Total Internal Reflection Fluorescence Illumination Research Articles

Correlative single-molecule and structured illumination microscopy of fast dynamics at the plasma membrane

Total internal reflection fluorescence (TIRF) microscopy offers powerful means to uncover the functional organization of proteins in the plasma membrane with very high spatial and temporal resolution. Traditional TIRF illumination, however, shows a Gaussian intensity profile, which is typically deteriorated by overlaying interference fringes hampering precise quantification of intensities—an important requisite for quantitative analyses in single-molecule localization microscopy (SMLM). Here, we combine flat-field illumination by using a standard πShaper with multi-angular TIR illumination by incorporating a spatial light modulator compatible with fast super-resolution structured illumination microscopy (SIM). This distinct combination enables quantitative multi-color SMLM with a highly homogenous illumination. By using a dual camera setup with optimized image splitting optics, we achieve a versatile combination of SMLM and SIM with up to three channels. We deploy this setup for establishing robust detection of receptor stoichiometries based on single-molecule intensity analysis and single-molecule Förster resonance energy transfer (smFRET). Homogeneous illumination furthermore enables long-term tracking and localization microscopy (TALM) of cell surface receptors identifying spatial heterogeneity of mobility and accessibility in the plasma membrane. By combination of TALM and SIM, spatially and molecularly heterogenous diffusion properties can be correlated with nanoscale cytoskeletal organization and dynamics.

Indicator-dependent differences in detection of local intracellular Ca2+ release events evoked by voltage-gated Ca2+ entry in pancreatic β-cells

Genetically encoded Ca2+ indicators have become widely used in cell signalling studies as they offer advantages over cell-loaded dye indicators in enabling specific cellular or subcellular targeting. Comparing responses from dye and protein-based indicators may provide information about indicator properties and cell physiology, but side-by-side recordings in cells are scarce. In this study, we compared cytoplasmic Ca2+ concentration ([Ca2+]i) changes in insulin-secreting β-cells recorded with commonly used dyes and indicators based on circularly permuted fluorescent proteins. Total internal reflection fluorescence (TIRF) imaging of K+ depolarization-triggered submembrane [Ca2+]i increases showed that the dyes Fluo-4 and Fluo-5F mainly reported stable [Ca2+]i elevations, whereas the proteins R-GECO1 and GCaMP5G more often reported distinct [Ca2+]i spikes from an elevated level. [Ca2+]i spiking occurred also in glucose-stimulated cells. The spikes reflected Ca2+ release from the endoplasmic reticulum, triggered by autocrine activation of purinergic receptors after exocytotic release of ATP and/or ADP, and the spikes were consequently prevented by SERCA inhibition or P2Y1-receptor antagonism. Widefield imaging, which monitors the entire cytoplasm, increased the spike detection by the Ca2+ dyes. The indicator-dependent response patterns were unrelated to Ca2+ binding affinity, buffering and mobility, and probably reflects the much slower dissociation kinetics of protein compared to dye indicators. Ca2+ dyes thus report signalling within the submembrane space excited by TIRF illumination, whereas the protein indicators also catch Ca2+ events originating outside this volume. The study highlights that voltage-dependent Ca2+ entry in β-cells is tightly linked to local intracellular Ca2+ release mediated via an autocrine route that may be more important than previously reported direct Ca2+ effects on phospholipase C or on intracellular channels mediating calcium-induced calcium release.

MesoTIRF: A prism-based Total Internal Reflection Fluorescence illuminator for high resolution, high contrast imaging of large cell populations

