Commercial Confocal Microscope Research Articles

Visualization of Intracellular Hydrogen Peroxide with the Genetically Encoded Fluorescent Probe HyPer in NIH-3T3 Cells.

Reactive oxygen species (ROS) are involved in regulating normal physiological cell functions as second messengers as well as nonspecific damage of biomolecules in a pathological process known as oxidative stress. The HyPer family of genetically encoded probes are a useful noninvasive tool for monitoring the real-time dynamics of ROS in individual cells or model organisms. HyPer, the first genetically encoded probe for detection of hydrogen peroxide (H2O2), is oxidized with high specificity and sensitivity by H2O2, leading to ratiometric changes in the fluorescence excitation spectrum of the probe. These changes can be detected with a wide range of commercial confocal and wide-field microscope systems. Here we describe a detailed protocol for ratiometric monitoring of H2O2 produced by D-amino acid oxidase (DAAO) or by NADPH oxidase (NOX) in NIH-3T3 cells using the HyPer probe.

Accessible quantitative phase imaging in confocal microscopy with sinusoidal-phase synthetic optical holography.

We present a technically simple implementation of quantitative phase imaging in confocal microscopy based on synthetic optical holography with sinusoidal-phase reference waves. Using a Mirau interference objective and low-amplitude vertical sample vibration with a piezo-controlled stage, we record synthetic holograms on commercial confocal microscopes (Nikon, model: A1R; Zeiss: model: LSM-880), from which quantitative phase images are reconstructed. We demonstrate our technique by stain-free imaging of cervical (HeLa) and ovarian (ES-2) cancer cells and stem cell (mHAT9a) samples. Our technique has the potential to extend fluorescence imaging applications in confocal microscopy by providing label-free cell finding, monitoring cell morphology, as well as non-perturbing long-time observation of live cells based on quantitative phase contrast.

Method of thickness measurement for transparent specimens with chromatic confocal microscopy.

In this paper, a new method for measuring the thickness of transparent specimens using chromatic confocal microscopy (CCM) is presented. The conventional CCM thickness measurement model relies on capturing the focal points on the upper and lower surfaces of a transparent specimen. This model has strict specimen placement tolerance and a limited measurement range. In order to overcome these limitations, a new thickness measurement model was developed by adding an auxiliary reflector below the specimen. The thickness of the specimen can be determined by comparing the wavelengths of light focused on the auxiliary reflector before and after placing the measurement specimen. Theoretical analysis and simulation showed that the proposed method has twice the measurement range of the conventional model. In order to verify the proposed CCM measurement model, a laboratory thickness measurement system was developed by the authors' team. A commercial laser scanning confocal microscope (Carl Zeiss LSM780) was used as the reference system. A set of quartz glasses was measured using both the proposed system and the reference system. Experimental comparison showed that the proposed method was able to achieve a measurement accuracy of 0.25μm. In addition, repeated measurements conducted at different heights showed negligible variation. Thus, it can be concluded that the specimen placement tolerance was improved significantly compared with the conventional model.

Circular intensity differential scattering (CIDS) scanning microscopy to image chromatin-DNA nuclear organization

Circular dichroism imaging has proved a powerful and simple method for extracting information on chiral molecules without specific fluorescent labels. Numerous mathematical models show that outside the absorption band, the circular dichroism signal comes from the scattering interaction and brings additional information about the organization of biopolymers. With this article, we propose a fast method to control the polarization states without moving parts, by means of a photoelastic modulator. We implemented the technique on a modified commercial confocal microscope realizing a multimodal configuration. We demonstrate its imaging capabilities by studying the organization of chromatin DNA inside isolated cell nuclei.

Optical adaptation of confocal microscopes for arbitrary imaging angles—and its application to sedimentation/creaming in dispersions

Confocal microscopy has become a powerful tool to obtain high-resolution, three-dimensional images after reconstruction of a point-by-point scan of the local fluorescence properties of a sample. For technical reasons, commercial confocal microscopes perform this scan in a manner that the x-y-plane of the scan is perpendicular to gravity and can be several square centimetres large (defined by the movement of displacement stages). The depth- or z-scan, on the other hand, is parallel to gravity and typically limited to 10–1000 micrometres depth. This is a constraint that limits the number of experimental scenarios in which confocal microscopy can be employed, since many experimental systems ask for images in which the main extension of the scan has an angle with gravity which is not 90 degrees. Using the example of different experimental systems (particle dispersions, emulsions, foams, foamed emulsions) that sediment or cream under gravity, we show here how the optical path of any confocal microscope can be inverted to obtain any imaging angle with respect to gravity without losing all pre-programmed scanning and reconstruction facilities.

