Erich E. Hoover

Simultaneous imaging of multiple focal planes using a two-photon scanning microscope

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.

Akinetic all-semiconductor programmable swept-source at 1550 nm and 1310 nm with centimeters coherence length

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.

Cell deformation cytometry using diode-bar optical stretchers

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.

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(WO 4 ) 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.

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.

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.

High-resolution mosaic imaging with multifocal, multiphoton photon-counting microscopy

High-resolution mosaic imaging is performed for the first time to our knowledge with a multifocal, multiphoton, photon-counting imaging system. We present a novel design consisting of a home-built femtosecond Yb-doped KGdWO(4) laser with an optical multiplexer, which is coupled with a commercial Olympus IX-71 microscope frame. Photon counting is performed using single-element detectors and an inexpensive electronic demultiplexer and counters.

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.

Phase-stable swept source OCT angiography in human skin using an akinetic source

We demonstrate noninvasive structural and microvascular contrast imaging of human skin in vivo, using phase difference swept source OCT angiography (pOCTA). The pOCTA system employs an akinetic, all-semiconductor, highly phase-stable swept laser source which operates at 1340 nm central wavelength, with 37 nm bandwidth (at 0 dB region) and 200 kHz A-scan rate. The phase sensitive detection does not need any external phase stabilizing implementations, due to the outstanding high phase linearity and sweep phase repeatability within 2 mrad. We compare the performance of phase based OCTA to speckle based OCTA for visualizing human vascular networks. pOCTA shows better contrast especially for deeper vascular details as compared to speckle based OCTA. The phase stability of the akinetic source allows the OCTA system to show decent vascular contrast only with 2 B-scans. We compare the performance of using 2 versus 4 B-scans for calculating the vascular contrast. Finally, the performance of a 100 nm bandwidth akinetic laser at 1310 nm is investigated for both OCT and OCTA.