Super-resolution wide-field optical microscopy by use of Evanescent standing waves

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2007.Vita.Includes bibliographical references.The development of high resolution, high speed imaging techniques allows the study of dynamical processes in biological systems. Optical fluorescence microscopy is an essential tool for investigations in many disciplines in biology and medicine with molecular specificity. The resolution of optical far-field microscopy has been limited by the wave nature of light. In this thesis, a microscopy technique, standing wave total internal reflection fluorescence (SW-TIRF), has been developed with resolution beyond the classical diffraction limit. The SW-TIRF approach modifies the point-spread function to effectively decrease the excitation wavelength by utilizing an evanescent standing wave, carrying high spatial frequency information near the interface between the specimen and a high refractive index substrate. Evanescent standing wave illumination is used to generate a sinusoidal, high-spatial frequency, fringe pattern on the specimen providing lateral resolution enhancement. Furthermore, the less than 100 nm penetration depth of the evanescent field from the substrate ensures a thin excitation region resulting in low background fluorescence. The first experimental realization of SW-TIRF in an objective-launched geometry demonstrates the potential for super-resolution imaging at high speed in wide-field microscopy.(cont.) The super-resolution has been realized with the effective point-spread function providing better than a fifth of the emission wavelength or approximately 100 nm, which is better than twice that of conventional microscopy. In addition, imaging biological specimens with SW-TIRF demonstrated the performance revealing the fine actin cytoskeleton structures of fibroblasts. On the other hand, the surface plasmons induced by evanescent fields at a specific angle can generate an enhanced electric field which can effectively excite fluorophores near a metal coated surface. We observed a unique doughnut-shaped point-spread function of surface plasmon coupled emission and explained it with theoretical modeling using vector field theory. The combination of surface plasmon resonance fluorescence imaging and SW-TIRF resulted in a novel high-resolution microscopy, the standing wave surface plasmon resonance fluorescence (SW-SPRF) microscopy. These findings may allow super-resolution imaging with even higher sensitivity and signal-to-noise ratio at high imaging speed.by Euiheon Chung.Ph.D

Tumor detection and treatment by means of thermography and laser irradiation

While passive thermal imaging of temperature difference between tumor and neighboring tissue provides limited contrast, active thermography with external thermal modulation may provide higher contrast presumably due to the distinct thermal response from the tumor tissue. We have investigated physiologically relevant parameters such as response rate with respect to thermal modulation and relaxation time. This new imaging modality in the infrared regime may prove useful as a label-free and non-invasive screening tool. On the other hand, we propose a novel use of controlled thermal injury from non-ablative fractional laser irradiation for early treatment of tumor growth with a fiber-based thulium laser at 1927 nm. We investigated the potential cancer prevention effect with mouse model of early tumor. This laser treatment could potentially be an alternative anticancer modality for early tumorigenesis in a minimally invasive manner. Please click Additional Files below to see the full abstract

Multiphoton tissue imaging by using moxifloxacin

Multiphoton microscopy has been widely used for in-vivo tissue imaging of various biological studies. However, its application to clinical studies has been limited due to either lack of clinically compatible exogenous contrast agents or weak autofluorescence of tissues. We investigated moxifloxacin as a contrast agent of cells for multiphoton tissue imaging. Moxifloxacin is an FDA approved antibiotic with relatively good pharmacokinetic properties for tissue penetration and intrinsic fluorescence. Two-photon microscopy (TPM) of moxifloxacin treated mouse corneas showed good tissue penetration and high concentration inside the corneal cells [1]. Cell labeling of moxifloxacin was tested in both cultured cells and isolated immune cells. Moxifloxacin tissue applications were tested in various mouse organs such as the skin, small intestine, and brain. Most of tissues were labeled well via topical administration, and only the skin required additional gentle removal of the outermost stratum corneum by tape stripping. TPM of these tissues showed non-specific cell labeling of moxifloxacin and fluorescence enhancement [2]. Although most of experimental results were from mouse tissues, its clinical application would be possible. Clinical application is promising since imaging based on moxifloxacin labeling could be 10 times faster than imaging based on endogenous fluorescence. Moxifloxacin labeling of cultured cells was demonstrated by comparing TPM images with and without moxifloxacin treatment. Bright fluorescence inside cells were observed only with moxifloxacin at the same imaging condition. TPM of the skin dermis visualized many dermal cells with increased fluorescence, and TPM of the villus in the small intestine showed the covering epithelial cells and cells inside the villus clearly. Please click Additional Files below to see the full abstract

Relation between speckle decorrelation and optical phase conjugation (OPC)- based turbidity suppression through dynamic scattering media: a study on in vivo mouse skin

Light scattering in biological tissue significantly limits the accessible depth for localized optical interrogation and deep-tissue optical imaging. This challenge can be overcome by exploiting the time-reversal property of optical phase conjugation (OPC) to reverse multiple scattering events or suppress turbidity. However, in living tissue, scatterers are highly movable and the movement can disrupt time-reversal symmetry when there is a latency in the OPC playback. In this paper, we show that the motion-induced degradation of the OPC turbidity-suppression effect through a dynamic scattering medium shares the same decorrelation time constant as that determined from speckle intensity autocorrelation – a popular conventional measure of scatterer movement. We investigated this decorrelation characteristic time through a 1.5-mm-thick dorsal skin flap of a living mouse and found that it ranges from 50 ms to 2.5 s depending on the level of immobilization. This study provides information on relevant time scales for applying OPC to living tissue