Fast and accurate: high-speed metrological large-range AFM for surface and nanometrology

Low measurement speed remains a major shortcoming of the scanning probe microscopic technique. It not only leads to a low measurement throughput, but a significant measurement drift over the long measurement time needed (up to hours or even days). To overcome this challenge, PTB, the national metrology institute of Germany, has developed a high-speed metrological large-range atomic force microscope (HS Met. LR-AFM) capable of measuring speeds up to 1 mm s−1. This paper has introduced the design concept in detail. After modelling scanning probe microscopic measurements, our results suggest that the signal spectrum of the surface to be measured is the spatial spectrum of the surface scaled by the scanning speed. The higher the scanning speed , the broader the spectrum to be measured. To realise an accurate HS Met. LR-AFM, our solution is to combine different stages/sensors synchronously in measurements, which provide a much larger spectrum area for high-speed measurement capability.Two application examples have been demonstrated. The first is a new concept called reference areal surface metrology. Using the developed HS Met. LR-AFM, surfaces are measured accurately and traceably at a speed of 500 µm s−1 and the results are applied as a reference 3D data map of the surfaces. By correlating the reference 3D data sets and 3D data sets of tools under calibration, which are measured at the same surface, it has the potential to comprehensively characterise the tools, for instance, the spectrum properties of the tools. The investigation results of two commercial confocal microscopes are demonstrated, indicating very promising results. The second example is the calibration of a kind of 3D nano standard, which has spatially distributed landmarks, i.e. special unique features defined by 3D-coordinates. Experimental investigations confirmed that the calibration accuracy is maintained at a measurement speed of 100 µm s−1, which improves the calibration efficiency by a factor of 10.

Effective scheme of photolysis of GFP in live cell as revealed with confocal fluorescence microscopy

We proposed an effective kinetics scheme of photolysis of green fluorescent protein (GFP) observed in live cells with a commercial confocal fluorescence microscope. We investigated the photolysis of GFP-tagged heterochromatin protein, HP1β-GFP, in live nucleus with the pulse position modulation approach, which has several advantages over the classical pump-and-probe method. At the basis of the proposed scheme lies a process of photoswitching from the native fluorescence state to the intermediate fluorescence state, which has a lower fluorescence yield and recovers back to native state in the dark. This kinetics scheme includes four effective parameters (photoswitching, reverse switching, photodegradation rate constants, and relative brightness of the intermediate state) and covers the time scale from dozens of milliseconds to minutes of the experimental fluorescence kinetics. Additionally, the applicability of the scheme was demonstrated in the cases of continuous irradiation and the classical pump-and-probe approach using numerical calculations and analytical solutions. An interesting finding of experimental data analysis was that the overall photodegradation of GFP proceeds dominantly from the intermediate state, and demonstrated approximately the second-order reaction versus irradiation power. As a practical example, the proposed scheme elucidates the artifacts of fluorescence recovery after the photobleaching method, and allows us to propose some suggestions on how to diminish them.

Mapping the dynamical organization of the cell nucleus through fluorescence correlation spectroscopy.

The hierarchical organization of the cell nucleus into specialized open reservoirs and the nucleoplasm overcrowding impose restrictions to the mobility of biomolecules and their interactions with nuclear targets. These properties determine that many nuclear functions such as transcription, replication, splicing or DNA repair are regulated by complex, dynamical processes that do not follow simple rules. Advanced fluorescence microscopy tools and, in particular, fluorescence correlation spectroscopy (FCS) provide complementary and exquisite information on the dynamics of fluorescent labeled molecules moving through the nuclear space and are helping us to comprehend the complexity of the nuclear structure. Here, we describe how FCS methods can be applied to reveal the dynamical organization of the nucleus in live cells. Specifically, we provide instructions for the preparation of cellular samples with fluorescent tagged proteins and detail how FCS can be easily instrumented in commercial confocal microscopes. In addition, we describe general rules to set the parameters for one and two-color experiments and the required controls for these experiments. Finally, we review the statistical analysis of the FCS data and summarize the use of numerical simulations as a complementary approach that helps us to understand the complex matrix of molecular interactions network within the nucleus.

