidence of fluorescence antibleaching in quantum dot samples.

Endoscopic optical coherence tomography angiography using a forward imaging piezo scanner probe.

A forward imaging endoscope for optical coherence tomography angiography (OCTA) featuring a piezoelectric fiber scanner is presented. Imaging is performed with an optical coherence tomography (OCT) system incorporating an akinetic light source with a center wavelength of 1300 nm, bandwidth of 90 nm and A-line rate of 173 kHz. The endoscope operates in contact mode to avoid motion artifacts, in particular, beneficial for OCTA measurements, and achieves a transversal resolution of 12 µm in air at a rigid probe size of 4 mm in diameter and 11.3 mm in length. A spiral scan pattern is generated at a scanning frequency of 360 Hz to sample a maximum field of view of 1.3 mm. OCT images of a human finger as well as visualization of microvasculature of the human palm are presented both in two and three dimensions. The combination of morphological tissue contrast with qualitative dynamic blood flow information within this endoscopic imaging approach potentially enables improved early diagnostic capabilities of internal organs for diseases such as bladder cancer.

Simultaneous multifocal, multiphoton, photon counting microscopy.

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.

Advancing multifocal nonlinear microscopy: development and application of a novel multibeam Yb:KGd(WO4)2 oscillator.

We present a novel Yb:KGd(WO(4))(2) oscillator design that generates six beams of temporally delayed, 253 fs, 11 nJ pulses. This allows multifocal nonlinear microscopy to be performed without the need for complicated optical multiplexers. We demonstrate our design with twelve simultaneously acquired two-photon, second-harmonic and/or third-harmonic images generated from six laterally separated foci.

Advances in multiphoton microscopy technology.

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.

Eliminating the scattering ambiguity in multifocal, multimodal, multiphoton imaging systems.

In this work we present how to entirely remove the scattering ambiguity present in existing multiphoton multifocal systems. This is achieved through the development and implementation of single-element detection systems that incorporate high-speed photon-counting electronics. These systems can be used to image entire volumes in the time it takes to perform a single transverse scan (four depths simultaneously at a rate of 30 Hz). In addition, this capability is further exploited to accomplish single-element detection of multiple modalities (two photon excited fluorescence and second harmonic generation) and to perform efficient image deconvolution. Finally, we demonstrate a new system that promises to significantly simplify this promising technology.

Remote focusing for programmable multi-layer differential multiphoton microscopy.

We present the application of remote focusing to multiphoton laser scanning microscopy and utilize this technology to demonstrate simultaneous, programmable multi-layer imaging. Remote focusing is used to independently control the axial location of multiple focal planes that can be simultaneously imaged with single element detection. This facilitates volumetric multiphoton imaging in scattering specimens and can be practically scaled to a large number of focal planes. Further, it is demonstrated that the remote focusing control can be synchronized with the lateral scan directions, enabling imaging in orthogonal scan planes.