Scanning Near-field Optical/Atomic Force Microscopy detection of fluorescence in situ hybridization signals beyond the optical limit

Fluorescence in situ hybridization (FISH) is widely used in molecular biological study. However, high-resolution analysis of fluorescent signals is theoretically limited by the 300-nm resolution optical limit of light microscopy. As an alternative to detection by light microscopy, we used Scanning Near-field Optical/Atomic Force Microscopy (SNOM/AFM), which can simultaneously obtain topographic and fluorescent images with nanometer-scale resolution. In this study, we demonstrated high-resolution SNOM/AFM imaging of barley chromosome (Hordeum vulgare, cv. Minorimugi) FISH signals using telomeric DNA probes. Besides detecting the granular structures on chromosomes in a topographic image, we clearly detected fluorescent signals in telomeric regions with low-magnification imaging. The high-resolution analysis suggested that one of the telomeric signals could be observed by expanded imaging as two fluorescent regions separated by approximately 250 nm. This result indicated that the fluorescent signals beyond the optical limit were detected with higher resolution scanning by SNOM/AFM.     

High-resolution near-field optical imaging of single nuclear pore complexes under physiological conditions

Scanning near-field optical microscopy (SNOM) circumvents the diffraction limit of conventional light microscopy and is able to achieve optical resolutions substantially below 100 nm. However, in the field of cell biology SNOM has been rarely applied, probably because previous techniques for sample-distance control are less sensitive in liquid than in air. Recently we developed a distance control based on a tuning fork in tapping mode, which is also well-suited for imaging in solution. Here we show that this approach can be used to visualize single membrane protein complexes kept in physiological media throughout. Nuclear envelopes were isolated from Xenopus laevis oocytes at conditions shown recently to conserve the transport functions of the nuclear pore complex (NPC). Isolated nuclear envelopes were fluorescently labeled by antibodies against specific proteins of the NPC (NUP153 and p62) and imaged at a resolution of approximately 60 nm. The lateral distribution of epitopes within the supramolecular NPC could be inferred from an analysis of the intensity distribution of the fluorescence spots. The different number densities of p62- and NUP153-labeled NPCs are determined and discussed. Thus we show that SNOM opens up new possibilities for directly visualizing the transport of single particles through single NPCs and other transporters.     

近场光学显微镜结合量子点标记人胃腺癌SGC-7901细胞靶点的观察

扫描近场光学显微镜突破衍射极限,具有纳米量级的空间分辨率,量子点(QD s)标记有荧光强度高且抗光漂白能力强等优点。结合上述两种技术,对人胃腺癌SGC-7901细胞膜表面特异性结合的叶酸受体(FR)进行成像探测,获得了叶酸受体在SGC-7901细胞膜表面上的分布,以及细胞内化外源性叶酸过程中叶酸受体在细胞膜表面的分布变化,成像的光学分辨率达到120 nm。实验结果表明:特异性结合的叶酸受体在SGC-7901细胞膜表面的分布,绝大部分是以聚集体的形式存在。随着SGC-7901细胞内化叶酸量的增加,叶酸受体在细胞膜表面的分布密度逐渐降低,并在经过120 m in左右趋于稳定。上述方法和手段为实现单细胞水平上靶点分布和变化的长期监测,肿瘤细胞内化受体的机制研究提供了新的技术途径。     

Measurements of multiply scattered light for on-line monitoring of changes in size distribution of cell debris suspension

Dual wavelength frequency-domain measurements of photon migration (FDPM) are conducted on filtrate samples obtained from an industrial centrifugation process designed to separate Escherichia coli cell debris from the inclusion bodies. FDPM measurements consist of detecting phase delay of intensity-modulated light at 670 and 820 (or 830) nm. Optical properties of isotropic scattering and absorption are obtained from the regression of phase delay data to the optical diffusion equation. We show that the corresponding intensity-based measurements alone cannot provide accurate and independent estimates for these optical properties. However, FDPM-derived scattering coefficients of filtrate solutions (primarily consisting of 0.1-0.2 micrometer E. coli cell debris) are sensitive to approximately 1 vol % of added inclusion bodies (of 1-2 micrometer size). The technique, theory, and future adaptation of FDPM as an on-line monitor to detect the loss of inclusion bodies in centrifugation following homogenization are presented and contrasted to conventional, intensity-based measurements.     

