Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength

Current far-field fluorescence nanoscopes provide subdiffraction resolution by exploiting a mechanism of fluorescence inhibition. This mechanism is implemented such that features closer than the diffraction limit emit separately when simultaneously exposed to excitation light. A basic mechanism for such transient fluorescence inhibition is the depletion of the fluorophore ground state by transferring it (via a triplet) in a dark state, a mechanism which is workable in most standard dyes. Here we show that microscopy based on ground state depletion followed by individual molecule return (GSDIM) can effectively provide multicolor diffraction-unlimited resolution imaging of immunolabeled fixed and SNAP-tag labeled living cells. Implemented with standard labeling techniques, GSDIM is demonstrated to separate up to four different conventional fluorophores using just two detection channels and a single laser line. The method can be expanded to even more colors by choosing optimized dichroic mirrors and selecting marker molecules with negligible inhomogeneous emission broadening.     

Imaging living cells and tissues by two-photon excitation microscopy

Two-photon excitation microscopy provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging. Since two-photon excitation occurs only at the focal point of the microscope, it inherently provides three-dimensional resolution. This localization of excitation also minimizes photobleaching and photodamage, which are the ultimate limiting factors in imaging living cells. Furthermore, no pinhole is required to attain three-dimensional discrimination, so the efficiency of fluorescence collection is increased. These advantages allow experiments on thick living samples that would not be possible with other imaging techniques. The cost and complexity of the lasers required for two-photon excitation microscopy have limited its use, but appropriate turn-key lasers have now been introduced, and their cost should decrease. Finally, the recent introduction of commercial two-photon excitation laser-scanning microscope systems allows a much larger group of researchers access to this state-of-the-art methodology.     

Live-cell 3D super-resolution imaging in thick biological samples

We demonstrate three-dimensional (3D) super-resolution live-cell imaging through thick specimens (50-150 μm), by coupling far-field individual molecule localization with selective plane illumination microscopy (SPIM). The improved signal-to-noise ratio of selective plane illumination allows nanometric localization of single molecules in thick scattering specimens without activating or exciting molecules outside the focal plane. We report 3D super-resolution imaging of cellular spheroids.     

Visualizing single fluorophores in live cells

The methods have been described that can be used to visualize single fluorescent molecules in live cells: laser epifluorescent, confocal, near-field, two-photon, and total internal reflection microscopy. Each method has its own advantages and limitations. We showed that total internal reflection microscopy is a method of choice for single fluorophore visualisation near substrate-medium interface. It can be used to study receptors, ion channels, and many cytoskeleton or signalling molecules located at or in close proximity to basal cell membrane. It was shown that it is very important to use rigorous criteria for single fluorophore identification since these objects emit a limited number of photons before irreversible photo-bleaching, and their fluorescence is often obscured by cell auto-fluorescence and out-of-focus fluorescence. Methods used for lateral mobility studies of single molecules floating on cell membrane were also described.     

Visualization of single fluorophores in living cells

Methods applicable to visualizing single fluorophores in living cells are described, namely, laser epifluorescence, confocal, near-field, two-photon, and total internal reflection microscopy. It is demonstrated that total internal reflection microscopy is the most appropriate for visualizing single fluorophores near the substrate-medium interface. This method can be used for studying receptors, ion channels, and numerous cytoskeletal and signal molecules located on or near the basal cell membrane. It is demonstrated that stringent criteria are necessary when identifying single molecules, as these objects emit a limited number of photons before irreversible photobleaching and their fluorescence is obscured by autofluorescence or out-of-focus fluorescence. The methods used for studying the lateral mobility of single molecules floating on the cell membrane are also described.     

