Eliminating unwanted far-field excitation in objective-type TIRF. Part I. identifying sources of nonevanescent excitation light.
ABSTRACT: Total internal reflection fluorescence microscopy (TIRFM) achieves subdiffraction axial sectioning by confining fluorophore excitation to a thin layer close to the cell/substrate boundary. However, it is often unknown how thin this light sheet actually is. Particularly in objective-type TIRFM, large deviations from the exponential intensity decay expected for pure evanescence have been reported. Nonevanescent excitation light diminishes the optical sectioning effect, reduces contrast, and renders TIRFM-image quantification uncertain. To identify the sources of this unwanted fluorescence excitation in deeper sample layers, we here combine azimuthal and polar beam scanning (spinning TIRF), atomic force microscopy, and wavefront analysis of beams passing through the objective periphery. Using a variety of intracellular fluorescent labels as well as negative staining experiments to measure cell-induced scattering, we find that azimuthal beam spinning produces TIRFM images that more accurately portray the real fluorophore distribution, but these images are still hampered by far-field excitation. Furthermore, although clearly measureable, cell-induced scattering is not the dominant source of far-field excitation light in objective-type TIRF, at least for most types of weakly scattering cells. It is the microscope illumination optical path that produces a large cell- and beam-angle invariant stray excitation that is insensitive to beam scanning. This instrument-induced glare is produced far from the sample plane, inside the microscope illumination optical path. We identify stray reflections and high-numerical aperture aberrations of the TIRF objective as one important source. This work is accompanied by a companion paper (Pt.2/2).
Project description:Azimuthal beam scanning makes evanescent-wave (EW) excitation isotropic, thereby producing total internal reflection fluorescence (TIRF) images that are evenly lit. However, beam spinning does not fundamentally address the problem of propagating excitation light that is contaminating objective-type TIRF. Far-field excitation depends more on the specific objective than on cell scattering. As a consequence, the excitation impurities in objective-type TIRF are only weakly affected by changes of azimuthal or polar beam angle. These are the main results of the first part of this study (Eliminating unwanted far-field excitation in objective-type TIRF. Pt.1. Identifying sources of nonevanescent excitation light). This second part focuses on exactly where up beam in the illumination system stray light is generated that gives rise to nonevanescent components in TIRF. Using dark-field imaging of scattered excitation light we pinpoint the objective, intermediate lenses and, particularly, the beam scanner as the major sources of stray excitation. We study how adhesion-molecule coating and astrocytes or BON cells grown on the coverslip surface modify the dark-field signal. On flat and weakly scattering cells, most background comes from stray reflections produced far from the sample plane, in the beam scanner and the objective lens. On thick, optically dense cells roughly half of the scatter is generated by the sample itself. We finally show that combining objective-type EW excitation with supercritical-angle fluorescence (SAF) detection efficiently rejects the fluorescence originating from deeper sample regions. We demonstrate that SAF improves the surface selectivity of TIRF, even at shallow penetration depths. The coplanar microscopy scheme presented here merges the benefits of beam spinning EW excitation and SAF detection and provides the conditions for quantitative wide-field imaging of fluorophore dynamics at or near the plasma membrane.
Project description:Total internal reflection fluorescence microscopy (TIRFM) has been proven to be an extremely powerful technique in animal cell research for generating high contrast images and dynamic protein conformation information. However, there has long been a perception that TIRFM is not feasible in plant cells because the cell wall would restrict the penetration of the evanescent field and lead to scattering of illumination. By comparative analysis of epifluorescence and TIRF in root cells, it is demonstrated that TIRFM can generate high contrast images, superior to other approaches, from intact plant cells. It is also shown that TIRF imaging is possible not only at the plasma membrane level, but also in organelles, for example the nucleus, due to the presence of the central vacuole. Importantly, it is demonstrated for the first time that this is TIRF excitation, and not TIRF-like excitation described as variable-angle epifluorescence microscopy (VAEM), and it is shown how to distinguish the two techniques in practical microscopy. These TIRF images show the highest signal-to-background ratio, and it is demonstrated that they can be used for single-molecule microscopy. Rare protein events, which would otherwise be masked by the average molecular behaviour, can therefore be detected, including the conformations and oligomerization states of interacting proteins and signalling networks in vivo. The demonstration of the application of TIRFM and single-molecule analysis to plant cells therefore opens up a new range of possibilities for plant cell imaging.
Project description:Azimuthal beam scanning eliminates the uneven excitation field arising from laser interference in through-objective total internal reflection fluorescence (TIRF) microscopy. The same principle can be applied to scanning angle interference microscopy (SAIM), where precision control of the scanned laser beam presents unique technical challenges for the builders of custom azimuthal scanning microscopes. Accurate synchronization between the instrument computer, beam scanning system and excitation source is required to collect high quality data and minimize sample damage in SAIM acquisitions. Drawing inspiration from open-source prototyping systems, like the Arduino microcontroller boards, we developed a new instrument control platform to be affordable, easily programmed, and broadly useful, but with integrated, precision analog circuitry and optimized firmware routines tailored to advanced microscopy. We show how the integration of waveform generation, multiplexed analog outputs, and native hardware triggers into a single central hub provides a versatile platform for performing fast circle-scanning acquisitions, including azimuthal scanning SAIM and multiangle TIRF. We also demonstrate how the low communication latency of our hardware platform can reduce image intensity and reconstruction artifacts arising from synchronization errors produced by software control. Our complete platform, including hardware design, firmware, API, and software, is available online for community-based development and collaboration.
