Conventional Confocal Fluorescence Microscopy Research Articles

High-Resolution, High-Contrast Optical Interface for Defect Qubits

Point defects in crystals provide important building blocks for quantum applications. To initialize, control, and read-out their quantum states, an efficient optical interface for addressing defects with photons is required. However, conventional confocal fluorescence microscopy with high refractive index crystals has limited photon collection efficiency and spatial resolution. Here, we demonstrate high resolution, high contrast imaging for defects qubits using microsphere-assisted confocal microscopy. A microsphere provides an excellent optical interface for point defects with a magnified virtual image that improves spatial resolution up to ~${\lambda}$/5 as well as an optical signal-to-noise ratio by four times. These features enable individual optical addressing of single photons and single spins of spatially-unresolved defects in conventional confocal microscopy with improved signal contrast. The combined optical tweezers show the possibility of positioning or scanning the microspheres for deterministic coupling and wide-field imaging of defects. The approach does not require any complicated fabrication and additional optical system but uses simple micro-optics off-the-shelf. From these distinctive advantages of the microspheres, our approach can provide an efficient way for imaging and addressing closely-spaced defects with higher resolution and sensitivity.

Parallel detecting super-resolution microscopy using correlation based image restoration

A novel approach to achieve the image restoration is proposed in which each detector’s relative position in the detector array is no longer a necessity. We can identify each detector’s relative location by extracting a certain area from one of the detector’s image and scanning it on other detectors’ images. According to this location, we can generate the point spread functions (PSF) for each detector and perform deconvolution for image restoration. Equipped with this method, the microscope with discretionally designed detector array can be easily constructed without the concern of exact relative locations of detectors. The simulated results and experimental results show the total improvement in resolution with a factor of 1.7 compared to conventional confocal fluorescence microscopy. With the significant enhancement in resolution and easiness for application of this method, this novel method should have potential for a wide range of application in fluorescence microscopy based on parallel detecting.

Method of super-resolution based on array detection and maximum-likelihood estimation.

Conventional confocal fluorescence microscopy has demonstrated its ability to gain enhanced image resolution after the employment of pinholes, but the contradiction between resolution and signal to noise ratio hinders the further improvement of resolution. However, an array detector scheme for confocal microscopes solves this problem and is therefore of interest in restoring the data of array detectors to its original image. In this paper, we apply maximum-likelihood estimation (MLE), which is a restoration method based on the Poisson distribution of the fluorescence sample, to restore the array-detection image. We set up a simulation and apply it to fluorescent beads and fixed cell samples to verify its effectiveness. The results show that, after the restoration, the lateral resolution is highly enhanced and the signal to noise ratio is remarkably improved.

Vorinostat differentially alters 3D nuclear structure of cancer and non-cancerous esophageal cells.

The histone deacetylase (HDAC) inhibitor vorinostat has received significant attention in recent years as an ‘epigenetic’ drug used to treat solid tumors. However, its mechanisms of action are not entirely understood, particularly with regard to its interaction with the aberrations in 3D nuclear structure that accompany neoplastic progression. We investigated the impact of vorinostat on human esophageal epithelial cell lines derived from normal, metaplastic (pre-cancerous), and malignant tissue. Using a combination of novel optical computed tomography (CT)-based quantitative 3D absorption microscopy and conventional confocal fluorescence microscopy, we show that subjecting malignant cells to vorinostat preferentially alters their 3D nuclear architecture relative to non-cancerous cells. Optical CT (cell CT) imaging of fixed single cells showed that drug-treated cancer cells exhibit significant alterations in nuclear morphometry. Confocal microscopy revealed that vorinostat caused changes in the distribution of H3K9ac-marked euchromatin and H3K9me3-marked constitutive heterochromatin. Additionally, 3D immuno-FISH showed that drug-induced expression of the DNA repair gene MGMT was accompanied by spatial relocation toward the center of the nucleus in the nuclei of metaplastic but not in non-neoplastic cells. Our data suggest that vorinostat’s differential modulation of 3D nuclear architecture in normal and abnormal cells could play a functional role in its anti-cancer action.

Continuous-Wave Stimulated Emission Depletion Microscope for Imaging Actin Cytoskeleton in Fixed and Live Cells.