Automatic detection of single fluorophores in live cells

Recent developments in light microscopy enable individual fluorophores to be observed in aqueous conditions. Biological molecules, labeled with a single fluorophore, can be localized as isolated spots of light when viewed by optical microscopy. Total internal reflection fluorescence microscopy greatly reduces background fluorescence and allows single fluorophores to be observed inside living cells. This advance in live-cell imaging means that the spatial and temporal dynamics of individual molecules can be measured directly. Because of the stochastic nature of single molecule behavior a statistically meaningful number of individual molecules must be detected and their separate trajectories in space and time stored and analyzed. Here, we describe digital image processing methods that we have devised for automatic detection and tracking of hundreds of molecules, observed simultaneously, in vitro and within living cells. Using this technique we have measured the diffusive behavior of pleckstrin homology domains bound to phosphoinositide phospholipids at the plasma membrane of live cultured mammalian cells. We found that mobility of these membrane-bound protein domains is dominated by mobility of the lipid molecule to which they are attached and is highly temperature dependent. Movement of PH domains isolated from the tail region of myosin-10 is consistent with a simple random walk, whereas, diffusion of intact PLC-delta1 shows behavior inconsistent with a simple random walk. Movement is rapid over short timescales but much slower at longer timescales. This anomalous behavior can be explained by movement being restricted to membrane regions of 0.7 microm diameter.     

Optical‐resolution photoacoustic microscopy with ultrafast dual‐wavelength excitation

Fast functional and molecular photoacoustic microscopy requires pulsed laser excitations at multiple wavelengths with enough pulse energy and short wavelength‐switching time. Recent development of stimulated Raman scattering in optical fiber offers a low‐cost laser source for multiwavelength photoacoustic imaging. In this approach, long fibers temporally separate different wavelengths via optical delay. The time delay between adjacent wavelengths may eventually limits the highest A‐line rate. In addition, a long‐time delay in fiber may limit the highest pulse energy, leading to poor image quality. In order to achieve high pulse energy and ultrafast dual‐wavelength excitation, we present optical‐resolution photoacoustic microscopy with ultrafast dual‐wavelength excitation and a signal separation method. The signal separation method is validated in numerical simulation and phantom experiments. We show that when two photoacoustic signals are partially overlapped with a 50‐ns delay, they can be recovered with 98% accuracy. We apply this ultrafast dual‐wavelength excitation technique to in vivo OR‐PAM. Results demonstrate that A‐lines at two wavelengths can be successfully separated, and sO₂ values can be reliably computed from the separated data. The ultrafast dual‐wavelength excitation enables fast functional photoacoustic microscopy with negligible misalignment among different wavelengths and high pulse energy, which is important for in vivo imaging of microvascular dynamics.     

Characterizing locus specific chromatin structure and dynamics with correlative conventional and super-resolution imaging in living cells

The dynamic rearrangement of chromatin is critical for gene regulation, but mapping both the spatial organization of chromatin and its dynamics remains a challenge. Many structural conformations are too small to be resolved via conventional fluorescence microscopy and the long acquisition time of super-resolution photoactivated localization microscopy (PALM) precludes the structural characterization of chromatin below the optical diffraction limit in living cells due to chromatin motion. Here we develop a correlative conventional fluorescence and PALM imaging approach to quantitatively map time-averaged chromatin structure and dynamics below the optical diffraction limit in living cells. By assigning localizations to a locus as it moves, we reliably discriminate between bound and unbound dCas9 molecules, whose mobilities overlap. Our approach accounts for changes in DNA mobility and relates local chromatin motion to larger scale domain movement. In our experimental system, we show that compacted telomeres move faster and have a higher density of bound dCas9 molecules, but the relative motion of those molecules is more restricted than in less compacted telomeres. Correlative conventional and PALM imaging therefore improves the ability to analyze the mobility and time-averaged nanoscopic structural features of locus specific chromatin with single molecule sensitivity and yields unprecedented insights across length and time scales.     

Analysis of replication factories in human cells by super-resolution light microscopy

Background

Retinoid-inducible gene 1 (RIG1), also known as tazarotene-induced gene 3 or retinoic-acid receptor responder 3, is a growth regulator, which induces apoptosis and differentiation. RIG1 is classified into the NC protein family. This study investigated functional domains and critical amino acids associated with RIG1-mediated cell death and apoptosis.