Total Internal Reflection Fluorescence (TIRF) illumination bypasses the axial diffraction limit of light by using an evanescent field to excite fluorophores close to a sample substrate. However, standard TIRF imaging through the objective requires a high numerical aperture (NA) to generate the evanescent wave. Available lenses have a high magnification with a correspondingly small field of view—ranging from ∼50 μm to 1 mm in diameter. Switching to the older prism-TIRF configuration introduced by Axelrod in the 1980s might seem to remove the requirement for high objective NA and allow the use of existing large-field objectives. Unfortunately, these lenses are unsuitable because their throughput of light is too low for TIRF imaging. As such, high sensitivity TIRF imaging over a much larger mesoscopic field has yet to be demonstrated. We have developed a prism-based TIRF illuminator for the Mesolens—a highly corrected objective lens with an unparalleled ratio of NA to magnification. The imaging field of the Mesolens is 204 times larger than that of the TIRF objectives previously described, increasing the optical throughput of the optical system by a factor of 25 compared to an off-the-shelf microscope objective of the same magnification. We demonstrate MesoTIRF imaging of cell specimens and show the multi-wavelength capability of the modality across more than 700 cells in a single image.

Construction and characterization of a versatile multimodal single-molecule super-resolution microscope for 3D whole cell studies.

The cellular environment is dense and complex, and imaging with 3D single-molecule super-resolution microscopy requires careful consideration of appropriate illumination methods to use in relation to the goals of the study. There is, however, typically a tradeoff between performance and simplicity when selecting illumination schemes. Epi-illumination is a common and simple technique, but it generates high unwanted background fluorescence and causes photodamage and photobleaching throughout the imaged cell. Total internal reflection fluorescence (TIRF) is another technique which provides excellent contrast, but it is limited to imaging a thin volume at the bottom of a sample and is thus incompatible with whole-cell imaging. Light sheet illumination is an alternative, whole-cell compatible approach where a thin sheet of light is directed orthogonally to the detection axis to optically section a cell and reduce the fluorescence background, photobleaching, and photodamage. In this work we demonstrate a versatile microscopy platform which integrates the sectioning and background reducing capability of light sheet illumination with homogeneous, flat-field epi- and TIRF illumination. A flat-field intensity profile improves the control of fluorophore blinking kinetics and the resolution across the entire field of view when compared to a standard Gaussian intensity profile. Additionally, this microscope is designed to allow very fast switching between illumination modalities and laser wavelengths, using primarily commercially available parts. When combined with point spread function engineering, our system conveniently enables 3D single-molecule super-resolution imaging and molecular counting throughout whole cells with optimized and adjustable illumination modalities.

Uniform, Universal, Shadowless TIRF Microscopy Via Annular Fiber Bundle

Total internal reflection fluorescence (TIRF) illumination is a popular excitation method in fluorescence microscopy. TIRF illumination is generated when the excitation beam strikes the glass/water interface of the sample at an angle greater than the critical angle, creating an evanescent excitation field with a shallow penetration depth that is well-suited to studying single-molecules and surface features of a biological sample with a high signal-to-background ratio. TIRF is typically achieved by focusing the excitation laser beam to the outermost edge of the back focal plane (BFP) of the imaging objective; however, this approach suffers from imaging artifacts such as interference fringes from the coherent excitation source and shadowing artifacts caused by the unidirectional light and scattering objects in cells.

Single-shot, shadowless total internal reflection fluorescence microscopy via annular fiber bundle.

We demonstrate a method of generating instantaneous and uniform total internal reflection fluorescence (TIRF) excitation by using an annular fiber bundle and spatially incoherent light sources. We show the flexibility of our method in that it can generate TIRF excitation with either a laser light source or an LED of different wavelengths, and facilitate switching between TIRF and epi illumination. In this report we detail the design of the fiber bundle, then demonstrate the performance via single-molecule imaging in the presence of high background and high throughput, and uniform TIRF imaging of cells over a large field of view. Our versatile method will enable quantitative shadowless TIRF imaging.

TIRF Microscope Image Sequences of Fluorescent IgE-FcεRI Receptor Complexes inside a FcεRI-Centric Synapse in RBL-2H3 Cells.

