In recent years, microlenses (ML), which are micro-scale spheres, have been used to overcome physical diffraction limit of optical microscopy (~200 nm). Although the use of such ML has provided highly resolved images of objects beyond the Abbe optical diffraction limit, the process needs to be refined before it can be applied widespread in materials, biological and clinical research. In this research work, we have implemented experiments on super-resolution imaging utilizing MLs of different refractive indices (n) and diameters to provide the scientific and engineering communities with practical guidelines for obtaining high resolution images with ease. With the support from experimental imaging data as well as FDTD simulations, we have shown that optimal super-resolution imaging with microspheres was accomplished under specific parameter range. We have identified ML with n=1.51 as a preferable choice over those MLs with n=1.4, 1.93, and 2.2, because of high reliability and high magnification for ML with n=1.51. With n=1.51 in mind, we have identified a diameter range from 15 μm to 50 μm provides high resolution and magnification for practical purposes. We show that other ML diameters provided high resolution as well; we believe that ML diameters between 15 μm and 50 μm are practically preferred. We were able to achieve <150 nm resolution and further refinement of this tool can potentially yield higher quality imaging results. Ideally, MLs will eventually be directly incorporated as a modular device in an optical microscope providing the researchers an effective, noninvasive, and economical alternative to complex super resolution microscopy techniques. To improve scanning efficiency, we also proposed microtubule (MT) based imaging. With the demonstration of theoretical optics, we conclude, at present time, that there are some practical concerns for MT-based imaging technique that may limit its application as super-resolution imaging technique. For example, MT-based imaging appears to possess a lower contrast than ML-based technique. Thus, although the concept of MT-based imaging is theoretically possible, we think that more work is needed to utilization of this tool for practical applications.

Quantitative phase Imaging(QPI) has emerged as a valuable tool for imaging specimens with weak scattering and absorbing abilities such as cells and tissues. It is complementary to fluorescence microscopy, as such, it can be applied to unlabelled specimens without the need for fluorescent tagging. By quantitatively mapping the phase changes induced in the incident light field by the optical path length delays of the specimen, QPI provides objective measurement of the cellular dynamics and enables imaging the specimen with high contrast. Transport of Intensity Equation(TIE) is a powerful computational tool for QPI because of its experimental and computational simplicity. Using TIE, the phase can be quantitatively retrieved from defocused intensity images. However, the resolution of the phase image computed using TIE is limited by the diffraction limit of the imaging system used to capture the intensity images. In this thesis, we have developed a super resolution phase imaging technique by applying the principles of Structured Illumination Microscopy(SIM) to Transport of Intensity phase retrieval. The modulation from the illumination shifts the high frequency components of the phase object into the system pass-band. This enables phase imaging with resolutions exceeding the diffraction limit. The proposed method is experimentally validated using a custom-made upright microscope. Because of its experimental and computational simplicity, the method in this thesis should find application in biomedical laboratories where super resolution phase imaging is required / Master of Science / Transport of Intensity Equation is a quantitative phase microscopy technique that enables imaging thin transparent specimens with high phase contrast using a through focus intensity stack. It provides speckle free imaging, compatibility with bright field microscopes and valid under partial coherence. However, the Optical Transfer Function(OTF) of the imaging system or the microscope acts a low pass filter, effectively limiting the maximum spatial frequency that can pass through the system. This reduces the spatial resolution of the computed phase image to the spatial diffraction limit. There has been a continuous drive to develop Super resolution techniques that will provide sub-diffraction resolutions because it will provide better insight into the cellular structure, morphology and composition. Structured Illumination Microscopy(SIM) is one such established technique. Existing work in super resolution phase imaging using SIM is exclusively limited to holography and interferometry based techniques. However, such methods require two-beam interference, illumination sources with high coherence, high experimental stability and phase unwrapping in the postprocessing step to retrieve the true object phase. In this work, we demonstrate a single beam propagation based super resolution phase imaging technique by applying structured illumination to Transport of Intensity Equation. It is valid under partial coherence, and does not require interference, simplifying the experimental and computational requirement. We have designed an upright microscope to demonstrate high resolution phase imaging of human cheek cells.