Results

Using enhanced green fluorescence protein (EGFP)-tagged RIG1 variants, RIG1 proteins with deletion at the NC domain significantly decreased cell death induced by RIG1, and fusion variants containing only the NC domain significantly induced apoptosis of HtTA cervical cancer cells. The EGFP-RIG1-induced apoptosis was significantly decreased in cells expressing N¹¹²C¹¹³ motif double- (NC→FG) or triple- (NCR→FGE) mutated RIG1 variants. Using dodecapeptides, nuclear localization and profound cell death was observed in HtTA cells expressing wild type RIG1_111–123 or Leu¹²¹-mutated RIG1_111–123:L→ C peptide, but peptides double- or triple-mutated at the NC motif alone, RIG1_111–123:NC→FG or RIG1_111–123:NCR→FGE, were cytoplasmically localized and did not induce apoptosis. The RIG1_111–123 also induced apoptosis of A2058 melanoma cells but not normal human fibroblasts.

Conclusion

The NC domain, especially the NC motif, plays the major role in RIG1-mediated pro-apoptotic activity. The RIG1_111–123 dodecapeptide exhibited strong pro-apoptotic activity and has potential as an anticancer drug.     

Analysis of replication factories in human cells by super-resolution light microscopy

Background  

DNA replication in human cells is performed in discrete sub-nuclear locations known as replication foci or factories. These factories form in the nucleus during S phase and are sites of DNA synthesis and high local concentrations of enzymes required for chromatin replication. Why these structures are required, and how they are organised internally has yet to be identified. It has been difficult to analyse the structure of these factories as they are small in size and thus below the resolution limit of the standard confocal microscope. We have used stimulated emission depletion (STED) microscopy, which improves on the resolving power of the confocal microscope, to probe the structure of these factories at sub-diffraction limit resolution.     

Mapping molecular complexes with super-resolution microscopy and single-particle analysis

Understanding the structure of supramolecular complexes provides insight into their functional capabilities and how they can be modulated in the context of disease. Super-resolution microscopy (SRM) excels in performing this task by resolving ultrastructural details at the nanoscale with molecular specificity. However, technical limitations, such as underlabelling, preclude its ability to provide complete structures. Single-particle analysis (SPA) overcomes this limitation by combining information from multiple images of identical structures and producing an averaged model, effectively enhancing the resolution and coverage of image reconstructions. This review highlights important studies using SRM–SPA, demonstrating how it broadens our knowledge by elucidating features of key biological structures with unprecedented detail.     

3D super-resolution microscopy reflects mitochondrial cristae alternations and mtDNA nucleoid size and distribution

3D super-resolution microscopy based on the direct stochastic optical reconstruction microscopy (dSTORM) with primary Alexa-Fluor-647-conjugated antibodies is a powerful method for accessing changes of objects that could be normally resolved only by electron microscopy. Despite the fact that mitochondrial cristae yet to become resolved, we have indicated changes in cristae width and/or morphology by dSTORM of ATP-synthase F₁ subunit α (F₁α). Obtained 3D images were analyzed with the help of Ripley's K-function modeling spatial patterns or transferring them into distance distribution function. Resulting histograms of distances frequency distribution provide most frequent distances (MFD) between the localized single antibody molecules. In fasting state of model pancreatic β-cells, INS-1E, MFD between F₁α were ~80?nm at 0 and 3?mM glucose, whereas decreased to 61?nm and 57?nm upon glucose-stimulated insulin secretion (GSIS) at 11?mM and 20?mM glucose, respectively. Shorter F₁α interdistances reflected cristae width decrease upon GSIS, since such repositioning of F₁α correlated to average 20?nm and 15?nm cristae width at 0 and 3?mM glucose, and 9?nm or 8?nm after higher glucose simulating GSIS (11, 20?mM glucose, respectively). Also, submitochondrial entities such as nucleoids of mtDNA were resolved e.g. after bromo-deoxyuridine (BrDU) pretreatment using anti-BrDU dSTORM. MFD in distances distribution histograms reflected an average nucleoid diameter (<100?nm) and average distances between nucleoids (~1000?nm). Double channel PALM/dSTORM with Eos-lactamase-β plus anti-TFAM dSTORM confirmed the latter average inter-nucleoid distance. In conclusion, 3D single molecule (dSTORM) microscopy is a reasonable tool for studying mitochondrion.     

