Two-color Fluorescence Imaging Research Articles

Two-site microendoscopic imaging probe for simultaneous three-dimensional imaging at two anatomic locations in tissues.

Systems that can image in three dimensions at cellular resolution and across different locations within an organism may enable insights into complex biological processes, such as immune responses, for which a single location measurement may be insufficient. In this Letter, we describe an in vivo two-site imaging probe (TIP) that can simultaneously image two anatomic sites with a maximum separation of a few centimeters. The TIP consists of two identical bendable graded index (GRIN) lenses and is demonstrated by a two-photon two-color fluorescence imaging system. Each GRIN lens has a field of view of 162 × 162 × 170 µm3, a nominal numerical aperture of 0.5, a magnification of 0.7, and working distances of 0.2 mm in air for both ends. A blind linear unmixing algorithm is applied to suppress bleedthrough between channels. We use this system to successfully demonstrate two-site two-photon two-color imaging of two biomedically relevant samples, i.e., (1) a mixture of two autofluorescent anti-cancer drugs and (2) a live hybrid tumor consisting of two spectrally distinct fluorescent cell lines.

Polarity-Sensitive Probe: Dual-Channel Visualization of the "Chameleon" Migration with the Assistance of Reactive Oxygen Species.

The real-time and differentiated visualization of the organelles is favorable for exploring the distribution and interaction. However, most visual probes emit monochromatic fluorescence and target a single organelle, which impedes the in-depth study of their interplay. To overcome this obstacle, we tactfully conceived a polarity-sensitive fluorescent DPDO-C that could accurately discriminate polarity changes in the cellular environment, exhibiting distinct fluorescence in lipid droplets (LDs) and mitochondria. Remarkably, the probe DPDO-C could migrate from mitochondria to LDs with the assistance of reactive oxygen species, which was conducive to further monitoring of the number and size of LDs as well as the interactions between LDs and other organelles. Moreover, the nuanced difference between normal and fatty liver tissues was also distinguished by two-color fluorescence imaging, which could act as a promising candidate for the early diagnosis of fatty liver.

A new cognate aptamer pair-based sandwich-type electrochemical biosensor for sensitive detection of Staphylococcus aureus

A pair of aptamers for Staphylococcus aureus (S. aureus) is immensely needed for developing sandwich-type signal-on electrochemical aptasensors. In this study, we have successfully developed a cognate pair of aptamers that bind to S. aureus simultaneously, among many aptamer candidates screened out after a total of ten rounds of bacterial cell-based systemic evolution of ligands by exponential enrichment (SELEX). The obtained aptamer candidates have been estimated by using flow cytometry and confocal microscope, to evaluate their binding affinity and specificity to the target cells. The screening for sandwich-type binding of cognate pair of aptamers with S. aureus was conducted by enzyme-based colorimetric assay and confirmed by circular dichroism (CD), two-color fluorescence imaging analysis, additionally. The cognate pair of two aptamers, named SA37 and SA81, showed very good affinity and specificity to S. aureus with their dissociation constants (Kd) of 16.5 ± 3.4 nM and 14.47 ± 8.18 nM, respectively. These newly discovered cognate pair of aptamers have been very successfully implemented to develop a sandwich-type signal-on electrochemical biosensor with the limit of detection (LOD) of 39 CFUs and 414 CFUs in buffer and spiked tap water samples, respectively. This study showed that this cognate pair of aptamers-based detection of S. aureus enables simple, rapid, and robust biosensors for food safety management.

Simultaneous Two-Color Visualization of Lipid Droplets and Endoplasmic Reticulum and Their Interplay by Single Fluorescent Probes in Lambda Mode.

In living systems, subcellular organelles mutually cooperate and closely contact to form organelle interaction networks. Thus, the simultaneous and discriminative visualization of different organelles is extremely valuable for elucidating their distribution and interplay. However, such meaningful investigations remain a great challenge due to the lack of advanced single fluorescent probes (SF-probes) capable of simultaneous and two-color imaging of two targets. Herein, for the first time, we present two excited-state intramolecular proton transfer (ESIPT) based SF-probes (PPC and EPC) for simultaneous two-color fluorescence imaging of lipid droplets (LDs) and the endoplasmic reticulum (ER) under single-wavelength excitation. Due to the strong electron-donating ability of the side substituents, the fluorescence spectra and colors of these ESIPT probes are highly sensitive to the nuance of water contents between LDs and ER, leading to orange and green fluorescence in LDs and ER, respectively, in the Lambda imaging mode. Using the probe PPC or EPC, the morphology, size, and distribution of LDs and ER have been investigated in live cells and tissues. With the aid of in situ and real-time fluorescence imaging in Lambda mode, we observed the generation of newborn LDs near the ER regions and their close apposition and shared identical fluorescence colors, probably providing a valuable proof for the mainstream hypothesis that LDs originate from the ER. The remarkable imaging performances render these SF-probes as powerful tools to decipher LD-ER related biological processes.