Near-field optical imaging of abasic sites on a single DNA molecule

Scanning near-field optical microscopy (SNOM) imaging was performed to allow for the direct visualization of damaged sites on individual DNA molecules to a scale of a few tens of nanometers. Fluorescence in situ hybridization on extended DNA molecules was modified to detect a single abasic site. Abasic sites were specifically labelled with a biotinlylated aldehyde-reactive probe and fluorochrome-conjugated streptavidin. By optimizing the performance of the SNOM technique, we could obtain high contrast near-field optical images that enabled high-resolution near-field fluorescence imaging using optical fiber probes with small aperture sizes. High-resolution near-field fluorescence imaging demonstrated that two abasic sites within a distance of 120 nm are clearly obtainable, something which is not possible using conventional fluorescence in situ hybridization combined with far-field fluorescence microscopy.     

Optical imaging of nanoscale cellular structures

Visualization of subcellular structures and their temporal evolution is of utmost importance to understand a vast range of biological processes. Optical microscopy is the method of choice for imaging live cells and tissues; it is minimally invasive, so processes can be observed over extended periods of time without generating artifacts due to intense light irradiation. The use of fluorescence microscopy is advantageous because biomolecules or supramolecular structures of interest can be labeled specifically with fluorophores, so the images reveal information on processes involving only the labeled molecules. The key restriction of optical microscopy is its moderate resolution, which is limited to about half the wavelength of light (～200 nm) due to fundamental physical laws governing wave optics. Consequently, molecular processes taking place at spatial scales between 1 and 100 nm cannot be studied by regular optical microscopy. In recent years, however, a variety of super-resolution fluorescence microscopy techniques have been developed that circumvent the resolution limitation. Here, we present a brief overview of these techniques and their application to cellular biophysics.     

Recent developments of genetically encoded optical sensors for cell biology

Optical sensors are powerful tools for live cell research as they permit to follow the location, concentration changes or activities of key cellular players such as lipids, ions and enzymes. Most of the current sensor probes are based on fluorescence which provides great spatial and temporal precision provided that high‐end microscopy is used and that the timescale of the event of interest fits the response time of the sensor. Many of the sensors developed in the past 20 years are genetically encoded. There is a diversity of designs leading to simple or sometimes complicated applications for the use in live cells. Genetically encoded sensors began to emerge after the discovery of fluorescent proteins, engineering of their improved optical properties and the manipulation of their structure through application of circular permutation. In this review, we will describe a variety of genetically encoded biosensor concepts, including those for intensiometric and ratiometric sensors based on single fluorescent proteins, Forster resonance energy transfer‐based sensors, sensors utilising bioluminescence, sensors using self‐labelling SNAP‐ and CLIP‐tags, and finally tetracysteine‐based sensors. We focus on the newer developments and discuss the current approaches and techniques for design and application. This will demonstrate the power of using optical sensors in cell biology and will help opening the field to more systematic applications in the future.     

Spatial Distribution of Optical Near-Fields in Plasmonic Gold Sphere Segment Voids

We present a comprehensive experimental and computational study on the electromagnetic field distribution in sphere segment void arrays. Surface plasmon polaritons can be excited in these void arrays, resulting in greatly enhanced electromagnetic fields. With the scanning near-field optical microscope (SNOM) we are able to measure the electromagnetic field distribution at the sample surface. For this purpose, an array of relatively large voids with a sphere diameter of 900 nm was fabricated, allowing for an easy access of the scanning glass-fibre tip and yielding very detailed scans. Complementary, finite-difference time-domain (FDTD) calculations on a complete void array have been performed and compared with the SNOM intensity maps and experimental reflectivity data. We show in a direct way both the existence of extended and localised modes in the Au void array for three different void depths. We also show and discuss the changes that the modes undergo for the different void depths and excitation wavelengths. Moreover, since the simulations were performed for two different void geometries, one containing perfectly spherical void surfaces and another more realistic one, which considers the presence of interstitial wall holes and other imperfections, as observed in scanning electron micrographs, we were able to determine by comparison with the experiment under which conditions an array of idealised sphere segment voids is a meaningful model. This demonstrates that both SNOM and FDTD simulations are powerful tools for understanding the plasmonic response of metallic nanostructures, thus enabling, for instance, a design for applications in ultra-sensitive optical detection.     