Myosin monomer density and exchange in synthetic thick filaments investigated using fluorescence microscopy with single molecule sensitivity

The number of myosin molecules in synthetic thick filaments, prepared by dialysis at 0.12 M NaCl and pH 7.0, was estimated to be between 400 and 800 molecules per micrometre under conditions appropriate for in vitro motility assays. This estimate was based on a number count of Cy3-labelled myosin molecules incorporated into filaments at a nominal ratio of 1:1000. At this dilution, single fluorescent spots were resolved corresponding to individual labelled myosins. The spots usually bleached with a one- or two-step profile but, in around 30% of the cases, fluctuations were observed indicating that additional photophysical or photochemical events had occurred. Myosin molecules were shown to exchange between filaments in suspension on a time-scale of several hours at 4 degrees C, but the reaction was only 75% complete after 48 h, suggesting a non-exchangeable core. However, myosin exchange does not appear to be the predominant source of the fluctuations in fluorescence intensity in the single molecule assays.     

X-ray microscopy enables multiscale high-resolution 3D imaging of plant cells,tissues, and organs

Capturing complete internal anatomies of plant organs and tissues within their relevant morphological context remains a key challenge in plant science. While plant growth and development are inherently multiscale, conventional light, fluorescence, and electron microscopy platforms are typically limited to imaging of plant microstructure from small flat samples that lack a direct spatial context to, and represent only a small portion of, the relevant plant macrostructures. We demonstrate technical advances with a lab-based X-ray microscope (XRM) that bridge the imaging gap by providing multiscale high-resolution three-dimensional (3D) volumes of intact plant samples from the cell to the whole plant level. Serial imaging of a single sample is shown to provide sub-micron 3D volumes co-registered with lower magnification scans for explicit contextual reference. High-quality 3D volume data from our enhanced methods facilitate sophisticated and effective computational segmentation. Advances in sample preparation make multimodal correlative imaging workflows possible, where a single resin-embedded plant sample is scanned via XRM to generate a 3D cell-level map, and then used to identify and zoom in on sub-cellular regions of interest for high-resolution scanning electron microscopy. In total, we present the methodologies for use of XRM in the multiscale and multimodal analysis of 3D plant features using numerous economically and scientifically important plant systems.

Lab-based X-ray microscopy allows high-resolution 3D imaging of intact plant samples over a wide range of sample types and sizes, filling the imaging gap between light and electron microscopy.     

Two-photon excitation and photoconversion of EosFP in dual-color 4Pi confocal microscopy

Recent years have witnessed enormous advances in fluorescence microscopy instrumentation and fluorescent marker development. 4Pi confocal microscopy with two-photon excitation features excellent optical sectioning in the axial direction, with a resolution in the 100 nm range. Here we apply this technique to cellular imaging with EosFP, a photoactivatable autofluorescent protein whose fluorescence emission wavelength can be switched from green (516 nm) to red (581 nm) by irradiation with 400-nm light. We have measured the two-photon excitation spectra and cross sections of the green and the red species as well as the spectral dependence of two-photon conversion. The data reveal that two-photon excitation and photoactivation of the green form of EosFP can be selectively performed by choosing the proper wavelengths. Optical highlighting of small subcellular compartments was shown on HeLa cells expressing EosFP fused to a mitochondrial targeting signal. After three-dimensionally confined two-photon conversion of EosFP within the mitochondrial networks of the cells, the converted regions could be resolved in a 3D reconstruction from a dual-color 4Pi image stack.     

Mouse transgenesis in a single locus with independent regulation for multiple fluorophores

A major barrier to complex experimental design in mouse genetics is the allele problem: combining three or more alleles is time-consuming and inefficient. Here, we solve this problem for transgenic animals with a simple modification of existing BAC transgenesis protocols, and generate triple-colored 'prism' mice in which the major cell types of the brain: neurons, astrocytes, and oligodendrocytes, are each labeled with a distinct fluorophore. All three fluorophores are expressed from the same locus, yet each fluorophore is expressed in an independent temporal and spatial pattern. All three transgenes are generally co-inherited across multiple generations with stable genomic copy number and expression patterns. This generic solution should permit more sophisticated experimental manipulations to assess functional interactions amongst populations of cell types in vivo in a more rapid and efficient manner.