Keng C. Chou

Review of Super-Resolution Fluorescence Microscopy for Biology

Several methodologies have been developed over the past several years for super-resolution fluorescence microscopy including saturated structured-illumination microscopy (SSIM), stimulated emission depletion microscopy (STED), photoactivated localization microscopy (PALM), fluorescence photoactivation localization microscopy (FPALM), and stochastic optical reconstruction microscopy (STORM). While they have shown great promise for biological research, these techniques all have individual strengths and weaknesses. This review will describe the basic principles for achieving super resolution, demonstrate some applications in biology, and provide an overview of technical considerations for implementing these methods.

Subdiffraction-Limit Two-Photon Fluorescence Microscopy for GFP-Tagged Cell Imaging

We report applications of two-photon excitation fluorescence (2PEF) microscopy with subdiffraction-limit resolution for green-fluorescent-protein-tagged cell imaging. The microscope integrates 2PEF microscopy and stimulated emission depletion microscopy in one microscope that has the benefits of both techniques: intrinsic three-dimensional resolution, confined photobleaching, and subdiffraction-limit resolution. The subdiffraction-limit resolution was demonstrated by resolving green-fluorescent-protein-tagged caveolar vesicles located within a distance shorter than the diffraction limit of a regular 2PEF microscope, which is approximately 250 nm even with the best optics. The full width at half-maximum of the effective point-spread function for the 2PEF microscope was estimated to be approximately 54 nm.

Why Do Sulfuric Acid Coatings Influence the Ice Nucleation Properties of Mineral Dust Particles in the Atmosphere

Laboratory studies with supermicrometer particles have shown that mineral particles coated with sulfuric acid are relatively poor ice nuclei. We investigated this phenomenon, which is of atmospheric relevance, by probing the structure of water at the mineral-aqueous acid interface as a function of the sulfuric acid concentration using sum frequency generation vibrational spectroscopy. We found that ordered water structures at water/mica interfaces drastically diminished at molarities of sulfuric acid equal to 0.5 M and totally disappeared when the molarities reached 5 M. The decrease in ordered water structures at the interface was caused by a combined effect of the decreased mica surface potential at low pH, the adsorption of sulfates on mica, and the lack of free water molecules in high concentrations of acidic solution. The good ice nucleation ability above liquid water saturation is correlated with the presence of structured water, suggesting that structured water at the interface may be needed for efficient heterogeneous ice nucleation.

Surface Structure Relaxation of Poly(methyl methacrylate)

Surface structure relaxations caused by temperature changes at the free surface of poly(methyl methacrylate) were studied using IR-visible sum-frequency generation (SFG). A polarization-rotating technique was introduced to enhance the sensitivity of SFG for monitoring the surface structure relaxations during a cooling process. A new surface structure relaxation was observed at 67 degrees C. This temperature does not match any known structure relaxation temperatures for the bulk and is 40 degrees C below the bulk glass transition temperature. As expected for a free-surface phenomenon, the surface relaxation temperature was found to be independent of film thickness in the range of 0.1-0.5 microm.

Role of interfacial water on protein adsorption at cross-linked polyethylene oxide interfaces.

Sum frequency generation (SFG) vibrational spectroscopy was used to study the structure of water at cross-linked PEO film interfaces in the presence of human serum albumin (HSA) protein. Although PEO is charge neutral, the PEO film/water interface exhibited an SFG signal of water similar to that of a highly charged water/silica interface, signifying the presence of ordered water. Ordered water molecules were observed not only at the water/PEO interface, but also within the PEO film. It indicates that the PEO and water form an ordered hydrogen-bonded network extending from the bulk PEO film into liquid water, which can provide an energy barrier for protein adsorption. Upon exposure to the protein solution, the SFG spectra of water at the water/PEO interface remained nearly unperturbed. For comparison, the SFG spectra of water/silica and water/polystyrene interfaces were also studied with and without HSA in the solution. The SFG spectra of the interfacial water were correlated with the amount of protein adsorbed on the surfaces using fluorescence microscopy, which showed that the amount of protein adsorbed on the PEO film was about 10 times less than that on a polystyrene film and 3 times less than that on silica.

Investigation of Sub-100 nm Gold Nanoparticles for Laser-Induced Thermotherapy of Cancer

Specialized gold nanostructures are of interest for the development of alternative treatment methods in medicine. Photothermal therapy combined with gene therapy that supports hyperthermia is proposed as a novel multimodal treatment method for prostate cancer. In this work, photothermal therapy using small (<100 nm) gold nanoparticles and near-infrared (NIR) laser irradiation combined with gene therapy targeting heat shock protein (HSP) 27 was investigated. A series of nanoparticles: nanoshells, nanorods, core-corona nanoparticles and hollow nanoshells, were synthesized and examined to compare their properties and suitability as photothermal agents. In vitro cellular uptake studies of the nanoparticles into prostate cancer cell lines were performed using light scattering microscopy to provide three-dimensional (3D) imaging. Small gold nanoshells (40 nm) displayed the greatest cellular uptake of the nanoparticles studied and were used in photothermal studies. Photothermal treatment of the cancer cell lines with laser irradiation at 800 nm at 4 W on a spot size of 4 mm (FWHM) for 6 or 10 min resulted in an increase in temperature of ~12 °C and decrease in cell viability of up to 70%. However, in vitro studies combining photothermal therapy with gene therapy targeting HSP27 did not result in additional sensitization of the prostate cancer cells to hyperthermia.