Video-rate large-scale imaging with Multi-Z confocal microscopy.

Fast, volumetric imaging over large scales has been a long-standing challenge in biological microscopy. To address this challenge, we report an augmented variant of confocal microscopy that uses a series of reflecting pinholes axially distributed in the detection space, such that each pinhole probes a different depth within the sample. We thus obtain simultaneous multiplane imaging without the need for axial scanning. Our microscope technique is versatile and configured here to provide two-color fluorescence imaging with a field of view larger than a millimeter at video rate. Its general applicability is demonstrated with neuronal imaging of both Caenorhabditis elegans and mouse brains in vivo.

A bright organic NIR-II nanofluorophore for three-dimensional imaging into biological tissues

Fluorescence imaging of biological systems in the second near-infrared (NIR-II, 1000–1700 nm) window has shown promise of high spatial resolution, low background, and deep tissue penetration owing to low autofluorescence and suppressed scattering of long wavelength photons. Here we develop a bright organic nanofluorophore (named p-FE) for high-performance biological imaging in the NIR-II window. The bright NIR-II >1100 nm fluorescence emission from p-FE affords non-invasive in vivo tracking of blood flow in mouse brain vessels. Excitingly, p-FE enables one-photon based, three-dimensional (3D) confocal imaging of vasculatures in fixed mouse brain tissue with a layer-by-layer imaging depth up to ~1.3 mm and sub-10 µm high spatial resolution. We also perform in vivo two-color fluorescence imaging in the NIR-II window by utilizing p-FE as a vasculature imaging agent emitting between 1100 and 1300 nm and single-walled carbon nanotubes (CNTs) emitting above 1500 nm to highlight tumors in mice.

Selective Imaging of Gram-Negative and Gram-Positive Microbiotas in the Mouse Gut.

The diverse gut microbial communities are crucial for host health. How the interactions between microbial communities and between host and microbes influence the host, however, is not well understood. To facilitate gut microbiota research, selective imaging of specific groups of microbiotas in the gut is of great utility but remains technically challenging. Here we present a chemical approach that enables selective imaging of Gram-negative and Gram-positive microbiotas in the mouse gut by exploiting their distinctive cell wall components. Cell-selective labeling is achieved by the combined use of metabolic labeling of Gram-negative bacterial lipopolysaccharides with a clickable azidosugar and direct labeling of Gram-positive bacteria with a vancomycin-derivatized fluorescent probe. We demonstrated this strategy by two-color fluorescence imaging of Gram-negative and Gram-positive gut microbiotas in the mouse intestines. This chemical method should be broadly applicable to different gut microbiota research fields and other bacterial communities studied in microbiology.

Local Pulses of Rhoa Activation Assemble Polarized Network Architectures for Efficient Actomyosin Contractility

Spatiotemporal patterning of actomyosin contractility plays a key role in cell and tissue morphogenesis during early development. In embryonic cells, actomyosin arrays are highly dynamic structures that remodel on a time scale of 10s of seconds, through a combination of local actomyosin turnover and rapid spatial redistribution of filaments and motors caused by myosin activity or actin polymerization. Because of these dynamic and active properties, contractility is complex and intrinsically self-organizing.We used the C. elegans early embryo to understand how cells pattern force generation through local modulation of self-organized contractility, focusing on pulsed contractility in the C. elegans embryo.We combined two-color fluorescence imaging, live single-molecule imaging, particle tracking, image analysis, and numerical modeling to tease apart the mechanisms of pulse initiation and termination. Our results demonstrate that the mechanical component (advection) played little role in pulse initiation or termination, and that the process was mostly governed by Actin and Myosin turnover. In our system, autocatalytic RhoA activation/recruitment is responsible for pulse initiation, while the delayed recruitment of a RhoA inactivator (RGA-3/4) onto Actin filaments drives pulse termination. Using single-molecule imaging, we further show that this mode of actin assembly drives the formation of structural units displaying a characteristically polarized architecture with (1) a gradient of myosin accumulation, myosin being recruited at the center of the pulses and (2) a transient anisotropic organization of the actin network, where filaments elongate from the instide of the pulse, barbed ends pointing outwards, as visualized by displacement of the actin-barbed-end-tracking formins that processively elongate actin filaments.Based on these results, we propose here that pulsed contractions represent a mode of actomyosin assembly which structures the actomyosin network in functional modules to drive efficient cell contractility.

Detection of rhodopsin dimerization in situ by PIE-FCCS, a time-resolved fluorescence spectroscopy.