This thesis is focused on understanding the interactions between molecules and surface-enhanced Raman scattering (SERS) substrates that are typically unresolved due to the diffraction limit of light. Towards this end, we have developed and tested several different sub-diffraction-limited imaging techniques in order to observe these interactions. First, we utilize an isotope-edited bianalyte approach combined with super-resolution imaging via Gaussian point-spread function fitting to elucidate the role of Raman reporter molecules on the location of the SERS emission centroids. By using low concentrations of two different analyte molecules, we find that the location of the SERS emission centroid depends on the number and positions of the molecules present on the SERS substrate. It is also known that SERS enhancement partially results from the molecule coupling its emission into the far-field through the plasmonic nanostructure. This results in a particle-dictated, dipole-like emission pattern, which cannot be accurately modeled as a Gaussian, so we tested the applicability of super-resolution imaging using a dipole-emission fitting model to this data. To test this model, we first fit gold nanorod (AuNR) luminescence images, as AuNR luminescence is primarily coupled out through the longitudinal dipole plasmon mode. This study showed that a three-dimensional dipole model is necessary to fit the AuNR emission, with the model providing accurate orientation and emission wavelength parameters for the nanostructure, as confirmed using correlated AFM and spectroscopy. The dipole fitting technique was next applied to single- and multiple-molecule SERS emission from silver nanoparticle dimers. We again found that a three-dimensional dipole PSF was necessary to accurately model the emission and orientation parameters of the dimer, but that at the single molecule level, the movement of the molecule causes increased uncertainty in the orientation parameters determined by the fit. Finally, we describe progress towards using a combined atomic force/optical microscope system in order to position a carbon nanotube analyte at known locations on the nanoparticle substrate. This would allow for the simultaneous mapping of nanoparticle topography and exact locations of plasmonic enhancement around the nanostructure, but consistently low signal-to-noise kept this technique from being viable. / text

There has been significant progress in the optical resolution of microscopes over the last two decades. However, the majority of currently used methods (e.g. STED, PALM, STORM) have a number of drawbacks, including high intensities of light that result in damage to living specimens in STED, and long data acquisition time leading to limitations on live-cell imaging. Therefore, there is a niche for faster image acquisition at lower intensities while maintaining resolution beyond the diffraction limit. Here, we have developed a new methodology - Quantum Dot-based Optical Spectral Separation (QDOSS) - which relies on using Quantum Dots (QDs) as fluorophores, and on their separation and localisation based on their spectral signatures. We utilise the key advantages of QDs over the usual organic fluorophores: broad excitation, narrow emission spectra and high resistance to photobleaching. Besides, since QDOSS is based on spectral differences for separation, QDs can be deterministically localised in a relatively short time - milliseconds and, potentially, microseconds. Last but not least, QDOSS is suitable for obtaining super-resolution images using a standard confocal fluorescence microscope equipped with a single laser excitation wavelength and capable of spectral signal separation (e.g. Leica TCS SP series or Zeiss LSM series). First, we demonstrated resolution down to 60 nm using triangular DNA origami as a reference. Furthermore, we labelled and imaged the alpha-tubulin structure in HEK293T cells. We showed that using a mixture of standard off-the-shelf QDs of different sizes, resolution down to 40 nm could be achieved via spectroscopic separation of QDs. Finally, we demonstrated that QDOSS could also be used for multicolour imaging of synaptic proteins distributed around synapsis in neurons within diffraction limit. All in all, we believe that these features of QDOSS make it a potential method for long-term live super-resolution imaging, which is going to have a high impact in biological sciences.

Ground state depletion with individual molecule return (GSDIM) super-resolution microscopy is used to interrogate the location of individual fluorescence bursts from two different nanoparticle-fluorophore systems. The first system consists of fluorophore-labeled DNA molecules on gold nanowire surfaces. In this system carboxytetramethyl rhodamine-labeled double-stranded DNA molecules were bound to the surface of gold nanowires via gold-thiol linkages. The second system focuses on mesoporous silica coated nanorods with dye embedded into the silica coating. The dye molecule, Rhodamine 6G, was incorporated into the silica shell during the nanorod coating procedure. Individual fluorescence bursts were spatially localized using point spread function fitting and used to reconstruct the image of the underlying nanowire or nanorod. / text

No description available.

