Jeffrey J. Field

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.

Ultra-large field-of-view two-photon microscopy

We present a two-photon microscope that images the full extent of murine cortex with an objective-limited spatial resolution across an 8 mm by 10 mm field. The lateral resolution is approximately 1 µm and the maximum scan speed is 5 mm/ms. The scan pathway employs large diameter compound lenses to minimize aberrations and performs near theoretical limits. We demonstrate the special utility of the microscope by recording resting-state vasomotion across both hemispheres of the murine brain through a transcranial window and by imaging histological sections without the need to stitch.

Photon counting, censor corrections, and lifetime imaging for improved detection in two-photon microscopy

We present a high-speed photon counter for use with two-photon microscopy. Counting pulses of photocurrent, as opposed to analog integration, maximizes the signal-to-noise ratio so long as the uncertainty in the count does not exceed the gain-noise of the photodetector. Our system extends this improvement through an estimate of the count that corrects for the censored period after detection of an emission event. The same system can be rapidly reconfigured in software for fluorescence lifetime imaging, which we illustrate by distinguishing between two spectrally similar fluorophores in an in vivo model of microstroke.

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.

Quartic-phase-limited grism-based ultrashort pulse shaper

By replacing the dispersive element in a zero-dispersion pulse shaper with a grism, we have constructed a quartic-phase-limited pulse shaper. We demonstrate compensation of 4500 fs2 without the use of a dynamic element in the pulse shaping line, which is approximately the amount of dispersion induced by a typical multiphoton microscope. We also demonstrate that detuning the pulse shaper to compensate for quadratic phase induces negligible spatial chirp, thereby maintaining a high-quality focal spot for a microscopy setup.

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.

Superresolved multiphoton microscopy with spatial frequency-modulated imaging

Significance Superresolution microscopy is indispensable in biological sciences. The vast majority of superresolution imaging techniques exploit real energetic states of fluorescent molecules to break the diffraction limit. To date, superresolved imaging of second- and third-harmonic generation has been limited to specific sample preparations where the polarization state of the excitation laser can be manipulated to overcome the diffraction limit. Here, we describe a method for multiphoton superresolved imaging that does not place such restrictions on the sample and allows for simultaneous superresolved imaging of both coherent and incoherent signal light. Combined with single-element detection, this technique may allow for significant advances in multimodal multiphoton imaging of highly scattering biological tissues. Superresolved far-field microscopy has emerged as a powerful tool for investigating the structure of objects with resolution well below the diffraction limit of light. Nearly all superresolution imaging techniques reported to date rely on real energy states of fluorescent molecules to circumvent the diffraction limit, preventing superresolved imaging with contrast mechanisms that occur via virtual energy states, including harmonic generation (HG). We report a superresolution technique based on spatial frequency-modulated imaging (SPIFI) that permits superresolved nonlinear microscopy with any contrast mechanism and with single-pixel detection. We show multimodal superresolved images with two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) from biological and inorganic media. Multiphoton SPIFI (MP-SPIFI) provides spatial resolution up to 2η below the diffraction limit, where η is the highest power of the nonlinear intensity response. MP-SPIFI can be used to provide enhanced resolution in optically thin media and may provide a solution for superresolved imaging deep in scattering media.