Rhodopsin self-associates in the plasma membrane. At low concentrations, the interactions are consistent with a monomer-dimer equilibrium (Comar et al., J Am Chem Soc 136(23):8342-8349, 2014). At high concentrations in native tissue, higher-order clusters have been observed (Fotiadis et al., Nature 421:127-128, 2003). The physiological role of rhodopsin dimerization is still being investigated, but it is clear that a quantitative assessment is essential to determining the function of rhodopsin clusters in vision. To quantify rhodopsin interactions, I will outline the theory and methodology of a specialized time-resolved fluorescence spectroscopy for measuring membrane protein-protein interactions called pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). The strength of this technique is its ability to quantify rhodopsin interactions in situ (i.e., a live cell plasma membrane). There are two reasons for restricting the scope to live cell membranes. First, the compositional heterogeneity of the plasma membrane creates a complex milieu with thousands of lipid, protein, and carbohydrate species. This makes it difficult to infer quaternary interactions from detergent solubilized samples or construct a model phospholipid bilayer that recapitulates all of the interactions present in native membranes. Second, organizational structure and dynamics is a key feature of the plasma membrane, and fixation techniques like formaldehyde cross-linking and vitrification will modulate the interactions. PIE-FCCS is based on two-color fluorescence imaging with time-correlated single-photon counting (TCSPC) (Becker et al., Rev Sci Instrum 70:1835-1841, 1999). By time-tagging every detected photon, the data can be analyzed as a fluorescence intensity distribution, fluorescence lifetime histogram, or fluorescence (cross-)correlation spectra (FCS/FCCS) (Becker, Advanced time-correlated single-photon counting techniques, Springer, Berlin, 2005). These analysis tools can then be used to quantify protein concentration, mobility, clustering, and Förster resonance energy transfer (FRET). In this paper I will focus on PIE-FCCS, which interleaves two wavelength excitation events in time so that the effects of spectral cross-talk and FRET can be isolated. In this way it is possible to characterize monomer-dimer-oligomer equilibria with high accuracy (Müller et al., Biophys J 89:3508-3522, 2005). Currently, PIE-FCCS requires a customized equipment configuration that will be described below. There is an excellent protocol that outlines traditional FCCS on a commercially available instrument (Bacia and Schwille, Nat Protoc 2:2842-2856, 2007). The PIE-FCCS approach is a relatively recent advance in FCCS that has been used in live cell assays to quantify lipid-anchored protein clustering (Triffo et al., J Am Chem Soc 134:10833-10842, 2012), epidermal growth factor receptor dimerization (Endres et al., Cell 152:543-556, 2013), and recently the dimerization of opsin (Comar et al., J Am Chem Soc 136(23):8342-8349, 2014). This paper will outline the theory and instrumentation requirements for PIE-FCCS, as well as the data collection and analysis process.

Nonperturbative imaging of nucleoid morphology in live bacterial cells during an antimicrobial peptide attack.

Studies of time-dependent drug and environmental effects on single, live bacterial cells would benefit significantly from a permeable, nonperturbative, long-lived fluorescent stain specific to the nucleoids (chromosomal DNA). The ideal stain would not affect cell growth rate or nucleoid morphology and dynamics, even during laser illumination for hundreds of camera frames. In this study, time-dependent, single-cell fluorescence imaging with laser excitation and a sensitive electron-multiplying charge-coupled-device (EMCCD) camera critically tested the utility of "dead-cell stains" (SYTOX orange and SYTOX green) and "live-cell stains" (DRAQ5 and SYTO 61) and also 4',6-diamidino-2-phenylindole (DAPI). Surprisingly, the dead-cell stains were nearly ideal for imaging live Escherichia coli, while the live-cell stains and DAPI caused nucleoid expansion and, in some cases, cell permeabilization and the halting of growth. SYTOX orange performed well for both the Gram-negative E. coli and the Gram-positive Bacillus subtilis. In an initial application, we used two-color fluorescence imaging to show that the antimicrobial peptide cecropin A destroyed nucleoid-ribosome segregation over 20 min after permeabilization of the E. coli cytoplasmic membrane, reminiscent of the long-term effects of the drug rifampin. In contrast, the human cathelicidin LL-37, while similar to cecropin A in structure, length, charge, and the ability to permeabilize bacterial membranes, had no observable effect on nucleoid-ribosome segregation. Possible underlying causes are suggested.