Imaging of cellular samples has for several hundred years been a way for scientists to investigate biological systems. With the discovery of immunofluorescence labeling in the 1940’s and later genetic fluorescent protein labeling in the 1980’s the most important part in imaging, contrast and specificity, was drastically improved. Eversince, we have seen a increased use of fluorescence imaging in biological research, and the application and tools are constantly being developed further. Specific ion imaging has long been a way to discern signaling events in cell systems. Through use of fluorescent ion reporters, ionic concentrations can be measured inliving cells as result of applied stimuli. Using Ca2+ imaging we have demonstrated that there is a inverse influence by plasma membrane voltage gated calcium channels on angiotensin II type 1 receptor (a protein involved in blood pressure regulation). This has direct implications in treatment of hypertension (high blood pressure),one of the most common serious diseases in the western civilization today with approximately one billion afflicted adults world wide in 2016. Extending from this more lower resolution live cell bioimaging I have moved into super resolution imaging. This thesis includes works on the interpretation of super resolution imaging data of the neuronal Na+, K+ - ATPase α3, a receptor responsible for maintaining cell homeostasis during brain activity. The imaging data is correlated with electrophysiological measurements and computer models to point towards possible artefacts in super resolution imaging that needs to be taken into account when interpreting imaging data. Moreover, I proceeded to develop a software for single-molecule localization microscopy analysis aimed for the wider research community and employ this software to identify expression artifacts in transiently transfected cell systems. In the concluding work super-resultion imaging was used to map out the early steps of the intrinsic apoptotic signaling cascade in space and time. Using superresoultion imaging, I mapped out in intact cells at which time points and at which locations the various proteins involved in apoptotic regulation are activated and interact. / Avbildning av biologiska prover har i flera hundra år varit ett sätt för forskare att undersöka biologiska system. Med utvecklingen av immunofluoresens inmärkn-ing och fluoresens-mikroskopi förbättrades de viktigaste aspekterna av mikroskopi,kontrast och specificitet. Sedan 1941 har vi sett kontinuerligt mer mångsidigt och frekvent användning av fluorosense-mikroskopi i biologisk forskning. Jon-mikroskopi har länge varit en metod att studera signalering i cell-system. Genom användning av fluorosenta jon-sensorer går det att mäta variationer avjon koncentrationer i levande celler som resultat av yttre påverkan. Genom att använda Ca2+ mikroskopi har jag visat att det finns en omvänd koppling mellan kalcium-kanaler i plasma-membran och angiotensin II typ 1 receptorn (ett proteininvolverat i blodtrycksreglering). Detta har direkta implikationer för behandlingav högt blodtryck, en av de mer vanliga sjukdomarna i västvärlden idag med överen miljard drabbade patienter i världen 2016. Efter detta projekt vidgades mitt fokus till att inkludera superupplösnings-mikroskopi. Denna avhandling inkluderar ett arbete fokuserat på tolkningen av superupplösnings-mikroskopi data från neuronal Na+, K+ - ATPase α3, en jon-pump som återställer cellernas jonbalans i samband med cell signalering. Mikroskopi-datan korreleras mot elektrofysiologi experiment och modeller för att illustrera möjliga artefakter i superupplösnings-mikroskopi som måste tas i beaktande i samband med tolkning av data. Jag fortsatte med att utveckla mjukvara för analys av data från singel-molekyl-lokalisations-mikroskopi där fokuset för mjukvaran framförallt varit på användarvänligheten. Detta då jag hoppas att den kommer vara användbar för ett bredare forskingsfält. Mjukvaran användes även i ett separat projekt för att identifiera överuttrycks-artefakter i transfekterade celler. I det avslutande arbetet använder jag superupplösnings-mikroskopi för att karakterisera de tidiga stegen i mitokondriell apoptos. Jag identifierar när och var i cellen de olika proteinerna involverade i apoptos signaleringen är aktiverade och interagerar. / <p>QC 20171003</p>