Total internal reflection fluorescence (TIRF) microscope image sequences are commonly used to study receptors in live cells. The dataset presented herein facilitates the study of the IgE-FcεRI receptor signaling complex (IgE-RC) in rat basophilic leukemia (RBL-2H3) cells coming into contact with a supported lipid bilayer with 25 mol% N-dinitrophenyl-aminocaproyl phosphatidylethanolamine, modeling an immunological synapse. TIRF microscopy was used to image IgE-RCs within this FcεRI-centric synapse by loading RBL-2H3 cells with fluorescent anti-dinitrophenyl (anti-DNP) immunoglobulin E (IgE) in suspension for 24 h. Fluorescent anti-DNP IgE (IgE488) concentrations of this suspension increased from 10% to 100% and corresponding non-fluorescent anti-DNP IgE concentrations decreased from 90% to 0%. After the removal of unbound anti-DNP IgE, multiple image sequences were taken for each of these ten conditions. Prior to imaging, anti-DNP IgE-primed RBL-2H3 cells were either kept for a few minutes, for about 30 min, or for about one hour in Hanks buffer. The dataset contains 482 RBL-2H3 model synapse image stacks, dark images to correct for background intensity, and TIRF illumination profile images to correct for non-uniform TIRF illumination. After background subtraction, non-uniform illumination correction, and conversion of pixel units from analog-to-digital units to photo electrons, the average pixel intensity was calculated. The average pixel intensity within FcεRI-centric synapses for all three Hanks buffer conditions increased linearly at a rate of 0.42 ± 0.02 photo electrons per pixel per % IgE488 in suspension. RBL-2H3 cell degranulation was tested by detecting β-hexosaminidase activity. Prolonged RBL-2H3 cell exposure to Hanks buffer inhibited exocytosis in RBL-2H3 cells.

Visualization of ligand-induced dopamine D2S and D2L receptor internalization by TIRF microscopy

G protein-coupled receptors (GPCRs), including the dopamine receptors, represent a group of important pharmacological targets. Upon agonist binding, GPCRs frequently undergo internalization, a process that is known to attenuate functional responses upon prolonged exposure to agonists. In this study, internalization was visualized by means of total internal reflection fluorescence (TIRF) microscopy at a level of discrete single events near the plasma membrane with high spatial resolution. A novel method has been developed to determine the relative extent of internalized fluorescent receptor-ligand complexes by comparative fluorescence quantification in living CHO cells. The procedure entails treatment with the reducing agent sodium borohydride, which converts cyanine-based fluorescent ligands on the membrane surface to a long-lived reduced form. Because the highly polar reducing agent is not able to pass the cell membrane, the fluorescent receptor-ligand complexes located in internalized compartments remain fluorescent under TIRF illumination. We applied the method to investigate differences of the short (D2S) and the long (D2L) isoforms of dopamine D2 receptors in their ability to undergo agonist-induced internalization.

Clearer view for TIRF and oblique illumination microscopy.

In Total Internal Reflection Fluorescence (TIRF) microscopy, the sample is illuminated with an evanescent field that yields a thin optical section. However, its widefield detection has no rejection mechanism against out-of-focus blur from scattered light that can compromise TIRF images. Here I demonstrate that via structured illumination, out-of-focus blur can be effectively suppressed in TIRF microscopy, yielding strikingly clearer images. The same mechanism can also be applied to oblique illumination schemes that extend the reach of TIRF microscopy beyond the basal surface of the cell. The two imaging modes are used to image a biosensor, clathrin coated vesicles and the actin cytoskeleton in different cell types with improved contrast.

Focus on lasers and imaging

Focus on lasers and imaging

Synaptophysin 1 Clears Synaptobrevin 2 from the Presynaptic Active Zone to Prevent Short-Term Depression.

Release site clearance is an important process during synaptic vesicle (SV) recycling. However, little is known about its molecular mechanism. Here we identify self-assembly of exocytosed Synaptobrevin 2 (Syb2) and Synaptophysin 1 (Syp1) by homo- and hetero-oligomerization into clusters as key mechanisms mediating release site clearance for preventing cis-SNARE complex formation at the active zone (AZ). In hippocampal neurons from Syp1 knockout mice, neurons expressing a monomeric Syb2 mutant, or after acute block of the ATPase N-ethylmaleimide-sensitive factor (NSF), responsible for cis-SNARE complex disassembly, we found strong frequency-dependent short-term depression (STD), whereas retrieval of Syb2 by compensatory endocytosis was only affected weakly. Defects in Syb2 endocytosis were stimulus- and frequency-dependent, indicating that Syp1 is not essential for Syb2 retrieval, but for its efficient clearance upstream of endocytosis. Our findings identify an SV protein as a release site clearance factor.