In Situ Localization of Enzyme Activity in Live Cells by a Molecular Probe Releasing a Precipitating Fluorochrome

AbstractCurrent enzyme‐responsive, fluorogenic probes fail to provide in situ information because the released fluorophores tend to diffuse away from the reaction sites. The problem of diffusive signal dilution can be addressed by designing a probe that upon enzyme conversion releases a fluorophore that precipitates. An excited‐state intramolecular proton transfer (ESIPT)‐based solid‐state fluorophore HTPQ was developed that is strictly insoluble in water and emits intense fluorescence in the solid state, with λex/em=410/550 nm, thus making it far better suited to use with a commercial confocal microscope. HTPQ was further utilized in the design of an enzyme‐responsive, fluorogenic probe (HTPQA), targeting alkaline phosphatase (ALP) as a model enzyme. HTPQA makes possible diffusion‐resistant in situ detection of endogenous ALP in live cells. It was also employed in the visualizing of different levels of ALP in osteosarcoma cells and tissue, thus demonstrating its interest for the diagnosis of this type of cancer.

In Situ Localization of Enzyme Activity in Live Cells by a Molecular Probe Releasing a Precipitating Fluorochrome.

Current enzyme-responsive, fluorogenic probes fail to provide in situ information because the released fluorophores tend to diffuse away from the reaction sites. The problem of diffusive signal dilution can be addressed by designing a probe that upon enzyme conversion releases a fluorophore that precipitates. An excited-state intramolecular proton transfer (ESIPT)-based solid-state fluorophore HTPQ was developed that is strictly insoluble in water and emits intense fluorescence in the solid state, with λex/em =410/550 nm, thus making it far better suited to use with a commercial confocal microscope. HTPQ was further utilized in the design of an enzyme-responsive, fluorogenic probe (HTPQA), targeting alkaline phosphatase (ALP) as a model enzyme. HTPQA makes possible diffusion-resistant in situ detection of endogenous ALP in live cells. It was also employed in the visualizing of different levels of ALP in osteosarcoma cells and tissue, thus demonstrating its interest for the diagnosis of this type of cancer.

Multidimensional fluorescence imaging and super-resolution exploiting ultrafast laser and supercontinuum technology

This thesis centres on the development of multidimensional fluorescence imaging tools, with a particular emphasis on fluorescence lifetime imaging (FLIM) microscopy for application to biological research. The key aspects of this thesis are the development and application of tunable supercontinuum excitation sources based on supercontinuum generation in microstructured optical fibres and the development of stimulated emission depletion (STED) microscope capable of fluorescence lifetime imaging beyond the diffraction limit. The utility of FLIM for biological research is illustrated by examples of experimental studies of the molecular structure of sarcomeres in muscle fibres and of signalling at the immune synapse. The application of microstructured optical fibre to provide tunable supercontinuum excitation source for a range of FLIM microscopes is presented, including wide-field, Nipkow disk confocal and hyper-spectral line scanning FLIM microscopes. The application of supercontinuum generation to the first super-resolved FLIM microscope is then described. This novel microscope exploited the concept of STED with a femtosecond mode-locked Ti:Sapphire laser providing a tunable excitation beam by pumping microstructured optical fibre for supercontinuum generation and directly providing the (longer wavelength) STED beam. This STED microscope was implemented in a commercial scanning confocal microscope to provide compatibility with standard biological imaging, and exploited digital holography using a spatial light modulator (SLM) to provide the appropriate phase manipulation for shaping the STED beam profile and to compensate for aberrations. The STED microscope was shown to be capable of recording superresolution images in both the lateral and axial planes, according to the settings of the SLM.

Immersion Meta-Lenses at Visible Wavelengths for Nanoscale Imaging.

Immersion objectives can focus light into a spot smaller than what is achievable in free space, thereby enhancing the spatial resolution for various applications such as microscopy, spectroscopy, and lithography. Despite the availability of advanced lens polishing techniques, hand-polishing is still required to manufacture the front lens of a high-end immersion objective, which poses major constraints for lens design. This limits the shape of the front lens to spherical. Therefore, several other lenses need to be cascaded to correct for spherical aberration, resulting in significant challenges for miniaturization and adding design complexity for different immersion liquids. Here, by using metasurfaces, we demonstrate liquid immersion meta-lenses free of spherical aberration at various design wavelengths in the visible spectrum. We report water and oil immersion meta-lenses of various numerical apertures (NA) up to 1.1 and show that their measured focal spot sizes are diffraction-limited with Strehl ratios of approximately 0.9 at 532 nm. By integrating the oil immersion meta-lens (NA = 1.1) into a commercial scanning confocal microscope, we achieve an imaging spatial resolution of approximately 200 nm. These meta-lenses can be easily adapted to focus light through multilayers of different refractive indices and mass-produced using modern industrial manufacturing or nanoimprint techniques, leading to cost-effective high-end optics.

