Supercontinuum (SC) stimulated emission depletion (STED) fluorescence lifetime imaging is demonstrated by using time-correlated single-photon counting (TCSPC) detection. The spatial resolution of the developed STED instrument was measured by imaging monodispersed 40-nm fluorescent beads and then determining their fwhm, and was 36 ± 9 and 40 ± 10 nm in theX and Y coordinates, respectively. The same beads measured by confocal microscopy were 450 ± 50 and 430 ± 30 nm, which is larger than the diffraction limit of light due to underfilling the microscope objective. Underfilling the objective and time gating the signal were necessary to achieve the stated STED spatial resolution. The same fluorescence lifetime (2.0 ± 0.1 ns) was measured for the fluorescent beads by using confocal or STED lifetime imaging. The instrument has been applied to study Alexa Fluor 594-phalloidin labeled F-actin-rich projections with dimensions smaller than the diffraction limit of light in cultured cells. ABSTRACT: Supercontinuum (SC) stimulated emission depletion (STED) fluorescence lifetime imaging is demonstrated by using time-correlated single-photon counting (TCSPC) detection. The spatial resolution of the developed STED instrument was measured by imaging monodispersed 40-nm fluorescent beads and then determining their fwhm, and was 36 ± 9 and 40 ± 10 nm in the X and Y coordinates, respectively. The same beads measured by confocal microscopy were 450 ± 50 and 430 ± 30 nm, which is larger than the diffraction limit of light due to underfilling the microscope objective. Underfilling the objective and time gating the signal were necessary to achieve the stated STED spatial resolution. The same fluorescence lifetime (2.0 ± 0.1 ns) was measured for the fluorescent beads by using confocal or STED lifetime imaging. The instrument has been applied to study Alexa Fluor 594-phalloidin labeled F-actin-rich projections with dimensions smaller than the diffraction limit of light in cultured cells. Fluorescence lifetimes of the actin-rich projections range from 2.2 to 2.9 ns as measured by STED lifetime imaging.

Subdiffraction spatial resolution luminescence depletion imaging was performed with giant CdSe/14CdS nanocrystal quantum dots (g-NQDs) dispersed on a glass slide. Luminescence depletion imaging used a Gaussian shaped excitation laser pulse overlapped with a depletion pulse, shaped into a doughnut profile, with zero intensity in the center. Luminescence from a subdiffraction volume is collected from the central portion of the excitation spot, where no depletion takes place. Up to 92% depletion of the luminescence signal was achieved. An average full width at half-maximum of 40 ± 10 nm was measured in the lateral direction for isolated g-NQDs at an air interface using luminescence depletion imaging, whereas the average full width at half-maximum was 450 ± 90 nm using diffraction-limited, confocal luminescence imaging. Time-gating of the luminescence depletion data was required to achieve the stated spatial resolution. No observable photobleaching of the g-NQDs was present in the measurements, which allowed imaging with a dwell time of 250 ms per pixel to obtain images with a high signal-to-noise ratio. The mechanism for luminescence depletion is likely stimulated emission, stimulated absorption, or a combination of the two. The g-NQDs fulfill a need for versatile, photostable tags for subdiffraction imaging schemes where high laser powers or long exposure times are used.

Interest in realizing conjugated polymer-based films with controlled morphology for efficient electronic devices, including photovoltaics, requires a parallel effort to characterize these films. Scanning angle (SA) Raman spectroscopy is applied to measure poly(3-hexylthiophene) (P3HT):phenyl-C61-butyric acid methyl ester (PCBM)-blend morphology on sapphire, gold, and indium tin oxide interfaces, including functional organic photovoltaic devices. Nonresonant SA Raman spectra are collected in seconds with signal-to-noise ratios that exceed 80, which is possible due to the reproducible SA signal enhancement. Raman spectra are collected as the incident angle of the 785 nm excitation laser is precisely varied upon a prism/sample interface from approximately 35 to 70°. The width of the ∼1447 cm(-1) thiophene C═C stretch is sensitive to P3HT order, and polymer order varied depending on the underlying substrate. This demonstrates the importance of performing the spectroscopic measurements on substrates and configurations used in the functioning devices, which is not a common practice. The experimental measurements are modeled with calculations of the interfacial mean square electric field to determine the distance dependence of the SA Raman signal. SA Raman spectroscopy is a versatile method applicable whenever the chemical composition, structure, and thickness of interfacial polymer layers need to be simultaneously measured.