<div>Light in heavily scattering media such as tissue can be modeled with a diffusion equation. A diffusion equation forward model in a computational imaging framework can be used to form images of deep tissue, an approach called diffuse optical tomography, which is important for biomedical studies. However, severe attenuation of high-spatial-frequency information occurs as light propagates through scattering media, and this limits image resolution. Here, we introduce a super-resolution approach based on a point emitter localization method that enables an improvement in spatial resolution of over two orders of magnitude. We demonstrate this experimentally by localizing a small fluorescent inhomogeneity in a highly scattering slab and characterize the localization uncertainty. The approach allows imaging in deep tissue with a spatial resolution of tens of microns, enabling cells to be resolved.</div><div><br></div><div>We also propose a localization-based method that relies on separation in time of the temporal responses of fluorescent signals, as would occur with biological reporters. By localizing each emitter individually, a high-resolution spatial image can be achieved. We develop a statistical detection method for localization based on temporal switching and characterization of multiple fluorescent emitters in a tissue-like domain. By scaling the spatial dimensions of the problem, the scope of applications is widened beyond tissue imaging to other scattering domains. </div><div><br></div><div>Finally, we demonstrate that motion of an object in structured illumination and intensity-based measurements provide sensitivity to material and subwavelength-scale-dimension information. The approach is illustrated as retrieving unknown parameters of interest, such as the refractive index and thickness of a film on a substrate, by utilizing measured power data as a function of object position. </div>

