Eric V. Chandler

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.

Inhomogeneities in the nonlinear tensorial responses of arrays of gold nanodots

We use second- and third-harmonic-generation microscopy to address the tensorial nonlinear responses of individual particles in an array of cylindrical gold nanodots. The responses in both orders exhibit widely-variable, polarization-dependent differences between individual nanodots and thereby indicate tensorial inhomogeneities in the sample. The result provides clear evidence that the second-order response, which is forbidden by symmetry for ideal particles, must arise from small-scale, symmetry-breaking features. A similar result for the third-order response, which is allowed for ideal particles, suggests that both nonlinear responses are dominated by strong variations in field localization around the small-scale features differing among individual nanodots.

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.

Integrated spectrometer design with application to multiphoton microscopy

We present a prism-based spectrometer integrated into a multifocal, multiphoton microscope. The multifocal configuration facilitates interrogation of samples under different excitation conditions. Notably, the image plane of the microscope and the image plane of the spectrometer are coincident eliminating the need for an intermediate image plane containing an entrance slit. An EM-CCD detector provides sufficient gain for spectral interrogation of single-emitters. We employ this spectrometer to observe spectral shifts in the two-photon excitation fluorescence emission of single CdSe nanodots as a function of excitation polarization.

Utilising Ultrafast Lasers for Multiphoton Biomedical Imaging

This chapter covers the benefits and applications of ultrafast laser scanning microscopes from a biomedical perspective. The basic architecture of a laser microscope is discussed, including how to design a laser scanning system with lateral and axial control. Also investigated is the design of custom collection optics for optimizing the detection of emitted photons and maximizing that emitted fluorescence in the presence of photobleaching. In addition, this chapter addresses three techniques novel to the biomedical community. The first is the technique of temporal focusing and its application toward wide-field imaging and micromachining. Also investigated is the concept of photon counting in multiphoton microscopy and how this approach to imaging has become practical for everyday use. Finally, several different methods are revealed for implementing spectral imaging with a multiphoton microscope platform.

Remote Focusing Differential Multiphoton Microscopy: Application to Neuronal Imaging

We apply remote focusing to multi-focal multiphoton microscopy by simultaneously imaging multiple focal planes of Drosophila melanogaster olfactory neurons. This technology permits imaging the entire volume of the antennal lobe in a single scan.

Differential Multiphoton Microscopy

High-speed nonlinear imaging systems capable of dynamically imaging differences in depth, excitation polarization, excitation wavelength, beam shape, and pulse shape with single element detection are presented for the first time.