Dawn Schafer

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.

In situ quantitative measurement of concentration profiles in a microreactor with submicron resolution using multiplex CARS microscopy

In situ quantitative imaging of concentration profiles of reactants and products inside a microfluidic reactor is achieved, with submicron spatial resolution with mM sensitivity and on ms time scales, for a given position. The label-free approach relies on quantitative vibrational spectroscopy, using Coherent Anti-Stokes Raman scattering microscopy in a spectrally resolved fashion, and is demonstrated on an elementary acid-base reaction.

Microfluidic cell counter with embedded optical fibers fabricated by femtosecond laser ablation and anodic bonding

A simple fabrication technique to create all silicon/glass microfluidic devices is demonstrated using femtosecond laser ablation and anodic bonding. In a first application, we constructed a cell counting device based on small angle light scattering. The counter featured embedded optical fibers for multiangle excitation and detection of scattered light and/or fluorescence. The performance of the microfluidic cell counter was benchmarked against a commercial fluorescence-activated cell sorter.

Three-dimensional chemical concentration maps in a microfluidic device using two-photon absorption fluorescence imaging

Two-photon absorption fluorescence is employed within a microfluidic device to create a three-dimensional chemical concentration map for mixing uniformity characterization. This multiphoton technique images fluorescence intensity directly and provides a simple, rapid, and readily employed route to composition characterization within microfluidic systems.

Optically integrated microfluidic systems for cellular characterization and manipulation

Expanding interest in microfluidic techniques for biomedical applications has driven the recent need for micro-integrated optics capable of both traditional characterization and emerging optical manipulation techniques. We discuss here how ultrafast laser micromachining can be used to create optical waveguides directly within microfluidic systems. We then utilize this fabrication approach to create a unique microfluidic platform for optical characterization and sorting of cells and particles. This new platform employs optically fabricated waveguides to scatter and refract light from individual particles, allowing accurate in situ size detection and sorting within a microfluidic channel.

Coherent anti-Stokes Raman scattering microscopy for quantitative characterization of mixing and flow in microfluidics

We present an optical, noninvasive and label-free approach to characterize flow profiles in microfluidic devices. Coherent anti-Stokes Raman scattering signals were used to map the mass transport in a microfluidic device that was then related back to the local flow rate of dilute solutes having constant fluid properties. Flow characterization was demonstrated in two common types of microfluidic devices, polydimethylsiloxane/glass square channels and wet-etched glass tapered channels.

Linear spatio-temporal characterization of a UV microscope objective for nonlinear imaging and spectroscopy by using two-dimensional spectral interferometry.

Two‐dimensional Fourier transform spectral interferometry is used to characterize the spatio‐temporal aberrations of a UV microscope objective. The spatial and temporal profiles of a 420 nm, 38 fs pulse at the focus of a 0.32 NA UV objective are then deduced using a wave propagation code incorporating the measured aberrations.

Multifocal, multi-modal, photon counting, multiphoton microscopy

High-speed nonlinear imaging systems capable of dynamically imaging multiple focal planes simultaneously, in multiple modalities (two photon excitation fluorescence, second harmonic generation, and third harmonic generation), are demonstrated for the first time.

Two-photon absorption fluorescence imaging to characterize microfluidic device performance

Two-photon absorption fluorescence imaging is used to quantitatively measure 3D flow and mixing in microfluidics. This is an important characterization tool for developing optimal microfluidic devices for use in the study of biological molecular dynamics.

An optically integrated microfluidic cell counter fabricated by femtosecond laser ablation and anodic bonding

We describe a method for integrating fiber optics in substrates by femtosecond laser ablation. In a first demonstration, we fabricate an optically integrated microfluidic device that counts cells by small angle light scattering.