Recently, we realized a simple technique, which spontaneously bypasses the diffraction limit in a conventional confocal microscope by exploiting super-linear effects in nanoparticle bio-markers: super-linear excitation-emission (SEE) microscopy. Here, we present a theoretical framework and its practical implementation for optimizing and expanding this technique. We accurately predict the expected 3D super-resolution by accounting for all crucial parameters affecting the resolution: the empirically measured/modelled excitation-emission curve, the filling factor of the microscope objective back pupil, the polarization and the pinhole setting. The presented theoretical framework is a practical tool, which enables end-users to augment their own confocal microscopes with super-resolution capabilities.
We achieve spontaneous 3D super-resolution on a standard confocal microscope by exploiting bio-friendly fluorescent markers with super-linear excitation-emission dependence (upconversion nanoparticles of NaYF4: Yb, Tm). We refer to this approach as upconversion super-linear excitation-emission (uSEE) microscopy. To demonstrate the applicability of the method for biological applications, we image sugar-coated upconversion nanoparticles in neuronal cells and we achieve resolution twice better than the diffraction limit both in lateral and axial directions. We envision that due to the application simplicity of the developed methodological toolbox, uSEE microscopy can be widely incorporated as an everyday super-resolution method in biological laboratories.
KEYWORDS: 3D image processing, 3D modeling, Tissue optics, Neuroimaging, Brain, Optical imaging, 3D surface sensing, Optical sensing, Animal model studies, Brain cancer
Successful clinical translation of optical techniques and therapies that advance the detection and treatment of high-grade brain cancer, glioblastoma multiforme (GBM), needs controlled, ethical and practical GBM models that accurately represent the biological reality. However, the available test-beds are not biologically accurate (artificial phantoms); are hindered by complex physiology and ethical concerns (animal models); or involve practical complexity due to rapid biological degradation of the samples ex vivo (surgical biopsies). Here, we present the development and validation of an in vitro, biologically accurate, 3-dimensional living GBM tumour model produced by tissue engineering techniques.
Our 3D living equivalents of GBM tumour tissue are in the millimeter size range, consist of brain-specific extracellular matrix and living cells, and exhibit the relevant (often unfavorable) tissue optical properties such as scattering and tissue auto-fluorescence. The model also reproduces essential challenges in translational neurophotonics that are due to uneven tissue surface topography, variation in structural, optical and biochemical properties of matrix, heterogeneous cellular phenotypes and uneven distribution of exogenous contrast and therapeutic agents.
We will show results of depth-resolved and wide-field imaging of the living GBM-equivalents in laboratory microscopic and theatre-based imaging systems under normal and fluorescence-guided surgery conditions using the typical 5-ALA to fluorescent PpIX conversion by GBM cells, in addition to 3D mapping of exogenous contrast agents such as fluorescent cell viability markers. These results illustrate the versatility of our 3D-engineered GBM model as macroscopic test-bed for the development of optical tools to improve the detection and treatment of brain cancer.
Cytokines play critical roles in homeostatic control of health and they are integral for the creation and maintenance of a myriad of disease states. Their ultra-low concentration, often in the picomolar range, and extremely dynamic transient secretion process place stringent demands on cytokine quantification. We developed a nanoparticle-based strategy to detect trace cytokine secretion from individual, single live cells, for which we coined the term “OnCELISA”. Using a capture surface on the cell membrane and fluorescent magnetic nanoparticles as assay reporters, our universal OnCELISA assay achieved the sensitivity 0.1 pg mL-1, an over 10-fold enhancement, compared to state-of-the-art. The sensitive OnCELISA cell labelling made it possible to select and sort different cell types to determine highly cytokine - secreting cell subpopulations . The capture surfaces on cell membranes did not show noticeable effect on cell viability and their subsequent proliferation. The capability to specifically select such highly cytokine-secreting cells and purify their populations is pivotal for their use in multicellular pathologies such as atherosclerosis. Accordingly, we used this new approach to label cytokine secretion from vascular tissues of apolipoprotein E-/- mice; an in vivo model of atherosclerosis. In response to lipopolysaccharide, we observed increased capture of cytokine using this model. With the capacity of monitoring multiple cytokine secretions (IL-6 and IL-1β)), our OnCELISA method is able to probe how the individual cells and tissues secrete cytokines as they respond in real time to the surrounding signals.
Despite significant advancement in the methodology used to conjugate, incorporate and visualize fluorescent molecules at the cellular and tissue levels, biomedical imaging predominantly relies on the limitations of established fluorescent molecules such as fluorescein, cyanine and AlexaFluor dyes or genetic incorporation of fluorescent proteins by viral or other means. These fluorescent dyes and conjugates are highly susceptible to photobleaching and compete with cellular autofluorescence, making biomedical imaging unreliable, difficult and time consuming in many cases. In addition, some proteins have low copy numbers and/or poor antibody recognition, further making detection and imaging difficult. We are developing better methods for imaging central nervous system neuroinflammatory markers using targeted mRNA transcripts labelled with fluorescent nanodiamonds or lanthanide chelates. These tags have increased signal and photostability and can also discriminate against tissue/cell autofluorescence. Brains and spinal cords from BALB/c mice with a chronic constriction model of neuropathic pain (neuroinflammation group) or that have undergone sham surgeries (control group) were collected. A subset of brains and spinal cords were perfused and fixed with paraformaldehyde (n=3 sham and n=3 pain groups) prior to sectioning and in situ hybridization using nanodiamond or lanthanide chelate conjugated complementary RNA probes. Another subset of brains and spinal cords from the same cohort of animals were perfused and processed for CLARITY hydrogel based clearing prior to in situ hybridization with the same probes. We will present our findings on the photostability, sensitivity and discrimination from background tissue autofluorescence of our novel RNA probes, compared to traditional fluorophore tags.
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