Meaningful Interpretation of Subdiffusive Measurements in Living Cells (Crowded Environment) by Fluorescence Fluctuation Microscopy

In living cell or its nucleus, the motions of molecules are complicated due to the large crowding and expected heterogeneity of the intracellular environment. Randomness in cellular systems can be either spatial (anomalous) or temporal (heterogeneous). In order to separate both processes, we introduce anomalous random walks on fractals that represented crowded environments. We report the use of numerical simulation and experimental data of single-molecule detection by fluorescence fluctuation microscopy for detecting resolution limits of different mobile fractions in crowded environment of living cells. We simulate the time scale behavior of diffusion times τD(τ) for one component, e.g. the fast mobile fraction, and a second component, e.g. the slow mobile fraction. The less the anomalous exponent α the higher the geometric crowding of the underlying structure of motion that is quantified by the ratio of the Hausdorff dimension and the walk exponent d f /dw and specific for the type of crowding generator used. The simulated diffusion time decreases for smaller values of α ≠ 1 but increases for a larger time scale τ at a given value of α ≠ 1. The effect of translational anomalous motion is substantially greater if α differs much from 1. An α value close to 1 contributes little to the time dependence of subdiffusive motions. Thus, quantitative determination of molecular weights from measured diffusion times and apparent diffusion coefficients, respectively, in temporal auto- and crosscorrelation analyses and from time-dependent fluorescence imaging data are difficult to interpret and biased in crowded environments of living cells and their cellular compartments; anomalous dynamics on different time scales τ must be coupled with the quantitative analysis of how experimental parameters change with predictions from simulated subdiffusive dynamics of molecular motions and mechanistic models. We first demonstrate that the crowding exponent α also determines the resolution of differences in diffusion times between two components in addition to photophyscial parameters well-known for normal motion in dilute solution. The resolution limit between two different kinds of single molecule species is also analyzed under translational anomalous motion with broken ergodicity. We apply our theoretical predictions of diffusion times and lower limits for the time resolution of two components to fluorescence images in human prostate cancer cells transfected with GFP-Ago2 and GFP-Ago1. In order to mimic heterogeneous behavior in crowded environments of living cells, we need to introduce so-called continuous time random walks (CTRW). CTRWs were originally performed on regular lattice. This purely stochastic molecule behavior leads to subdiffusive motion with broken ergodicity in our simulations. For the first time, we are able to quantitatively differentiate between anomalous motion without broken ergodicity and anomalous motion with broken ergodicity in time-dependent fluorescence microscopy data sets of living cells. Since the experimental conditions to measure a selfsame molecule over an extended period of time, at which biology is taken place, in living cells or even in dilute solution are very restrictive, we need to perform the time average over a subpopulation of different single molecules of the same kind. For time averages over subpopulations of single molecules, the temporal auto- and crosscorrelation functions are first found. Knowing the crowding parameter α for the cell type and cellular compartment type, respectively, the heterogeneous parameter γ can be obtained from the measurements in the presence of the interacting reaction partner, e.g. ligand, with the same α value. The product α⋅γ=γ˜ is not a simple fitting parameter in the temporal auto- and two-color crosscorrelation functions because it is related to the proper physical models of anomalous (spatial) and heterogeneous (temporal) randomness in cellular systems. We have already derived an analytical solution for γ˜ in the special case of γ = 3/2 . In the case of two-color crosscorrelation or/and two-color fluorescence imaging (co-localization experiments), the second component is also a two-color species gr, for example a different molecular complex with an additional ligand. Here, we first show that plausible biological mechanisms from FCS/ FCCS and fluorescence imaging in living cells are highly questionable without proper quantitative physical models of subdiffusive motion and temporal randomness. At best, such quantitative FCS/ FCCS and fluorescence imaging data are difficult to interpret under crowding and heterogeneous conditions. It is challenging to translate proper physical models of anomalous (spatial) and heterogeneous (temporal) randomness in living cells and their cellular compartments like the nucleus into biological models of the cell biological process under study testable by single-molecule approaches. Otherwise, quantitative FCS/FCCS and fluorescence imaging measurements in living cells are not well described and cannot be interpreted in a meaningful way.

Two diverged human homeobox genes involved in the differentiation of human hematopoietic progenitors map to chromosome 1, bands q41-42.1.

Proteins encoded by homeobox containing genes are sequence-specific DNA binding proteins implicated in the control of gene expression in both developing and adult tissues. Two recently characterized human homeobox genes, HB9 and HB24, are highly expressed in CD34-positive marrow cells but not in CD34-depleted marrow cells. Their expression is readily down-regulated during the differentiation of hematopoietic progenitors to specific cell lineages. In this study, genomic DNA fragments isolated with HB9 (3 kb) and HB24 (6 kb) cDNAs were used to map their chromosomal location by fluorescence in situ hybridization. Both HB9 and HB24 DNA probes gave specific hybridization signals on chromosome 1. The hybridization loci were identified by combining fluorescence images of the probe signals with fluorescence banding patterns generated by cohybridization in situ with an Alu probe (R-like banding) and by DAPI staining (G-like). The results demonstrate that the loci of the HB24 and HB9 genes are within bands 1q41-q42.1. A cohybridization experiment utilizing both probes with two-color fluorescence imaging could not resolve separate loci for the two genes.