Session D19: Focus Session: New Frontiers in Imaging III

Tissue Imaging and Multidimensional Spectroscopy Using Shaped Femtosecond Laser Pulses

We use rapidly updatable, femtosecond pulse shaping and multidimensional spectroscopy to make new targets accessible by nonlinear optical imaging. For example, we observe two-photon absorption (TPA), sum frequency absorption (SFA) and self phase modulation (SPM)). Detection of TPA and related effects, such as the local quantum yield (fluorescence/absorption) permits direct observation of important endogenous molecular markers which are invisible in multiphoton fluorescence microscopy; it also permits excitation in the long-wavelength water windows which have significantly reduced scattering, but little endogenous two-photon fluorescence. The fundamental problem is that at the powers one might reasonably apply to tissue (e.g. 5 mW from a modelocked laser) typically $10^{-6}$of the light is removed by TPA, with the rest lost to scattering and linear absorption; and SPM does not broaden the spectrum in the dramatic way associated with (for example) continuum generation. A variety of solutions to these problems using femtosecond pulse shaping will be presented. The simplest solution, which uses amplitude modulation of a fs pulse train, has led to high quality microscopic images of the melanin distribution in melanotic lesions, and has led to discrimination between the different types of melanin in melanosomes. Shaping individual pulses instead of the envelope permits high sensitivity detection of both SPM and TPA via spectral hole refilling combined with heterodyne detection. We manufacture laser pulses with a narrow (ca. 3 nm) spectral hole, which can only be refilled by nonlinear processes; TPA causes refilling 180 degrees out of phase with the wings of the pulse, SPM is 90 degrees out of phase. By inserting a phase-coherent pedestal in the hole, then repeating the experiment with a different phase on a timescale rapid compared to any physiological processes, we can extract the phase of the refilling, hence the relative contributions of SPM and TPA. This method can extract excellent signatures from hemoglobin as well as melanin. We have also used it to image neurons firing in tissue, and to characterize off-diagonal peaks of contrast agents in two-dimensional spectra.   

Laser-detected Magnetic Resonance Imaging

Magnetic resonance imaging is often performed in the presence of a superconducting magnet for high polarization and sensitive detection. However the cost and immobility of the system impose some restrictions on its applications. To overcome these limiting factors, we present an alternative detection technique: laser-based atomic magnetometry. This technique detects nuclear magnetization at virtually room temperature with an excellent sensitivity at low fields, eliminating the necessity of cryogenics and a homogenous high magnetic field. We show the characteristics of a gradiometer based on two atomic magnetometers and its coupling to a low-field encoding setup. Various flow images are obtained, with spatial resolution reaching sub-millimeter regime. Additional applications and future developments are discussed.   

New Techniques for Signal Optimization in Harmonic and Multiphoton Absorption Fluorescence Microscopy

Nonlinear imaging with ultrafast lasers continues to broaden its application base as a significant tool for exploring and understanding biological structure and function at the microscopic level. The challenge is significant - the biological community needs to be able to quantitatively visualize 100 cubic micrometer volumes with a resolution of 50 nm, and do so in a dynamic fashion -- millisecond time scales are desirable. In order to achieve these demanding imaging requirements we need to strive to achieve new levels of efficiency -- improved resolution is a function of how many photons can be extracted from ever smaller volumes. Towards this end, in this talk we discuss new methods for fiber delivery of femtosecond pulses, spatio-temporal characterization of femtosecond pulses through high-numerical aperture optics, and adaptive spatio-temporal control of these pulses.   

Research Applications of Photoelectron Emission Microscopy

Photoelectron emission microscopy (PEEM) is a developing technique that images electrons emitted from conductor and semiconductor surfaces under UV, X-ray, or laser irradiation. Low energy PEEM can reveal surface morphology on a 10 nm scale and is sensitive material properties such as phase, adsorbed molecules, surface electronic structure, and other physical properties that affect work function and hence the photoelectron yield. We have used PEEM to study phase transformation in shape memory alloys diffusion of Cu in Cu/Ru bilayers and laser-induced oxygen vacancy creation on TiO$_{2}$. Femtosecond laser irradiation from a frequency-doubled Ti:sapphire oscillator was used to remove bridge-bonded oxygen atoms. To further illustrate the utility of PEEM, we will discuss applications in different fields such as thermal-induced structural phase transformation of shape memory alloys and diffusion of Cu through an Ru barrier layer.   