A method for imaging single molecules at the plasma membrane of live cells within tissue slices

Recent advances in light microscopy allow individual biological macromolecules to be visualized in the plasma membrane and cytosol of live cells with nanometer precision and ∼10-ms time resolution. This allows new discoveries to be made because the location and kinetics of molecular interactions can be directly observed in situ without the inherent averaging of bulk measurements. To date, the majority of single-molecule imaging studies have been performed in either unicellular organisms or cultured, and often chemically fixed, mammalian cell lines. However, primary cell cultures and cell lines derived from multi-cellular organisms might exhibit different properties from cells in their native tissue environment, in particular regarding the structure and organization of the plasma membrane. Here, we describe a simple approach to image, localize, and track single fluorescently tagged membrane proteins in freshly prepared live tissue slices and demonstrate how this method can give information about the movement and localization of a G protein–coupled receptor in cardiac tissue slices. In principle, this experimental approach can be used to image the dynamics of single molecules at the plasma membrane of many different soft tissue samples and may be combined with other experimental techniques.     

Non-Invasive Detection of Early Retinal Neuronal Degeneration by Ultrahigh Resolution Optical Coherence Tomography

Optical coherence tomography (OCT) has revolutionises the diagnosis of retinal disease based on the detection of microscopic rather than subcellular changes in retinal anatomy. However, currently the technique is limited to the detection of microscopic rather than subcellular changes in retinal anatomy. However, coherence based imaging is extremely sensitive to both changes in optical contrast and cellular events at the micrometer scale, and can generate subtle changes in the spectral content of the OCT image. Here we test the hypothesis that OCT image speckle (image texture) contains information regarding otherwise unresolvable features such as organelle changes arising in the early stages of neuronal degeneration. Using ultrahigh resolution (UHR) OCT imaging at 800 nm (spectral width 140 nm) we developed a robust method of OCT image analyses, based on spatial wavelet and texture-based parameterisation of the image speckle pattern. For the first time we show that this approach allows the non-invasive detection and quantification of early apoptotic changes in neurons within 30 min of neuronal trauma sufficient to result in apoptosis. We show a positive correlation between immunofluorescent labelling of mitochondria (a potential source of changes in cellular optical contrast) with changes in the texture of the OCT images of cultured neurons. Moreover, similar changes in optical contrast were also seen in the retinal ganglion cell- inner plexiform layer in retinal explants following optic nerve transection. The optical clarity of the explants was maintained throughout in the absence of histologically detectable change. Our data suggest that UHR OCT can be used for the non-invasive quantitative assessment of neuronal health, with a particular application to the assessment of early retinal disease.     

Single Molecule Probe Scanning Optical Force Imaging Microscopefor Viewing Live Cells

We have developed an imaging system that combines the soft compliance of an optical trap with the sensitivity of single particle tracking to image forces on/in live cells using a single molecule probe. The probe used is a single (or few) molecule of interest that is conjugated with a single 40 nm colloidalgold probe. The colloidal gold/membrane protein complex, freely diffusing on a live cell, is held in a laser trap while the cell is scanned underneath. Computer control allows for synchronization of the cell scan and capture of the probe position. Resistance to the dragging of the probe images a fine structure of barriers in the membrane of live cells.     

Sensitive and High Resolution Localization and Tracking of Membrane Proteins in Live Cells with BRET

Peripheral and integral membrane proteins can be located in several different subcellular compartments, and it is often necessary to determine the location of such proteins or to track their movement in living cells. Image‐based colocalization of labeled membrane proteins and compartment markers is frequently used for this purpose, but this method is limited in terms of throughput and resolution. Here we show that bioluminescence resonance energy transfer (BRET) between membrane proteins of interest and compartment‐targeted BRET partners can report subcellular location and movement of membrane proteins in live cells. The sensitivity of the method is sufficient to localize a few hundred protein copies per cell. The spatial resolution can be sufficient to determine membrane topology, and the temporal resolution is sufficient to track changes that occur in less than 1 second. BRET requires little user intervention, and is thus amenable to large‐scale experimental designs with standard instruments.     

