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

Improved resolution and speed in nonlinear microscopy

Department Head: Anthony A. Maciejewski.2010 Spring.Includes bibliographical references (pages 115-124).Optical microscopy is an important tool for biomedical research. New techniques for microscopy enable new capabilities for studying biological systems. Moreover, in optical microscopy, the polarization state of the focal field strongly influences the images formed due to the impact of focal spot size, adjusting the relative strength and phase of both transverse and longitudinal field components, and manipulating inter- action with the sample under study. In particular, coherent nonlinear microscopies, such as third harmonic generation (THG), and second harmonic generation (SHG), offer rich possibilities for new control over the imaging process. In the first part of this dissertation, I demonstrate that control over the spatial polarization state of the focal field can be used to improve the spatial resolution in a laser-scanning THG microscopy. First, we show a detailed design of our nonlinear scanning microscope, then we introduce a non-iterative algorithm for measurement of spatially inhomogeneous polarization distributions in third-harmonic generation microscopy. We also, show control of spatial polarization state of the focal field through imaging of a spatial light modulator to the focus of a microscope objective. Then, we introduced a novel technique for enhancing resolution in THG microscopy, through spatial polarization shaping at the focal field. In the second part of this dissertation, we show an alternative method to laser- scanning nonlinear microscopy in biological tissue, namely, nonlinear holographic microscopy. First, we introduce the foundation of nonlinear holographic microscopy by reviewing linear off-axis holography. We start by introducing digital recording in off- axis holography, its limitations, and show how through holography we can obtain 3-D images from 2-D data. We then explore numerical reconstruction of the object field from the recorded holograms. Finally, we expand this technique to SHG nonlinear holographic microscopy to construct 3-dimensional images of biological tissues

In vitro and in vivo antibiofilm activity of the synthetic antimicrobial peptide WLBU2 against multiple drug resistant Pseudomonas aeruginosa strains

Abstract Background The global crisis of antibiotic resistance increases the demand for the novel promising alternative drugs such as antimicrobial peptides (AMPs). Here, the antibiofilm activity of the WLBU2 peptide against Pseudomonas aeruginosa (P. aeruginosa) isolates was investigated in this study. Methods Two clinical MDR and carbapenem resistant P. aeruginosa (CRPA) isolates, and standard P. aeruginosa ATCC 27,853 were investigated. The MIC and MBC of WLBU2 were determined. The MBIC was determined to evaluate inhibitory activity of WLBU2 on biofilm formation and MBEC to dispersal activity on preformed biofilm. The relative expression levels of biofilm-associated genes including rhlI, rhlR, lasI and lasR were analyzed using RT-qPCR. In vivo evaluation of inhibitory effect of WLBU2 on biofilm formation was performed in the murine models of P. aeruginosa biofilm-associated subcutaneous catheter infection. Results MIC and MBC of WLBU2 for both MDR and ATCC 27,853 P. aeruginosa strains were 8 and 16 µg/mL, respectively, while both the MIC and MBC against the CR strain were 4 µg/mL. MBIC was estimated to be 64 µg/ml for all strains. MBEC against MDR and ATCC 27,853- P. aeruginosa strains was 128 µg/ml and against CRPA was 64 µg/ml. The bacterial adhesion to a static abiotic solid surface (the surface in the polypropylene microtiter wells) was significantly inhibited at 1/4× MIC in all P. aeruginosa strains and at 1/8× MIC in CRPA strain (P < 0.05). Following treatment with WLBU2 at 1/8× MIC, significant inhibition in biofilm formation was observed in all isolates (P < 0.05). Results of the colorimetric assay showed that WLBU2 at 4× MIC was able to disperse 69.7% and 81.3% of pre-formed biofilms on abiotic surface produced by MDR and standard (ATCC 27,853) P. aeruginosa, respectively (P < 0.03), while a 92.2% reduction in the CRPA biofilm was observed after treatment with 4× MIC WLBU2 (P < 0.03). The expression levels of all genes in isolates treated with 1/2 MIC of WLBU2 were down-regulated by more than four-fold compared to the untreated isolates (P < 0.05). WLBU2 significantly inhibited biofilm formation in murine catheter-associated CRPA infection model at 1/4×MIC, 1/2×MIC, and 1×MIC by 33%, 52%, and 67%, respectively. Conclusion Considering relatively strong inhibitory and eradication potency of WLBU2 on the P. aeruginosa biofilms in in vitro and in vivo conditions, the peptide can be considered as a promising candidate for designing an antibiofilm drug