Stimulated emission depletion (STED) microscopy provides a new opportunity to study fine sub-cellular structures and highly dynamic cellular processes, which are challenging to observe using conventional optical microscopy. Using actin as an example, we explored the feasibility of using a continuous wave (CW)-STED microscope to study the fine structure and dynamics in fixed and live cells. Actin plays an important role in cellular processes, whose functioning involves dynamic formation and reorganization of fine structures of actin filaments. Frequently used confocal fluorescence and STED microscopy dyes were employed to image fixed PC-12 cells (dyed with phalloidin- fluorescein isothiocyante) and live rat chondrosarcoma cells (RCS) transfected with actin-green fluorescent protein (GFP). Compared to conventional confocal fluorescence microscopy, CW-STED microscopy shows improved spatial resolution in both fixed and live cells. We were able to monitor cell morphology changes continuously; however, the number of repetitive analyses were limited primarily by the dyes used in these experiments and could be improved with the use of dyes less susceptible to photobleaching. In conclusion, CW-STED may disclose new information for biological systems with a proper characteristic length scale. The challenges of using CW-STED microscopy to study cell structures are discussed.

Nonlinear optical microscopy: Endogenous signals and exogenous probes

Nonlinear optical microscopy (NLOM) relies on nonlinear light–matter interactions to provide images from larger depths within biological structures compared to conventional confocal fluorescence microscopy. These nonlinear light–matter interactions include multiphoton excitation fluorescence (MPEF), second‐harmonic generation (SHG), coherent anti‐Stokes Raman scattering (CARS), and stimulated Raman scattering (SRS). This review discusses the theories of and instrumentation for various NLOM techniques, with a particular focus on endogenous signals and exogenous probes. These signals and probes expand the breadth of information that optical imaging can provide. We also discuss the application of NLOM in biomedical research, including tissue engineering, drug delivery and clinical diagnostics. Current technological limitations are also discussed. image

Evaluation of drug delivery to intact and porated skin by coherent Raman scattering and fluorescence microscopies

Stimulated Raman scattering microscopy was used to assess the permeation of topically applied drugs and formulation excipients into porcine skin. This chemically selective technique generates high-resolution 3D images, from which semi-quantitative information may be elucidated. Ibuprofen, applied as a close-to-saturated solution in propylene glycol, was directly observed to crystallise in/on the skin, as the co-solvent permeated more rapidly, resulting in precipitation of the drug. Coherent Raman scattering microscopy is also an excellent tool, in conjunction with more conventional confocal fluorescence microscopy, with which to image micro/nanoparticle-based formulations. Specifically, the uptake of particles into thermal ablation transport pathways in the skin has been examined.

Label-free and noninvasive optical detection of the distribution of nanometer-size mitochondria in single cells

A microfluidic flow cytometric technique capable of obtaining information on nanometer-sized organelles in single cells in a label-free, noninvasive optical manner was developed. Experimental two-dimensional (2D) light scattering patterns from malignant lymphoid cells (Jurkat cell line) and normal hematopoietic stem cells (cord blood CD34+ cells) were compared with those obtained from finite-difference time-domain simulations. In the simulations, we assumed that the mitochondria were randomly distributed throughout a Jurkat cell, and aggregated in a CD34+ cell. Comparison of the experimental and simulated light scattering patterns led us to conclude that distinction from these two types of cells may be due to different mitochondrial distributions. This observation was confirmed by conventional confocal fluorescence microscopy. A method for potential cell discrimination was developed based on analysis of the 2D light scattering patterns. Potential clinical applications using mitochondria as intrinsic biological markers in single cells were discussed in terms of normal cells (CD34+ cell and lymphocytes) versus malignant cells (THP-1 and Jurkat cell lines).

Emission enhancement within gold spherical nanocavity arrays

Gold nanocavity arrays were prepared on fluorine-doped tin oxide on glass by electrochemical deposition of gold through monolayers of polystyrene spheres. The impact of the resulting spherical cap architecture on the photophysics of solutions and self-assembled monolayers of luminophore encapsulated within the nanocavities is reported for the first time. From conventional confocal fluorescence microscopy, the emission intensity of solutions of [Ru(bpy)(2)(Qbpy)](2+) (where bpy is 2,2'-bipyridyl and Qbpy is 2,2':4,4'':4,4''-quarterpyridyl) and fullerene (C(60)) encapsulated within the 820 nm diameter nanocavities was demonstrated to increase by approximately an order of magnitude compared with that of the associated bulk solution. Comparison was also made with the emission observed for luminophore solution encapsulated in cobalt nanocavities of comparable dimensions, where plasmonic interactions are not anticipated. Again, approximately an order of magnitude enhancement was observed for the gold arrays. Luminescence lifetime imaging revealed that the enhancement of the emission intensity of this solution within the nanocavity was accompanied by a small but significant decrease in the luminescent lifetime for [Ru(bpy)(2)(Qbpy)](2+). Enhancement was, in addition, strongly influenced by the wavelength of excitation, suggesting that plasmonics may play a role in the enhancement of the excitation process. An important observation from confocal imaging studies was that the dimensions of the luminophore emission field from solution within the cavities were significantly smaller than the dimensions of the cavity aperture and corresponded to a little more than that of the point spread function of the microscope. This indicates that its origin is significantly smaller than the wavelength of the emitted light and suggests that luminescence enhancement is highly localised. When the array was filled with a solution of [Ru(bpy)(2)(Qbpy)](2+) the emission spectrum of this complex was red shifted and broadened compared with that of the bulk solution, typical of the formation of a luminescent surface film. In addition, significant enhancement was only observed when the solution was sonicated into the array. Both these observations suggest that the emission enhancement is localised near the bottom of the cavity. Self-assembled monolayers of [Ru(bpy)(2)(Qbpy)](2+) were formed on the array and approximately 7 orders of magnitude enhancement of the Raman signal was achieved. Significantly, the emission intensity was approximately 4-fold higher for the monolayer than for a solid film under the same conditions, but surface quenching is thought to play a significant role in the observed decrease in lifetime for the monolayer of this complex on the array.