Raman Tweezers Spectroscopy of Live,Single Red and White Blood Cells

An optical trap has been combined with a Raman spectrometer to make high-resolution measurements of Raman spectra of optically-immobilized, single, live red (RBC) and white blood cells (WBC) under physiological conditions. Tightly-focused, near infrared wavelength light (1064 nm) is utilized for trapping of single cells and 785 nm light is used for Raman excitation at low levels of incident power (few mW). Raman spectra of RBC recorded using this high-sensitivity, dual-wavelength apparatus has enabled identification of several additional lines; the hitherto-unreported lines originate purely from hemoglobin molecules. Raman spectra of single granulocytes and lymphocytes are interpreted on the basis of standard protein and nucleic acid vibrational spectroscopy data. The richness of the measured spectrum illustrates that Raman studies of live cells in suspension are more informative than conventional micro-Raman studies where the cells are chemically bound to a glass cover slip.     

Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of fluorescence can be changed by exposure to light of a specific wavelength. Optical highlighting allows noninvasive marking of a subpopulation of fluorescent molecules, and is therefore ideal for tracking single cells or organelles.Critical parameters for efficient photoconversion are the intensity and the exposure time of the photoconversion light. If the intensity is too low, photoconversion will be slow or not occur at all. On the other hand, too much intensity or too long exposure can photobleach the protein and thereby reduce the efficiency of photoconversion.This protocol describes a general approach how to set up a confocal laser scanning microscope for pc-FP photoconversion applications. First, we describe a procedure for preparing purified protein droplet samples. This sample format is very convenient for studying the photophysical behavior of fluorescent proteins under the microscope. Second, we will use the protein droplet sample to show how to configure the microscope for photoconversion. And finally, we will show how to perform optical highlighting in live cells, including dual-probe optical highlighting with mOrange2 and Dronpa.Download video file.^{(127M, mp4)}     

Phosphatidylinositol 4,5-bisphosphate functions as a second messenger that regulates cytoskeleton-plasma membrane adhesion

Binding interactions between the plasma membrane and the cytoskeleton define cell functions such as cell shape, formation of cell processes, cell movement, and endocytosis. Here we use optical tweezers tether force measurements and show that plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) acts as a second messenger that regulates the adhesion energy between the cytoskeleton and the plasma membrane. Receptor stimuli that hydrolyze PIP2 lowered adhesion energy, a process that could be mimicked by expressing PH domains that sequester PIP2 or by targeting a 5'-PIP2-phosphatase to the plasma membrane to selectively lower plasma membrane PIP2 concentration. Our study suggests that plasma membrane PIP2 controls dynamic membrane functions and cell shape by locally increasing and decreasing the adhesion between the actin-based cortical cytoskeleton and the plasma membrane.     

Condition-dependent cell volume and concentration of Escherichia coli to facilitate data conversion for systems biology modeling

Systems biology modeling typically requires quantitative experimental data such as intracellular concentrations or copy numbers per cell. In order to convert population-averaging omics measurement data to intracellular concentrations or cellular copy numbers, the total cell volume and number of cells in a sample need to be known. Unfortunately, even for the often studied model bacterium Escherichia coli this information is hardly available and furthermore, certain measures (e.g. cell volume) are also dependent on the growth condition. In this work, we have determined these basic data for E. coli cells when grown in 22 different conditions so that respective data conversions can be done correctly. First, we determine growth-rate dependent cell volumes. Second, we show that in a 1 ml E. coli sample at an optical density (600 nm) of 1 the total cell volume is around 3.6 μl for all conditions tested. Third, we demonstrate that the cell number in a sample can be determined on the basis of the sample's optical density and the cells' growth rate. The data presented will allow for conversion of E. coli measurement data normalized to optical density into volumetric cellular concentrations and copy numbers per cell--two important parameters for systems biology model development.     

