Emily A. Gibson

Efficient reflection grisms for pulse compression and dispersion compensation of femtosecond pulses

Efficient reflection grisms for pulse-compression and material-dispersion compensation have been designed and demonstrated in a 40 fs, 300 microJ, 5 kHz downchirped pulse amplification system for the first time to our knowledge. A grism design for 800 nm femtosecond laser pulse dispersion compensation applications is realized by using standard, commercial diffraction gratings.

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.

Multiphoton Microscopy for Ophthalmic Imaging

We review multiphoton microscopy (MPM) including two-photon autofluorescence (2PAF), second harmonic generation (SHG), third harmonic generation (THG), fluorescence lifetime (FLIM), and coherent anti-Stokes Raman Scattering (CARS) with relevance to clinical applications in ophthalmology. The different imaging modalities are discussed highlighting the particular strength that each has for functional tissue imaging. MPM is compared with current clinical ophthalmological imaging techniques such as reflectance confocal microscopy, optical coherence tomography, and fluorescence imaging. In addition, we discuss the future prospects for MPM in disease detection and clinical monitoring of disease progression, understanding fundamental disease mechanisms, and real-time monitoring of drug delivery.

Phase-matching techniques for coherent soft X-ray generation

Coherent beams at soft X-ray (SXR) wavelengths can be generated using extreme nonlinear optics by focusing an intense laser into a gas. In this paper, we discuss phase-matching and quasi-phase-matching techniques that use gas-filled modulated waveguides to enhance the frequency conversion process. This leads to the generation of SXR beams that are both spatially and temporally coherent.

Raman and coherent anti-Stokes Raman scattering microscopy studies of changes in lipid content and composition in hormone-treated breast and prostate cancer cells

Abstract. Increasing interest in the role of lipids in cancer cell proliferation and resistance to drug therapies has motivated the need to develop better tools for cellular lipid analysis. Quantification of lipids in cells is typically done by destructive chromatography protocols that do not provide spatial information on lipid distribution and prevent dynamic live cell studies. Methods that allow the analysis of lipid content in live cells are therefore of great importance. Using micro-Raman spectroscopy and coherent anti-Stokes Raman scattering (CARS) microscopy, we generated a lipid profile for breast (T47D, MDA-MB-231) and prostate (LNCaP, PC3) cancer cells upon exposure to medroxyprogesterone acetate (MPA) and synthetic androgen R1881. Combining Raman spectra with CARS imaging, we can study the process of hormone-mediated lipogenesis. Our results show that hormone-treated cancer cells T47D and LNCaP have an increased number and size of intracellular lipid droplets and higher degree of saturation than untreated cells. MDA-MB-231 and PC3 cancer cells showed no significant changes upon treatment. Principal component analysis with linear discriminant analysis of the Raman spectra was able to differentiate between cancer cells that were treated with MPA, R1881, and untreated.

Direct trabecular meshwork imaging in porcine eyes through multiphoton gonioscopy

Abstract. The development of technologies to characterize the ocular aqueous outflow system (AOS) is important for the understanding of the pathophysiology of glaucoma. Multiphoton microscopy (MPM) offers the advantage of high-resolution, label-free imaging with intrinsic image contrast because the emitted signals result from the specific biomolecular content of the tissue. Previous attempts to use MPM to image the murine irido-corneal region directly through the sclera have suffered from degradation in image resolution due to scattering of the focused laser light. As a result, transscleral MPM has limited ability to observe fine structures in the AOS. In this work, the porcine irido-corneal angle was successfully imaged through the transparent cornea using a gonioscopic lens to circumvent the highly scattering scleral tissue. The resulting high-resolution images allowed the detailed structures in the trabecular meshwork (TM) to be observed. Multimodal imaging by two-photon autofluorescence and second harmonic generation allowed visualization of different features in the TM without labels and without disruption of the TM or surrounding tissues. MPM gonioscopy is a promising noninvasive imaging tool for high-resolution studies of the AOS, and research continues to explore the potential for future clinical applications in humans.

High-throughput examination of fluorescence resonance energy transfer-detected metal-ion response in mammalian cells.

Fluorescence resonance energy transfer (FRET)-based genetically encoded metal-ion sensors are important tools for studying metal-ion dynamics in live cells. We present a time-resolved microfluidic flow cytometer capable of characterizing the FRET-based dynamic response of metal-ion sensors in mammalian cells at a throughput of 15 cells/s with a time window encompassing a few milliseconds to a few seconds after mixing of cells with exogenous ligands. We have used the instrument to examine the cellular heterogeneity of Zn(2+) and Ca(2+) sensor FRET response amplitudes and demonstrated that the cluster maps of the Zn(2+) sensor FRET changes resolve multiple subpopulations. We have also measured the in vivo sensor response kinetics induced by changes in Zn(2+) and Ca(2+) concentrations. We observed an ∼30 fold difference between the extracellular and intracellular sensors.

Using a genetically targeted sensor to investigate the role of presenilin-1 in ER Ca2+ levels and dynamics

The ER plays a fundamental role in storing cellular Ca(2+), generating Ca(2+) signals, and modulating Ca(2+) in both the cytosol and mitochondria. Genetically encoded Ca(2+) sensors can be explicitly targeted to the ER to directly define Ca(2+) levels and monitor fluxes of Ca(2+) within this organelle. In this study we use an ER-targeted Ca(2+) sensor to define both the level and dynamics of ER Ca(2+) in cells expressing mutant presenilin proteins. Growing evidence suggests the enigmatic presenilin-1 plays a role in regulating ER Ca(2+). Presenilin-1 was initially identified in a screen for genetic causes of inherited familial Alzheimers disease (fAD). The connection between presenilin-1, calcium regulation, and Alzheimers disease may provide the key to understanding the long-observed, but poorly understood, link between Alzheimers disease and Ca(2+) dysregulation. In this study we examined seven fAD-causing mutations in presenilin-1 to define how they influence ER Ca(2+) levels and dynamics. We observed that some, but not all, mutations in PS1 decrease the level of Ca(2+) within the ER and this difference depends on the enzymatic activity of PS1. Two mutations tested altered the kinetics of Ca(2+) release from the ER upon ATP stimulation, resulting in faster spiking. Combined, these results indicate that mutations in PS1 can alter the balance of Ca(2+) in cells and have the potential to influence the nature of Ca(2+) signals.

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.

Multi-kilohertz repetition rate Ti:sapphire amplifier based on down-chirped pulse amplification

We present a novel ultrafast multipass laser amplifier design optimized for sub-millijoule output energy and capable of being operated at repetition rates exceeding 40 kHz. This ti:sapphire based system makes use of a grism based stretcher, a cryogenically cooled ti:sapphire crystal and an astigmatically compensated multipass amplifier design that allows for pumping with significantly lower pump pulse energies than has been demonstrated to date. We also make use of the downchirped pulse amplification scheme to minimize loss in the pulse compression process. Preliminary experiments demonstrate an output pulse energy of 290 muJ at 10 kHz and 270 muJ at 15 kHz with a pulse duration of 36 fs.