Imaging TOF-SIMS analysis of oligonucleotide microarrays.

Single strand thiolated oligonucleotide (25-mer) was printed onto chemically modified glass and silicon surfaces. Confirmation of the level of attachment attained in each case was effected through detection by conventional confocal fluorescence microscopy. Both positive-ion and negative ion imaging time-of-flight mass spectra were recorded for the visualization of micro-patterned oligonucleotide arrays. This represents the first report of such detection by this form of mass spectrometry on glass. Ultimately, we are interested in the possibility that imaging time-of-flight secondary ion mass spectrometry can discern the orientation and conformation of DNA strands present on the surface of a substrate.

Two‐photon microscopy: Imaging in scattering samples and three‐dimensionally resolved flash photolysis

Two-photon molecular excitation microscopy has several advantages over conventional confocal fluorescence microscopy, including the ability to section deeper into scattering samples and to allow spatially resolved flash photolysis. We describe and examine the benefit of incorporating non-descanned fluorescence detection in our microscope system. In a scattering sample where almost no signal could be obtained at a depth of 50 μm with confocal detection, non-descanned detection resulted in an improvement of signal strength by more than an order of magnitude at depths >40 μm. The spatio-temporal properties of stationary spot two-photon excited flash photolysis (TPEFP) in drops of test solutions and cardiac myocytes were also examined. At input powers that produce >10% of the maximum rate of DM-nitrophen photolysis, serious photodestruction of the reporter fluorochrome (Fluo-3) at the photolysis spot occurred. At power levels of ∼4 mW for periods <50 ms, we were able to produce small repeatable calcium release events using DM-nitrophen in cardiac myocytes, which were similar to naturally occurring calcium sparks. The properties of these artifical calcium sparks were very similar to signals obtained from drops of test solutions, suggesting that the apparent rate of calcium diffusion in myocytes is similar to the rate of diffusion of Fluo-3 in solution. Using TPEFP, we also examined the ability of a combination of EGTA and a low-affinity calcium indicator to track the time course of calcium release. Although the addition of EGTA improved the temporal fidelity of the rise of the calcium signal, it did not significantly reduce the spread of the fluorescence signal from the photolysis spot. Microsc. Res. Tech. 47:182–195, 1999. © 1999 Wiley-Liss, Inc.

Two-photon microscopy: imaging in scattering samples and three-dimensionally resolved flash photolysis.

Two-photon molecular excitation microscopy has several advantages over conventional confocal fluorescence microscopy, including the ability to section deeper into scattering samples and to allow spatially resolved flash photolysis. We describe and examine the benefit of incorporating non-descanned fluorescence detection in our microscope system. In a scattering sample where almost no signal could be obtained at a depth of 50 microm with confocal detection, non-descanned detection resulted in an improvement of signal strength by more than an order of magnitude at depths >40 microm. The spatio-temporal properties of stationary spot two-photon excited flash photolysis (TPEFP) in drops of test solutions and cardiac myocytes were also examined. At input powers that produce >10% of the maximum rate of DM-nitrophen photolysis, serious photodestruction of the reporter fluorochrome (Fluo-3) at the photolysis spot occurred. At power levels of approximately 4 mW for periods <50 ms, we were able to produce small repeatable calcium release events using DM-nitrophen in cardiac myocytes, which were similar to naturally occurring calcium sparks. The properties of these artificial calcium sparks were very similar to signals obtained from drops of test solutions, suggesting that the apparent rate of calcium diffusion in myocytes is similar to the rate of diffusion of Fluo-3 in solution. Using TPEFP, we also examined the ability of a combination of EGTA and a low-affinity calcium indicator to track the time course of calcium release. Although the addition of EGTA improved the temporal fidelity of the rise of the calcium signal, it did not significantly reduce the spread of the fluorescence signal from the photolysis spot.