Kraig E. Sheetz

Invited Review Article: Imaging techniques for harmonic and multiphoton absorption fluorescence microscopy

We review the current state of multiphoton microscopy. In particular, the requirements and limitations associated with high-speed multiphoton imaging are considered. A description of the different scanning technologies such as line scan, multifoci approaches, multidepth microscopy, and novel detection techniques is given. The main nonlinear optical contrast mechanisms employed in microscopy are reviewed, namely, multiphoton excitation fluorescence, second harmonic generation, and third harmonic generation. Techniques for optimizing these nonlinear mechanisms through a careful measurement of the spatial and temporal characteristics of the focal volume are discussed, and a brief summary of photobleaching effects is provided. Finally, we consider three new applications of multiphoton microscopy: nonlinear imaging in microfluidics as applied to chemical analysis and the use of two-photon absorption and self-phase modulation as contrast mechanisms applied to imaging problems in the medical sciences.

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.

Ultrafast optics: Imaging and manipulating biological systems

The rapid evolution of ultrafast optics technology over the past two decades has opened the window to a broad range of applications in biology and medicine. Compact, reliable, and turn-key ultrafast laser systems are enabling cutting-edge science to take place in everyday laboratories and clinics. Led by the discovery of two-photon excitation fluorescence microscopy nearly 20 years ago, the biological imaging community is exploring unique image contrast mechanisms and pushing spatial and temporal resolution to new limits. Concurrent with advancements in imaging are developments in the precision application of extremely high peak intensities available in ultrashort pulses for disrupting or manipulating targeted locations in biological systems on the submicron scale while leaving surrounding tissue healthy. The ability for scientists to selectively discriminate structures of interest at the cellular and subcellular levels under relevant physiological conditions shows tremendous promise for accelerating the ...

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.

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.

Differential Multiphoton Laser Scanning Microscopy

Multifocal multiphoton laser scanning microscopy (mfMPLSM) in the biological and medical sciences has the potential to become a ubiquitous tool for obtaining high-resolution images at video rates. While current implementations of mfMPLSM 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 mfMPLSM in which whole-field detection with a single detector, rather than detection with a matrix of detectors, such as a charge-coupled device (CCD) camera, is implemented. This advance makes mfMPLSM fully compatible for use in imaging through scattering media. Further, we demonstrate that this method makes it possible to simultaneously obtain multiple images and view differences in excitation parameters in a single scan of the specimen.

A pragmatic guide to multiphoton microscope design.

Multiphoton microscopy has emerged as a ubiquitous tool for studying microscopic structure and function across a broad range of disciplines. As such, the intent of this paper is to present a comprehensive resource for the construction and performance evaluation of a multiphoton microscope that will be understandable to the broad range of scientific fields that presently exploit, or wish to begin exploiting, this powerful technology. With this in mind, we have developed a guide to aid in the design of a multiphoton microscope. We discuss source selection, optical management of dispersion, image-relay systems with scan optics, objective-lens selection, single-element light-collection theory, photon-counting detection, image rendering, and finally, an illustrated guide for building an example microscope.

What Kind of Stories Should a Virtual Human Swap

Telling stories is an important aspect virtual agents designed to interact with people socially over time. We describe an experiment designed to investigate the impact of the identity, presentation form, and perspective of a virtual storyteller on a human user who engages in a story-swapping activity with two virtual characters. For each interaction, the user was given 10 “ice-breaker” questions to ask a virtual character and respond to the character’s reciprocal request. Participants also filled out a post-interaction survey, measuring rapport with the character and impressions of the character’s personality. Results generally show that participants prefer characters who tell first person stories, however there were some interactions with presentation order. No significant preferences were established for the form or identity variables.