Secreted Gaussia Luciferase as a Biomarker for Monitoring Tumor Progression and Treatment Response of Systemic Metastases

Background: Currently, only few techniques are available for quantifying systemic metastases in preclinical model. Thus techniques that can sensitively detect metastatic colonization and assess treatment response in real-time are urgently needed. To this end, we engineered tumor cells to express a naturally secreted Gaussia luciferase (Gluc), and investigated its use as a circulating biomarker for monitoring viable metastatic or primary tumor growth and their treatment responses. Methodology/Principal Findings: We first developed orthotopic primary and metastatic breast tumors with derivative of MDA-MB-231 cells expressing Gluc. We then correlated tumor burden with Gluc activity in the blood and urine along with bioluminescent imaging (BLI). Second, we utilized blood Gluc assay to monitor treatment response to lapatinib in an experimental model of systemic metastasis. We observed good correlation between the primary tumor volume and Gluc concentration in blood (R2 = 0.84) and urine (R2 = 0.55) in the breast tumor model. The correlation deviated as a primary tumor grew due to a reduction in viable tumor fraction. This was also supported by our mathematical models for tumor growth to compare the total and viable tumor burden in our model. In the experimental metastasis model, we found numerous brain metastases as well as systemic metastases including bone and lungs. Importantly, blood Gluc assay revealed early growth of metastatic tumors before BLI could visualize their presence. Using secreted Gluc, we localized systemic metastases by BLI and quantitatively monitored the total viable metastatic tumor burden by blood Gluc assay during the course of treatment with lapatinib, a dual tyrosine kinase inhibitor of EGFR and HER2. Conclusion/Significance: We demonstrated secreted Gluc assay accurately reflects the amount of viable cancer cells in primary and metastatic tumors. Blood Gluc activity not only tracks metastatic tumor progression but also serves as a longitudinal biomarker for tumor response to treatments

Secreted Gaussia Luciferase as a Biomarker for Monitoring Tumor Progression and Treatment Response of Systemic Metastases

Currently, only few techniques are available for quantifying systemic metastases in preclinical model. Thus techniques that can sensitively detect metastatic colonization and assess treatment response in real-time are urgently needed. To this end, we engineered tumor cells to express a naturally secreted Gaussia luciferase (Gluc), and investigated its use as a circulating biomarker for monitoring viable metastatic or primary tumor growth and their treatment responses.We first developed orthotopic primary and metastatic breast tumors with derivative of MDA-MB-231 cells expressing Gluc. We then correlated tumor burden with Gluc activity in the blood and urine along with bioluminescent imaging (BLI). Second, we utilized blood Gluc assay to monitor treatment response to lapatinib in an experimental model of systemic metastasis. We observed good correlation between the primary tumor volume and Gluc concentration in blood (R(2) = 0.84) and urine (R(2) = 0.55) in the breast tumor model. The correlation deviated as a primary tumor grew due to a reduction in viable tumor fraction. This was also supported by our mathematical models for tumor growth to compare the total and viable tumor burden in our model. In the experimental metastasis model, we found numerous brain metastases as well as systemic metastases including bone and lungs. Importantly, blood Gluc assay revealed early growth of metastatic tumors before BLI could visualize their presence. Using secreted Gluc, we localized systemic metastases by BLI and quantitatively monitored the total viable metastatic tumor burden by blood Gluc assay during the course of treatment with lapatinib, a dual tyrosine kinase inhibitor of EGFR and HER2.We demonstrated secreted Gluc assay accurately reflects the amount of viable cancer cells in primary and metastatic tumors. Blood Gluc activity not only tracks metastatic tumor progression but also serves as a longitudinal biomarker for tumor response to treatments

Optical phase conjugation assisted scattering lens: variable focusing and 3D patterning

Variable light focusing is the ability to flexibly select the focal distance of a lens. This feature presents technical challenges, but is significant for optical interrogation of three-dimensional objects. Numerous lens designs have been proposed to provide flexible light focusing, including zoom, fluid, and liquid-crystal lenses. Although these lenses are useful for macroscale applications, they have limited utility in micron-scale applications due to restricted modulation range and exacting requirements for fabrication and control. Here, we present a holographic focusing method that enables variable light focusing without any physical modification to the lens element. In this method, a scattering layer couples low-angle (transverse wave vector) components into a full angular spectrum, and a digital optical phase conjugation (DOPC) system characterizes and plays back the wavefront that focuses through the scattering layer. We demonstrate micron-scale light focusing and patterning over a wide range of focal distances of 22–51 mm. The interferometric nature of the focusing scheme also enables an aberration-free scattering lens. The proposed method provides a unique variable focusing capability for imaging thick specimens or selective photoactivation of neuronal networks

Wide-field extended-resolution fluorescence microscopy with standing surface-plasmon-resonance waves

The resolution of conventional SPR imaging has been limited by the diffraction nature of light. A wide-field extended-resolution optical imaging technique, standing-wave surface plasmon resonance fluorescence (SW-SPRF) microscopy, has been developed. Based on evanescent SPR standing waves, SW-SPRF provides lateral resolution approaching 100 nm and offers the advantages of significant signal enhancement and background noise reduction. SW-SPRF has the potential for sensitive biomolecular detection, nanoscale imaging, and lithographic applications