High-pressure ammonia adsorption and dissociation on clean fe(111) and oxygen-precovered fe(111) studied by sum frequency generation vibrational spectroscopy.

The adsorption of gases N2, H2, O2, and NH3 that play a role in ammonia synthesis have been studied on the Fe(111) crystal surface by Sum Frequency Generation (SFG) vibrational spectroscopy using an integrated ultrahigh vacuum/high-pressure system. SFG spectra are presented for the dissociation intermediates, NH2 ( approximately 3325 cm-1) and NH ( approximately 3235 cm-1) under high pressure of ammonia (200 Torr) on the clean Fe(111) surface. Addition of 0.5 Torr of oxygen to 200 Torr of ammonia does not significantly change the bonding of dissociation intermediates to the surface. However, it leads to a phase change of nearly 180 degrees between the resonant and nonresonant second-order nonlinear susceptibility of the surface, demonstrated as a reversal of the SFG spectral features. Heating the surface in the presence of 200 Torr of ammonia and 0.5 Torr of oxygen reduces the oxygen coverage, which can be seen from the SFG spectra as another relative phase change of 180 degrees . The reduction of the oxide is also supported by Auger electron spectroscopy. The result suggests that the phase change of the spectral features could serve as a sensitive indicator of the chemical environment of the adsorbates. Clean Fe(111) is found to have a large SFG nonresonant signal. The magnitude of the nonresonant signal was dependent on the adsorption species; O2 and N2 decrease, while H2 and NH3 increase the SFG nonresonant signal. The change in nonresonant signal is correlated to the change in work function for Fe(111) upon adsorption. Adsorption-induced changes in the SFG nonresonant signal was used as an indicator of surface conditions and to monitor surface reactions.

Interactions of Sulfobetaine Zwitterionic Surfactants with Water on Water Surface

We carried out a combined study using surface tension, phase-sensitive frequency generation (SFG) vibrational spectroscopy, and MD simulations to investigate the industrially relevant zwitterionic surfactant N-dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate (DDAPS) on water surface. The SFG Im(χ(2)) spectra showed that the interaction between DDAPS and water was different from those between biologically relevant zwitterionic phospholipids and water. While zwitterionic phospholipids were found to be anionic-like and flipped water molecules with their OHs pointing toward the air, DDAPS oriented water molecules with their OHs mostly pointing toward the liquid water. We built a new force field for the MD simulation which produced the correct surface tension of water with various DDPAS coverage. The MD simulation showed that the head groups of DDPAS were nearly parallel to the water surface. When the surface coverage of DDPAS was increased, the averaged tilting angle of DDPASs tails decreased, but it had little effect on the orientation of the headgroup. The sulfobetaine zwitterionic surfactant was found to be more cationic-like because the positively charged group was more capable of orienting interfacial water.

Single molecule localization deep within thick cells; a novel super‐resolution microscope

A novel 3D imaging system based on single-molecule localization microscopy is presented to allow high-accuracy drift-free (<0.7 nm lateral; 2.5 nm axial) imaging many microns deep into a cell. When imaging deep within the cell, distortions of the point-spread function result in an inaccurate and very compressed Z distribution. For the system to accurately represent the position of each blink, a series of depth-dependent calibrations are required. The system and its allied methodology are applied to image the ryanodine receptor in the cardiac myocyte. Using the depth-dependent calibration, the receptors deep within the cell are spread over a Z range that is many hundreds of nanometers greater than implied by conventional analysis. We implemented a time domain filter to detect overlapping blinks that were not filtered by a stringent goodness of fit criterion. This filter enabled us to resolve the structure of the individual (30 nm square) receptors giving a result similar to that obtained with electron tomography.

Transient Phase of Ice Observed by Sum Frequency Generation at the Water/Mineral Interface During Freezing

We observed a transient noncentrosymmetric phase of ice at water/mineral interfaces during freezing, which enhanced the intensity of the IR-visible sum frequency generation intensity by up to 20-fold. The lifetime of the transient phase was several minutes. Since the most stable form of ice, hexagonal and cubic ice, are centrosymmetric, our study suggests the transient existence of stacking-disordered ice during the freezing process at water/mineral interfaces. Stacking-disordered ice, which has only been observed in bulk ice at temperatures lower than -20 °C, is a random mixture of layers of hexagonal ice and cubic ice. However, the transient phase at the ice/mineral interface was observed at temperatures as high as -1 °C. It suggests that the mineral surface may play a role in promoting and stabilizing the formation of stacking-disordered ice at the interface.