Time binning is used to increase the number of photon counts in the peak channel of stimulated emission depletion fluorescence lifetime decay curves to determine how it affects the resulting lifetime image. The fluorescence lifetime of the fluorophore, Alexa Fluor 594 phalloidin, bound to F-actin is probed in cultured S2 cells at a spatial resolution of ~40 nm. This corresponds to a 10-fold smaller probe volume compared to confocal imaging, and a reduced number of photons contributing to the signal. Pixel-by-pixel fluorescence lifetime measurements and error analysis show that an average of 40 ± 30 photon counts in the peak channel with a signal-to-noise ratio of 20 is enough to calculate a reliable fluorescence lifetime from a single exponential fluorescence decay. No heterogeneity in the actin cytoskeleton in different regions of the cultured cells was measured in the 40-400 nm spatial regime.

The photostability of bulk heterojunction organic photovoltaic films containing a polymer donor and a fullerene-derivative acceptor was examined using resonance Raman spectroscopy and controlled laser power densities. The polymer donors were poly(3-hexylthiophene-2,5-diyl) (P3HT), poly [[9-(1-octylnonylFour sample preparation methods were studied: (i) thin or (ii) thick films with fast solvent evaporation under nitrogen, (iii) thick films with slow solvent evaporation under nitrogen, and (iv) thin films dried under nitrogen followed by thermal annealing. Polymer order was assessed by monitoring a Raman peak's full width at half-maximum and location as a function of illumination time and laser power densities from 2.5 × 10 3 to 2.5 × 10 5 W cm −2. Resonance Raman spectroscopy measurements show that before prolonged illumination, PCDTBT and PTB7 have the same initial order for all preparation conditions, while P3HT order improves with slow solvent drying or thermal annealing. All films exhibited changes to bulk heterojunction structure with 2.5 × 10 5 Wcm −2 laser illumination as measured by resonance Raman spectroscopy, and atomic force microscopy images show evidence of sample heating that affects the polymer over an area greater than the illumination profile. Photostability data are important for proper characterization by techniques involving illumination and the development of devices suitable for real-world applications.

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CHAPTER 1 : INTRODUCTION TO IMAGING WITH AN EMPHASIS ON DEPLETION-BASED SUBDIFFRACTION TECHNIQUES Background on Imaging Fluorescence and Raman Imaging Subdiffraction Imaging Techniques Wide field imaging Raster imaging Axial resolution enhancement with total internal reflection Overview of Thesis References CHAPTER 2 : SUPER-CONTINUUM STIMULATED EMISSION DEPLETION (STED) FLUORESCENCE LIFETIME IMAGING Abstract Introduction Materials and Methods Instrumentation. Sample preparation. Imaging. Results and Discussion Instrumental spatial resolution: fluorescent beads. SC STED fluorescence lifetime imaging. Sub-diffraction fluorescence lifetime imaging of cultured cells. Conclusions Acknowledgements References CHAPTER 3 : SUBDIFFRACTION LUMINESCENCE-DEPLETION OF ISOLATED, GIANT, CdSe/CdS NANOCRYSTAL QUANTUM DOTS Abstract Experimental Sample preparation and characterization Imaging iv Results and Discussion Luminescence depletion efficiencies of g-NQDs Determination of G-NQDs on/off Time and photobleaching rates Subdiffraction spatial resolution using g-NQDs Conclusions Acknowledgements References