Medical Applications of X-Ray Phase Contrast Imaging

We report the use of an inline holographic x-ray imaging technique for medical purposes. In contrast to conventional x-ray radiography a phase-sensitive x-ray imaging method is employed. This phase-contrast x-ray imaging is fundamentally different from conventional x-ray shadowgraphy because the mechanism of image formation does not rely on differential absorption by tissues. Instead, x-ray beams undergo differential phase shifts in passing through an organ and subsequently interfere constructively or destructively at the x-ray camera. Hence, tissues are distinguished by their different indices of refraction rather than their absorptive properties. This imaging method is more than a thousand times more sensitive to density variations of tissues than conventional absorption methods and enables imaging of soft tissues with high contrast without the use of contrast agents. For example, we will present images of mouse livers yielding resolution of arterial capillaries as small as tens of micrometers. We also show the imaging technique operates in combination with ultrasound-induced, tissue-selective, differential movement of cancer tumors which highlights the tumor of interest and in some cases obviates the need for chemical contrasting agents.   

Optical/MRI Multimodality Molecular Imaging

Multimodality molecular imaging that combines anatomical and functional information has shown promise in development of tumor-targeted pharmaceuticals for cancer detection or therapy. We present a new multimodality imaging technique that combines fluorescence molecular tomography (FMT) and magnetic resonance imaging (MRI) for in vivo molecular imaging of preclinical tumor models. Unlike other optical/MRI systems, the new molecular imaging system uses parallel phase acquisition based on heterodyne principle. The system has a higher accuracy of phase measurements, reduced noise bandwidth, and an efficient modulation of the fluorescence diffuse density waves. Fluorescent Bombesin probes were developed for targeting breast cancer cells and prostate cancer cells. Tissue phantom and small animal experiments were performed for calibration of the imaging system and validation of the targeting probes.   

Compressed Sensing for Multispectral and Confocal Microscopy

Compressive sensing is an emerging field based on the revelation that a small number of random linear projections of a signal or an image contain enough information for reconstruction of a high resolution one. This technique has been applied to magnetic resonance imaging and neutron scattering. We have previously developed an optical camera based on this concept which is capable of megapixel images while utilizing a single photodiode for acquisition and implemented through the use of a digital micromirror device to randomly modulate and acquire the necessary projections of the image. In addition, this scheme allows for the rapid acquisition of multispectral information. We are now extending this scheme to imaging beyond the visible spectrum into the infrared and terahertz where high resolution image sensors are much more costly. Lastly we will present a scheme for utilizing this method in confocal microscopy similar to the flying pinhole concept except that the individual pinhole is replaced by a complex random projection and reconstructed via linear programming.   

Source localization of auditory evoked responses from a human brain with an atomic magnetometer

We report first measurements of auditory evoked fields (AEF) in a human brain with an atomic magnetometer system and discuss the techniques for magnetic source localization using this system. Until recent development of spin-exchange relaxation free (SERF) atomic magnetometers with a sensitivity of 0.5fT/Hz$^{1/2}$, only SQUID magnetometers had sufficient sensitivity to measure a magnetoencephalograph (MEG). With simple multi-channel operation and no cryogenic maintenance, the atomic magnetometer provides a promising alternative for brain activity measurements. A clear N100m feature in AEF was observed after averaging over 600 stimuli. Currently the intrinsic magnetic noise level is 3.5 fT/Hz$^{1/2}$ at 10 Hz. Optical detection of magnetic fields allows flexibility in magnetic mapping while in the same time imposing certain geometrical constraints. To investigate the magnetic source localization capabilities of the atomic MEG system we performed extensive numerical simulations and measurements with a brain phantom consisting of an artificial current source in a saline-filled sphere. We will discuss the results of numerical analysis and experimental implementation of magnetic source localization with atomic magnetometer.