Labeling cellular structures in vivo using confined primed conversion of photoconvertible fluorescent proteins.

The application of green-to-red photoconvertible fluorescent proteins (PCFPs) for in vivo studies in complex 3D tissue structures has remained limited because traditional near-UV photoconversion is not confined in the axial dimension, and photomodulation using axially confined, pulsed near-IR (NIR) lasers has proven inefficient. Confined primed conversion is a dual-wavelength continuous-wave (CW) illumination method that is capable of axially confined green-to-red photoconversion. Here we present a protocol to implement this technique with a commercial confocal laser-scanning microscope (CLSM); evaluate its performance on an in vitro setup; and apply primed conversion for in vivo labeling of single cells in developing zebrafish and mouse preimplantation embryos expressing the green-to-red photoconvertible protein Dendra2. The implementation requires a basic understanding of laser-scanning microscopy, and it can be performed within a single day once the required filter cube is manufactured.

In vivo optical interferometric imaging of human skin utilizing monochromatic light source.

We have demonstrated tomographic imaging of in vivo human skin with an optical interferometric imaging technique using a monochromatic light source. The axial resolution of this method is determined by the center wavelength and the NA of the objective and is irrelevant to the bandwidth of the light source in contrast to optical coherence tomography. Our imaging system is constructed with low-priced and small-sized compact disk optical pickup components, a laser diode, a high NA objective, and a voice coil actuator. In spite of its low cost and small size, our imaging system can visualize the structure of human skin as clearly as a commercial reflectance confocal microscope.

Multimodal and non‐linear optical microscopy applications in reproductive biology

A plethora of optical techniques is currently available to obtain non-destructive, contactless, real time information with subcellular spatial resolution to observe cell processes. Each technique has its own unique features for imaging and for obtaining certain biological information. However none of the available techniques can be of universal use. For a comprehensive investigation of biological specimens and events, one needs to use a combination of bioimaging methods, often at the same time. Some modern confocal/multiphoton microscopes provide simultaneous fluorescence, fluorescence lifetime imaging, and four-dimensional imaging. Some of them can also easily be adapted for harmonic generation imaging, and to permit cell manipulation technique. In this work we present a multimodal optical workstation that extends a commercially available confocal microscope to include nonlinear/multiphoton microscopy and optical manipulation/stimulation tools. The nonlinear microscopy capabilities were added to the commercial confocal microscope by exploiting all the flexibility offered by the manufacturer. The various capabilities of this workstation as applied directly to reproductive biology are discussed. Microsc. Res. Tech. 79:567-582, 2016. © 2016 Wiley Periodicals, Inc.

Efficient Cementing of CH3NH3PbBr3 Nanoparticles to Upconversion Nanoparticles Visualized by Confocal Microscopy

Upconversion‐perovskite nanohybrids are prepared by assembling CH3NH3PbBr3 perovskite nanoparticles (PK) with naked NaYF4:Yb3+,Tm3+ nanoparticles (UCn) via an innovative strategy consisting of using cucurbit[7]uril (CB[7]) to anchor the perovskite nanoparticles firmly and closely to the upconversion nanoparticles, thus leading to UCn@PKCB nanohybrids. A commercial multiphoton laser scanning confocal microscope is used to demonstrate the successful assembly. This technique proves to be useful to evaluate luminescence lifetime in the range of several tens of μs and allows visualization of the extraordinarily efficient nontrivial resonance energy transfer from the upconversion nanoparticle to the perovskite after near‐infrared (NIR) excitation of the nanohybrid as well as the homogeneity of the UCn@PKCB sample. The considerable photostability of the perovskite in these nanohybrids is demonstrated by prolonged irradiation of the nanohybrid under UV light as well as under NIR light.

A new hand-held microfluidic cytometer for evaluating irradiation damage by analysis of the damaged cells distribution.

Space radiation brings uneven damages to cells. The detection of the distribution of cell damage plays a very important role in radiation medicine and the related research. In this paper, a new hand-held microfluidic flow cytometer was developed to evaluate the degree of radiation damage of cells. The device we propose overcomes the shortcomings (e.g., large volume and high cost) of commercial flow cytometers and can evaluate the radiation damage of cells accurately and quickly with potential for onsite applications. The distribution of radiation-damaged cells is analyzed by a simultaneous detection of immunofluorescence intensity of γ-H2AX and resistance pulse sensor (RPS) signal. The γ-H2AX fluorescence intensity provides information of the degree of radiation damage in cells. The ratio of the number of cells with γ-H2AX fluorescence signals to the total numbers of cells detected by RPS indicates the percentage of the cells that are damaged by radiation. The comparison experiment between the developed hand-held microfluidic flow cytometer and a commercial confocal microscope indicates a consistent and comparable detection performance.