SCOPE-Seq: a scalable technology for linking live cell imaging and single-cell RNA sequencing

Optically decodable beads link the identity of a sample to a measurement through an optical barcode, enabling libraries of biomolecules to be captured on beads in solution and decoded by fluorescence. This approach has been foundational to microarray, sequencing, and flow-based expression profiling technologies. We combine microfluidics with optically decodable beads and show that phenotypic analysis of living cells can be linked to single-cell sequencing. As a proof-of-concept, we demonstrate the accuracy and scalability of our tool called Single Cell Optical Phenotyping and Expression sequencing (SCOPE-Seq) to combine live cell imaging with single-cell RNA sequencing.     

Ultra‐structural analysis of human spermatozoa by aperture scanning near‐field optical microscopy

Scanning near‐field optical microscopy (SNOM) represents a potential candidate for investigation of ultrastructure in human spermatozoa. It is a noninvasive optical technique that offers two main advantages: minimal sample preparation and simultaneous topographical and optical images acquisition with a spatial resolution beyond the diffraction limit. This enables the combination of surface characterization and information from the inner cellular organization in a single acquisition providing an immediate and comprehensive analysis of the cellular portions. In this work spermatozoa are immobilized on poly‐L‐lysine coated coverslips, fixed according to a standard protocol and imaged by aperture‐SNOM in air. In the SNOM images, all peculiar sperm portions show well‐resolved optical features, which exhibit good similarities with the structures revealed in transmission electron microscopy images, as compared with literature data. The optical features of anomalous spermatozoa are clearly different as respect with those observed for healthy ones. This analysis reveals the potentialities of SNOM and opens to its application to high‐resolution analysis of sperm morphological alterations, which might be relevant in reproductive medicine.     

Understanding laser desorption with circularly polarized light

We present aspects of emerging optical activity in thin racemic 1,1′-Bi-2-naphthol films upon irradiation with circularly polarized light and subsequent resonant two-photon absorption in the sample. Thorough analysis of the sample morphology is conducted by means of (polarization-resolved) optical microscopy and scanning electron microscopy (SEM). The influence of crystallization on the nonlinear probing technique (second harmonic generation circular dichroism [SHG-CD]) is investigated. Optical activity and crystallization are brought together by a systematic investigation in different crystallization regimes. We find crystallization to be responsible for two counter-acting effects, which arise for different states of crystallization. Measuring crystallized samples offers the best signal-to-noise ratio, but it limits generation of optical activity due to self-assembly effects. For suppression of crystallization on the other hand, there is a clear indication that enantiomeric selective desorption is responsible for the generation of optical activity in the sample. We reach the current resolution limit of probing with SHG-CD, as we suppress the crystallization in the racemic sample during desorption. In addition, intensity-dependent measurements on the induced optical activity reveal an onset threshold (≈0.7 TW cm⁻²), above which higher order nonlinear processes impair the generation of optical activity by desorption with CPL.     

Characterization of photodamage to escherichia coli in optical traps

Optical tweezers (infrared laser-based optical traps) have emerged as a powerful tool in molecular and cell biology. However, their usefulness has been limited, particularly in vivo, by the potential for damage to specimens resulting from the trapping laser. Relatively little is known about the origin of this phenomenon. Here we employed a wavelength-tunable optical trap in which the microscope objective transmission was fully characterized throughout the near infrared, in conjunction with a sensitive, rotating bacterial cell assay. Single cells of Escherichia coli were tethered to a glass coverslip by means of a single flagellum: such cells rotate at rates proportional to their transmembrane proton potential (. J. Mol. Biol. 138:541-561). Monitoring the rotation rates of cells subjected to laser illumination permits a rapid and quantitative measure of their metabolic state. Employing this assay, we characterized photodamage throughout the near-infrared region favored for optical trapping (790-1064 nm). The action spectrum for photodamage exhibits minima at 830 and 970 nm, and maxima at 870 and 930 nm. Damage was reduced to background levels under anaerobic conditions, implicating oxygen in the photodamage pathway. The intensity dependence for photodamage was linear, supporting a single-photon process. These findings may help guide the selection of lasers and experimental protocols best suited for optical trapping work.