Quantifying and optimizing single-molecule switching nanoscopy at high speeds.
ABSTRACT: Single-molecule switching nanoscopy overcomes the diffraction limit of light by stochastically switching single fluorescent molecules on and off, and then localizing their positions individually. Recent advances in this technique have greatly accelerated the data acquisition speed and improved the temporal resolution of super-resolution imaging. However, it has not been quantified whether this speed increase comes at the cost of compromised image quality. The spatial and temporal resolution depends on many factors, among which laser intensity and camera speed are the two most critical parameters. Here we quantitatively compare the image quality achieved when imaging Alexa Fluor 647-immunolabeled microtubules over an extended range of laser intensities and camera speeds using three criteria - localization precision, density of localized molecules, and resolution of reconstructed images based on Fourier Ring Correlation. We found that, with optimized parameters, single-molecule switching nanoscopy at high speeds can achieve the same image quality as imaging at conventional speeds in a 5-25 times shorter time period. Furthermore, we measured the photoswitching kinetics of Alexa Fluor 647 from single-molecule experiments, and, based on this kinetic data, we developed algorithms to simulate single-molecule switching nanoscopy images. We used this software tool to demonstrate how laser intensity and camera speed affect the density of active fluorophores and influence the achievable resolution. Our study provides guidelines for choosing appropriate laser intensities for imaging Alexa Fluor 647 at different speeds and a quantification protocol for future evaluations of other probes and imaging parameters.
Project description:To suppress optical aberrations caused by refractive index mismatch, we employ glycerol-immersion objectives in conjunction with fused silica cover glasses and imaging buffers with a high glycerol content. Here we demonstrate that the addition of glycerol to the buffer does not degrade the switching behaviour of the dyes Alexa Fluor 647 and Alexa Fluor 568 in dSTORM measurements, which shows that this approach is suitable for dSTORM. Additionally, we report evidence that sealed sample geometries as used in our experiments reduce photobleaching due to the lower influx of oxygen into the imaging buffer.
Project description:Noninvasive localized measurement of extracellular pH in cancer tissues can have a significant impact on the management of cancer. Despite its significance, there are limited approaches for rapid and noninvasive measurement of local pH in a clinical environment. In this study, we demonstrate the potential of noninvasive topical delivery of Alexa-647 labeled pHLIP (pH responsive peptide conjugated with Alexa Fluor(®) 647) to image changes in extracellular pH associated with head and neck squamous cell carcinoma using widefield and high resolution imaging. We report a series of preclinical analyses to evaluate the optical contrast achieved after topical delivery of Alexa-647 labeled pHLIP in intact fresh human tissue specimens using widefield and high-resolution fluorescence imaging. Using topical delivery, Alexa-647 labeled pHLIP can be rapidly delivered throughout the epithelium of intact tissues with a depth exceeding 700 µm. Following labeling with Alexa-647 labeled pHLIP, the mean fluorescent contrast increased four to eight fold higher in clinically abnormal tissues as compared to paired clinically normal biopsies. Furthermore, the imaging approach showed significant differences in fluorescence contrast between the cancer and the normal biopsies across diverse patients and different anatomical sites (unpaired comparison). The fluorescence contrast differences between clinically abnormal and normal tissues were in agreement with the pathologic evaluation. Topical application of fluorescently labeled pHLIP can detect and differentiate normal from cancerous tissues using both widefield and high resolution imaging. This technology will provide an effective tool to assess tumor margins during surgery and improve detection and prognosis of head and neck cancer.
Project description:Transcriptional profile of the uppS-RBS (A to C) mutant during log growth phase in LB medium. Bacillus subtilis W168, WT vs. uppS-RBS (A to C). The experiment was conducted two times using three independent total RNA preparations (biological triplicates). Each comparison was performed in dye-swap with the same RNA preparation. For each paried comparison, one sample was labeled with Alexa Fluor 555 and the other was with Alexa Fluor 647.
Project description:We introduce a method for registration and visualization of correlative super-resolution microscopy images from different microscopy techniques. We established an automated registration procedure based on the generalized Hough transform. We developed a software tool to apply this algorithm and visualize correlated images from structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). To demonstrate the potential of this super-resolution correlator, we visualize the distribution of the presynaptic protein bassoon in the active zones of synapses in the molecular layer of the mouse cerebellum. First, a multiple labeled sample is imaged by SIM, followed by imaging of one of the fluorescent labels by dSTORM. To avoid the use of artificial fiducial markers, we used the signal of Alexa Fluor 647 recorded in switching buffer on the two microscopes for image superposition. We recorded multicolor SIM images in 20-?m thick brain slices to identify synapses in the dendritic system of Purkinje cells and put higher-resolved dSTORM images of the synaptic distribution of bassoon in registry.
Project description:Finding the right combination of a fluorescent dye and a mounting medium is crucial for optimal microscopy of fixed samples. It was recently shown that Vectashield, one of the most commonly used mounting media for conventional microscopy, can also be applied to super-resolution direct stochastic optical reconstruction microscopy (dSTORM). dSTORM utilizes conventional dyes and starts with samples in a fluorescent "ON" state. This helps in identifying structures of interest. Subsequently, labelled samples are induced into blinking, which is necessary for determining the position of single molecules and reconstruction of super-resolution images. This is only possible with certain fluorescent dyes and imaging buffers. One of the most widely used dyes for dSTORM, Alexa Fluor 647 (AF647), blinks in Vectashield. However, after preparing immunocytochemical samples in Vectashield, we noticed that the fluorescence intensity of AF647 is quenched. This is particularly evident for dimmer immunostainings, such as stainings of some components of neuronal cytoskeleton and axonal initial segment. Because structures of interest cannot be identified in quenched samples, loss of fluorescence intensity hinders imaging of AF647 in Vectashield. This has consequences for both conventional and dSTORM imaging. To overcome this, we provide: 1) a quantitative analysis of AF647 intensity in different imaging media, 2) a quantitative analysis of the suitability of Vectashield for dSTORM imaging of high and low-abundance AF647-labelled targets. Furthermore, for the first time, we quantitatively analyse the performance of Alexa Fluor Plus 647, a new variant of AF647-conjugated antibody, in dSTORM imaging.