The nuclear pore complex (NPC) is a proteinaceous gateway embedded in the nuclear envelope (NE) that regulates nucleocytoplasmic transport of molecules in eukaryotes. The NPC is formed by hundreds of proteins that are classified into approximately thirty different types of proteins called nucleoporin (Nup), each presents in multiples of eight copies. These nucleoporins are divided into two categories: the scaffold Nups forming the main structure of the NPC and the phenylalanine-glycine (FG) Nups that contain multiple repeats of intrinsically disordered and hydrophobic FG domains. These FG-Nups constitute the selective permeability barrier in the central channel of the NPC, which mediates the nuclear import of proteins into the nucleus, and the nuclear export of mRNA and pre-ribosomal subunits out of the nucleus. However, the precise copies of these Nups and their specific roles in the nucleocytoplasmic transport mechanism remain largely unknown. Moreover, the dysfunctional nuclear transport and the mutations of Nups have been closely associated with numerous human diseases, such as cancer, tumor and liver cirrhosis. We have developed and employed live-cell high-speed single-molecule microscopy to elucidate these critical questions remained in the nuclear transport and provide the fundamental knowledge for developing therapies. In this dissertation, I will present my major findings for the following three research projects: 1) determine the dynamic components of FG-Nups in native NPCs; 2) track the nucleocytoplasmic transport of transcription factor Smad proteins under ligand-activated conditions; and 3) elucidate the relationship between the nuclear export of mRNA and the presence and absence of specific Nups in live cells.Determination of the dynamic components for FG-Nups in native NPCs. Scaffold Nups have been intensively studied with electron microscopy to reveal their spatial positions and architecture in the past decades. However, the spatial organization of FG-Nups remains obscure due to the challenge of probing these disordered and dynamic polypeptides in live NPCs. By employing high-speed single-molecule microscopy and a live cell HaloTag labeling technique, I have mapped the spatial distribution for all eleven known mammalian FG-Nups within individual NPCs. Results show that all FG-Nups within NPCs are distinct in conformations and organized to form a ~300nm long hourglass shaped toroidal channel through the nuclear envelope. Exceptionally, the two remaining Nups (Nup98 and hCG1) almost extend through the entire NPC and largely overlap with all other FG-Nups in their spatial distributions. These results provide a complete map of FG-Nup organization within the NPC and also offer structural and functional insights into nucleocytoplasmic transport models. Tracking of the nucleocytoplasmic transport of Smad proteins under ligand-activated conditions. The inducement of transforming growth factor β1 (TGF-β1) was reported to cause the nuclear accumulation of Smad2/Smad4 heterocomplexes. However, the relationship between nuclear accumulation and the nucleocytoplasmic transport kinetics of Smad proteins in the presence of TGF-β1 remains obscure. By combining a high-speed single-molecule tracking microscopy technique (FRET), I tracked the entire TGF-β1-induced process of Smad2/Smad4 heterocomplex formation, as well as their transport through nuclear pore complex in live cells. The FRET results have revealed that in TGF-β1-treated cells, Smad2/Smad4 heterocomplexes formed in the cytoplasm, imported through the nuclear pore complexes as entireties, and finally dissociated in the nucleus. Moreover, it was found that basal-state Smad2 or Smad4 cannot accumulate in the nucleus without the presence of TGF-β1, mainly because both of them have an approximately twofold higher nuclear export efficiency compared to their nuclear import. Remarkably and reversely, heterocomplexes of Smad2/Smad4 induced by TGF-β1 can rapidly concentrate in the nucleus because of their almost fourfold higher nuclear import rate in comparison with their nuclear export rate. Thus, these single-molecule tracking data elucidate the basic molecular mechanism to understand nuclear transport and accumulation of Smad protein. Elucidation of the relationship between the nuclear export of mRNA and the presence and absence of specific Nups in live cells. In addition to explore the dynamic organization of NPC, in vivo characterization of the exact copy number and the specific function of each nucleoporin in the nuclear pore complex (NPC) remains desirable and challenging. Using live-cell high-speed super-resolution single-molecule microscopy, we first quantify the native copies of nuclear basket FG-Nups (Nup153, Nup50 and Tpr). Second, with same imaging technique and the auxin-inducible degradation strategies, I track the nuclear export of mRNA through native NPCs in absence of these FG-Nups. I found that these FG-Nups proteins possess the stoichiometric ratio of 1:1:1 and play distinct roles in the nuclear export of mRNAs in live cells. Tpr’s absence in the NPC dominantly reduces nuclear mRNA’s probability of entering the NPC for export. Complete depletion of Nup153 causes mRNA’s successful nuclear export efficiency dropped approximately four folds. Remarkably, the relationship between mRNA’s successful export efficiency and the copy number of Nup153 is not linear but instead follows a sigmoid function, in which mRNA can gain its maximum successful export efficiency as Nup153 increased from zero to around half of their full copies in the NPC. Lastly, the absence of Tpr or Nup153 also alters mRNA’s export routes through the NPC, but the removal of only Nup50 has almost no impact upon mRNA export route and kinetics. / Biology

<p>Optical imaging methods are limited by the wavelength of light that they use and the amount of scatter that must be imaged through. Super-resolution imaging and sensing methods are those that bypass or mitigate such restrictions. Two super-resolution approaches are presented here using spatially or temporally structured light. Temporal intermittence or blinking of fluorescent emitters is exploited for localization through significant depths of heavy scatter to high resolution, and an efficient algorithm for doing so is presented.</p> <p>Such temporal structure of emission allows far greater resolution than previous comparable imaging methods, providing opportunities in biophotonics and environmental sensing. Spatial structure can be imposed on coherent light that passes through a heavily scattering medium, in the form of a speckle pattern. Speckle intensity correlations are sensitive to the motion of a moving object obscured by scatter, and we demonstrate that this scatter can act as an analyzer, enhancing this sensitivity as the amount of scatter increases. This increased sensitivity is studied using random matrix theory, and eigenchannel analysis is proposed as an explanation. Simulations demonstrate that a randomly scattering analyzer can give sub-wavelength geometric information about a translated, hidden object. Relative motion of structured illumination is explored, with simulations and mathematical analysis demonstrating far-subwavelength sensitivity using moving fields with multiple different types of structure. This work could enable a new approach for material inspection and characterization, and provide improvements in microscopy. </p>