Large apparent slip at a moving contact line

Two different particle tracking velocimetry techniques are used to measure the fluid velocities close to the substrate in the vicinity of both receding and advancing contact lines. The slip velocity is found to be as much as 60% of the substrate speed near the contact line and persists as far as 10 μm from the liquid-gas interface. The estimated slip length near the contact line singularity requires a measurement of the shear rate close the substrate which depends strongly on the spatial resolution of the measurement technique. The slip length is found to be approximately 5 μm when flood illumination is used and approximately 500 nm when total internal reflection fluorescence illumination is used.

Total Internal Reflection Fluorescence (TIRF) Microscopy Illuminator for Improved Imaging of Cell Surface Events

Total internal reflection fluorescence (TIRF) microscopy is a high-contrast imaging technique suitable for observing biological events that occur on or near the cell membrane. The improved contrast is accomplished by restricting the thickness of the excitation field to over an order of a magnitude narrower than the z-resolution of an epi-fluorescence microscope. This technique also increases signal-to-noise, making it a valuable tool for imaging cellular events such as vesicles undergoing exocytosis or endocytosis, viral particle formation, cell signaling, and dynamics of membrane proteins. This protocol describes the basic procedures for setting up a through-the-objective TIRF illuminator and a prism-based TIRF illuminator. In addition, an alternate protocol for incorporating an automated deflection system into through-the-objective TIRF is given. This system can be used to decrease aberrations in the illumination field, to quickly switch between epi- and TIRF illumination, and to adjust the penetration depth during multicolor TIRF applications. In the commentary, a description of the total internal reflection phenomenon is given, critical parameters of a TIRF microscope are discussed, and technical challenges and considerations are reviewed.

Probing the regulation of TASK potassium channels by PI(4,5)P2 with switchable phosphoinositide phosphatases

TASK channels are background K+ channels that contribute to the resting conductance in many neurons. A key feature of TASK channels is the reversible inhibition by Gq-coupled receptors, thereby mediating the dynamic regulation of neuronal activity by modulatory transmitters. The mechanism that mediates channel inhibition is not fully understood. While it is clear that activation of Gαq is required, the immediate signal for channel closure remains controversial. Experimental evidence pointed to either phospholipase C (PLC)-mediated depletion of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) as the cause for channel closure or to a direct inhibitory interaction of active Gαq with the channel. Here, we address the role of PI(4,5)P2 for G-protein-coupled receptor (GPCR)-mediated TASK inhibition by using recently developed genetically encoded tools to alter phosphoinositide (PI) concentrations in the living cell.When expressed in CHO cells, TASK-1- and TASK-3-mediated currents were not affected by depletion of plasma membrane PI(4,5)P2 either via the voltage-activated phosphatase Ci-VSP or via chemically triggered recruitment of a PI(4,5)P2-5'-phosphatase. Depletion of both PI(4,5)P2 and PI(4)P via membrane recruitment of a novel engineered dual-specificity phosphatase also did not inhibit TASK currents. In contrast, each of these methods produced robust inhibition of the bona fide PI(4,5)P2-dependent channel KCNQ4. Efficient depletion of PI(4,5)P2 and PI(4)P was further confirmed with a fluorescent phosphoinositide sensor. Moreover, TASK channels recovered normally from inhibition by co-expressed muscarinic M1 receptors when resynthesis of PI(4,5)P2 was prevented by depletion of cellular ATP. These results demonstrate that TASK channel activity is independent of phosphoinositide concentrations within the physiological range. Consequently, Gq-mediated inhibition of TASK channels is not mediated by depletion of PI(4,5)P2.