3D reconstruction and characterization of laser induced craters by in situ optical microscopy

A low-cost optical microscope was developed and coupled to an irradiation system in order to study the induced effects on material during a multipulse regime by an in situ visual inspection of the surface, in particular of the spot generated at different pulses. In the case of laser ablation, a reconstruction of the crater in 3D was made from the images of the sample surface taken during the irradiation process, and the subsequent profiles of ablated material were extracted. The implementation of this homemade optical device gives an added value to the irradiation system, providing information about morphology evolution of irradiated area when successive pulses are applied. In particular, the determination of ablation rates in real time can be especially useful for a better understanding and controlling of the ablation process in applications where removal of material is involved, such as laser cleaning and in-depth characterization of multilayered samples and diffusion processes. The validation of the developed microscope was made by a comparison with a commercial confocal microscope configured for the characterization of materials where similar results of crater depth and diameter were obtained for both systems.

Raman Spectroscopic Techniques for Planetary Exploration: Detecting Microorganisms through Minerals

Raman spectroscopy can provide highly specific chemical fingerprints of inorganic and organic materials and is therefore expected to play a significant role in interplanetary missions, especially for the search for life elsewhere in our solar system. A major challenge will be the unambiguous detection of low levels of biomarkers on a mineral background. In addition, these biomarkers may not be present at the surface but rather inside or underneath minerals. Strong scattering may prevent focusing deeper into the sample. In this paper, we report the detection of carotenoid-containing microorganisms behind mineral layers using time-resolved Raman spectroscopy (TRRS). Two extremophiles, the bacterium Deinococcus radiodurans and the cyanobacterium Chroococcidiopsis, were detected through translucent and transparent minerals using 440 nm excitation under resonance conditions to selectively enhance the detection of carotenoids. Using 3 ps laser pulses and a 250 ps gated intensified CCD camera provided depth selectivity for the subsurface microorganisms over the mineral surface layer and in addition lowered the contribution of the fluorescent background. Raman spectra of both organisms could be detected through 5 mm of translucent calcite or 20 mm of transparent halite. Multilayered mineral samples were used to further test the applied method. A separate tunable laser setup for resonance Raman and a commercial confocal Raman microscope, both with continuous (non-gated) detection, were used for comparison. This study demonstrates the capabilities of TRRS for the depth-selective analysis through scattering samples, which could be used in future planetary exploration to detect microorganisms or biomarkers within or behind minerals.

Upgrade of a Scanning Confocal Microscope to a Single-Beam Path STED Microscope.

By overcoming the diffraction limit in light microscopy, super-resolution techniques, such as stimulated emission depletion (STED) microscopy, are experiencing an increasing impact on life sciences. High costs and technically demanding setups, however, may still hinder a wider distribution of this innovation in biomedical research laboratories. As far-field microscopy is the most widely employed microscopy modality in the life sciences, upgrading already existing systems seems to be an attractive option for achieving diffraction-unlimited fluorescence microscopy in a cost-effective manner. Here, we demonstrate the successful upgrade of a commercial time-resolved confocal fluorescence microscope to an easy-to-align STED microscope in the single-beam path layout, previously proposed as “easy-STED”, achieving lateral resolution < λ/10 corresponding to a five-fold improvement over a confocal modality. For this purpose, both the excitation and depletion laser beams pass through a commercially available segmented phase plate that creates the STED-doughnut light distribution in the focal plane, while leaving the excitation beam unaltered when implemented into the joint beam path. Diffraction-unlimited imaging of 20 nm-sized fluorescent beads as reference were achieved with the wavelength combination of 635 nm excitation and 766 nm depletion. To evaluate the STED performance in biological systems, we compared the popular phalloidin-coupled fluorescent dyes Atto647N and Abberior STAR635 by labeling F-actin filaments in vitro as well as through immunofluorescence recordings of microtubules in a complex epithelial tissue. Here, we applied a recently proposed deconvolution approach and showed that images obtained from time-gated pulsed STED microscopy may benefit concerning the signal-to-background ratio, from the joint deconvolution of sub-images with different spatial information which were extracted from offline time gating.