Project description:Here, we measure the actin cytoskeleton arrangement of different morphological states of human platelets using a new protocol for photo-switching of rhodamine class fluorophores. A new medium composition was established for imaging the cytoskeleton using Alexa Fluor 488 conjugated to phalloidin. Morphological states of platelets bound to a glass substrate are visualized and quantified by two-dimensional localization microscopy at nanoscopic resolution. Marker-less drift correction yields localization of individual Alexa 488 conjugated to phalloidin with a positional accuracy of 12 nm.
Project description:Protease activity is frequently assayed using short peptides that are equipped with a Förster resonance energy transfer (FRET) reporter system. Many frequently used donor-acceptor pairs are excited in the ultraviolet range and suffer from low extinction coefficients and quantum yields, limiting their usefulness in applications where a high sensitivity is required. A large number of alternative chromophores are available that are excited in the visible range, for example, based on xanthene or cyanine core structures. These alternatives are not only larger in size but also more hydrophobic. Here, we show that the hydrophobicity of these chromophores not only affects the solubility of the resulting FRET-labeled peptides but also their kinetic parameters in a model enzymatic reaction. In detail, we have compared two series of 4-8 amino acid long peptides, designed to serve as substrates for the thermolysin-like protease (TLP-ste) from Geobacillus stearothermophilus. These peptides were equipped with a carboxyfluorescein donor and either Cy5 or its sulfonated derivative Alexa Fluor 647 as the acceptor. We show that the turnover rate k cat is largely unaffected by the choice of the acceptor fluorophore, whereas the K M value is significantly lower for the Cy5- than for the Alexa Fluor 647-labeled substrates. TLP-ste is a rather nonspecific protease with a large number of hydrophobic amino acids surrounding the catalytic site, so that the fluorophore itself may form additional interactions with the enzyme. This hypothesis is supported by the result that the difference between Cy5- and Alexa Fluor 647-labeled substrates becomes less pronounced with increasing peptide length, that is, when the fluorophore is positioned at a larger distance from the catalytic site. These results suggest that fluorophores may become an integral part of FRET-labeled peptide substrates and that K M and k cat values are generally only valid for a specific combination of the peptide sequence and FRET pair.
Project description:Transcriptional response of Bacillus subtilis to daptomycin in wild-type and in a daptomycin resistant mutant. Bacillus subtilis 168, WT (-DAP) vs. DapR1 (-DAP), WT (+DAP) vs. DapR1 (+DAP), DapR1 (+DAP) vs. DapR1 (-DAP). Each experiment was conducted at least twice using two independent total RNA preparations. For daptomycin untreated comparison between 168 WT and DapR1 mutant, DapR1 was labeled with Alexa Fluor 647 and WT was labeled with Alexa Fluor 555. For daptomycin treated experiments between WT and DapR1, DapR1 was labeled with Alexa Fluor 647 and WT with Alexa Fluor 555. For treated vs. untreated DapR1, the DAP treated samples were labeled with Alexa Fluor 647 and the untreated with Alexa Fluor 555. For dye swap, untreated DapR1 was labeled with Alexa Fluor 647 and DAP treated with Alexa Fluor 555.
Project description:To identify genes involved in the developmental process of Atlantic salmon smoltification, gene expression was compared between smolt and parr in tissues involved in osmoregulation (gill), metabolism (liver), imprinting (olfactory rosettes) and neuroendocrine control (hypothalamus and pituitary). Tissue samples were harvested from laboratory-reared parr and smolts on the same date. Smolts were distinguished from parr by size and appearance; developmental status was confirmed by physiological assays. Eight biological replicates (16 fish) balanced for sex and for dye were used in the liver, gill, olfactory rosette, and hypothalamus comparisons. Four male parr were compared to four male smolts and four female parr were compared to four female smolts; smolts were labeled with Alexa Fluor 555 on four arrays and with Alexa Fluor 647 on four arrays. Six biological replicates (12 fish) were used for the pituitary comparison (two female and four male).
Project description:BACKGROUND: Optical super-resolution imaging of fluorescently stained biological samples is rapidly becoming an important tool to investigate protein distribution at the molecular scale. It is therefore important to develop practical super-resolution methods that allow capturing the full three-dimensional nature of biological systems and also can visualize multiple protein species in the same sample. METHODOLOGY/PRINCIPAL FINDINGS: We show that the use of a combination of conventional near-infrared dyes, such as Alexa 647, Alexa 680 and Alexa 750, all excited with a 671 nm diode laser, enables 3D multi-colour super-resolution imaging of complex biological samples. Optically thick samples, including human tissue sections, cardiac rat myocytes and densely grown neuronal cultures were imaged with lateral resolutions of ?15 nm (std. dev.) while reducing marker cross-talk to <1%. Using astigmatism an axial resolution of ?65 nm (std. dev.) was routinely achieved. The number of marker species that can be distinguished depends on the mean photon number of single molecule events. With the typical photon yields from Alexa 680 of ?2000 up to 5 markers may in principle be resolved with <2% crosstalk. CONCLUSIONS/SIGNIFICANCE: Our approach is based entirely on the use of conventional, commercially available markers and requires only a single laser. It provides a very straightforward way to investigate biological samples at the nanometre scale and should help establish practical 4D super-resolution microscopy as a routine research tool in many laboratories.