Scanning Evanescent Fields in TIRF Microscopy Using a Single Point-Like Light Source and a DNA Worm Drive

Total internal reflection fluorescence (TIRF) microscopy is an elegant optical technique that limits the dimension of the volume, in which fluorophores are excited, along the z-direction to the hundred nanometer-scale. The method makes use of the evanescent field, which arises when light is totally internally reflected at the boundary to a medium of lower refractive index. Often the penetration depth of this exponentially decaying field is left undetermined, which sets limits to the reproducibility in different experiments. Here, we present a novel method to measure this quantity: We use a quantum dot as a point-like light source and a Holliday junction as a drive to move the fluorescent probe with nanometer precision perpendicular to the substrate surface. The junction serves as a worm drive, which couples rotation into translational movement, while the DNA pitch serves as a precise intrinsic ruler. The Holliday junction is forced to migrate by adding negative turns to the DNA stretched perpendicular to the surface using magnetic tweezers. This causes the quantum dot, which is attached upstream of the junction, to decrease its height above the surface by 3.4 nm per turn. Thus it can be moved continuously through the excitation field while monitoring its height-dependent fluorescent signal. Since the quantum dot is a point-like light source, the intensity decay of the evanescent field can be obtained by dividing the signal recorded in TIRF illumination by the one recorded in conventional epi-illumination without further corrections. Our assay should allow for probing the axial intensity profile of any electromagnetic field distribution with a point-like light source, without the necessity of attaching it to an interfering surface.

Substrate Binding Stoichiometry and Kinetics of the Norepinephrine Transporter

The human norepinephrine (NE) transporter (hNET) attenuates neuronal signaling by rapid NE clearance from the synaptic cleft, and NET is a target for cocaine and amphetamines as well as therapeutics for depression, obsessive-compulsive disorder, and post-traumatic stress disorder. In spite of its central importance in the nervous system, little is known about how NET substrates, such as NE, 1-methyl-4-tetrahydropyridinium (MPP+), or amphetamine, interact with NET at the molecular level. Nor do we understand the mechanisms behind the transport rate. Previously we introduced a fluorescent substrate similar to MPP+, which allowed separate and simultaneous binding and transport measurement (Schwartz, J. W., Blakely, R. D., and DeFelice, L. J. (2003) J. Biol. Chem. 278, 9768-9777). Here we use this substrate, 4-(4-(dimethylamino)styrl)-N-methyl-pyridinium (ASP+), in combination with green fluorescent protein-tagged hNETs to measure substrate-transporter stoichiometry and substrate binding kinetics. Calibrated confocal microscopy and fluorescence correlation spectroscopy reveal that hNETs, which are homomultimers, bind one substrate molecule per transporter subunit. Substrate residence at the transporter, obtained from rapid on-off kinetics revealed in fluorescence correlation spectroscopy, is 526 micros. Substrate residence obtained by infinite dilution is 1000 times slower. This novel examination of substrate-transporter kinetics indicates that a single ASP+ molecule binds and unbinds thousands of times before being transported or ultimately dissociated from hNET. Calibrated fluorescent images combined with mass spectroscopy give a transport rate of 0.06 ASP+/hNET-protein/s, thus 36,000 on-off binding events (and 36 actual departures) occur for one transport event. Therefore binding has a low probability of resulting in transport. We interpret these data to mean that inefficient binding could contribute to slow transport rates.

Evanescent field excitation of fluorescence by epi-illumination microscopy

By simple modification of the pattern of fluorescence excitation light in an epi-illumination inverted microscope, one can achieve conditions that produce total internal reflection fluorescence (TIRF) by evanescent wave excitation. Though traditionally requiring a collimated beam traversing through a special prism, TIRF also can be achieved by epi-illumination through the periphery of a 1.4 numerical aperture objective. An opaque disk of appropriate size is placed in the illumination path external to the microscope so as to cast a sharp, real-image shadow at the objective's back focal plane. This shadow allows a hollow cone of epiillumination rays traveling at only super-critical angles to reach the glass/water interface at the sample plane. Three kinds of TIRF illumination patterns can be produced by variations of this scheme: (1) a small spot of illumination of 1.5 microm radius by use of a laser light source, (2) a large region of illumination by use of a laser-illuminated diffusing screen located upbeam from the opaque disk, and (3) a large region of illumination by use of a conventional mercury arc.