Project description:Total-internal-reflection fluorescence (TIRF) microscopy provides high optical-sectioning capability and a good signal-contrast ratio for structures near the surfaces of cells. In recent years, several improvements have been developed, such as variable-angle TIRF (VA-TIRF) and spinning TIRF (sp-TIRF), which permit quantitative image analysis and address non-uniform scattering fringes, respectively. Here, we present a dual-color DMD-based shadowless-illuminated variable-angle TIRF (siva-TIRF) system that provides a uniform illumination field. By adjusting the incidence angle of the illuminating laser on the back focal plane (BFP) of the objective, we can rapidly illuminate biological samples in layers of various thicknesses in TIRF or hollow-cone epi-fluorescence mode. Compared with other methods of accomplishing VA-TIRF/sp-TIRF illumination, our system is simple to build and cost-effective, and it provides optimal multi-plane dual-color images. By showing spatiotemporal correlated movement of clathrin-coated structures with microtubule filaments from various layers of live cells, we demonstrate that cortical microtubules are important spatial regulators of clathrin-coated structures. Moreover, our system can be used to prove superb axial information of three-dimensional movement of structures near the plasma membrane within live cells.
Project description:Total internal reflection fluorescence (TIRF) microscopy, which has about 100-nm axial excitation depth, is the method of choice for nanometer-sectioning imaging for decades. Lately, several new imaging techniques, such as variable angle TIRF microscopy, supercritical-angle fluorescence microscopy, and metal-induced energy transfer imaging, have been proposed to enhance the axial resolution of TIRF. However, all of these methods use high numerical aperture (NA) objectives, and measured images inevitably have small field-of-views (FOVs). Small-FOV can be a serious limitation when multiple cells need to be observed. We propose large-FOV nanometer-sectioning microscopy, which breaks the complementary relations between the depth of focus and axial sectioning by using MIET. Large-FOV imaging is achieved with a low-magnification objective, while nanometer-sectioning is realized utilizing metal-induced energy transfer and biexponential fluorescence lifetime analysis. The feasibility of our proposed method was demonstrated by imaging nanometer-scale distances between the basal membrane of human aortic endothelial cells and a substrate.
Project description:We report superresolution optical sectioning using a multiangle total internal reflection fluorescence (TIRF) microscope. TIRF images were constructed from several layers within a normal TIRF excitation zone by sequentially imaging and photobleaching the fluorescent molecules. The depth of the evanescent wave at different layers was altered by tuning the excitation light incident angle. The angle was tuned from the highest (the smallest TIRF depth) toward the critical angle (the largest TIRF depth) to preferentially photobleach fluorescence from the lower layers and allow straightforward observation of deeper structures without masking by the brighter signals closer to the coverglass. Reconstruction of the TIRF images enabled 3D imaging of biological samples with 20-nm axial resolution. Two-color imaging of epidermal growth factor (EGF) ligand and clathrin revealed the dynamics of EGF-activated clathrin-mediated endocytosis during internalization. Furthermore, Bayesian analysis of images collected during the photobleaching step of each plane enabled lateral superresolution (<100 nm) within each of the sections.
Project description:Multifocal structured illumination microscopy (MSIM) provides a twofold resolution enhancement beyond the diffraction limit at sample depths up to 50 µm, but scattered and out-of-focus light in thick samples degrades MSIM performance. Here we implement MSIM with a microlens array to enable efficient two-photon excitation. Two-photon MSIM gives resolution-doubled images with better sectioning and contrast in thick scattering samples such as Caenorhabditis elegans embryos, Drosophila melanogaster larval salivary glands, and mouse liver tissue.
Project description:Total internal reflection fluorescence microscopy (TIRFM) is the method of choice to visualize a variety of cellular processes in particular events localized near the plasma membrane of live adherent cells. This imaging technique not relying on particular fluorescent probes provides a high sectioning capability. It is, however, restricted to a single plane. We present here a method based on a versatile design enabling fast multiwavelength azimuthal averaging and incidence angles scanning to computationally reconstruct 3D images sequences. We achieve unprecedented 50-nm axial resolution over a range of 800 nm above the coverslip. We apply this imaging modality to obtain structural and dynamical information about 3D actin architectures. We also temporally decipher distinct Rab11a-dependent exocytosis events in 3D at a rate of seven stacks per second.
Project description:Second-harmonic generation (SHG) is the most specific label-free indicator of collagen accumulation in widespread pathologies such as fibrosis, and SHG-based measurements hold important potential for biomedical analyses. However, efficient collagen SHG scoring in histological slides is hampered by the limited depth-of-field of usual nonlinear microscopes relying on focused Gaussian beam excitation. In this work we analyze theoretically and experimentally the use of Bessel beam excitation to address this issue. Focused Bessel beams can provide an axially extended excitation volume for nonlinear microscopy while preserving lateral resolution. We show that shaping the focal volume has consequences on signal level and scattering directionality in the case of coherent signals (such as SHG) which significantly differ from the case of incoherent signals (two-photon excited fluorescence, 2PEF). We demonstrate extended-depth SHG-2PEF imaging of fibrotic mouse kidney histological slides. Finally, we show that Bessel beam excitation combined with spatial filtering of the harmonic light in wave vector space can be used to probe collagen accumulation more efficiently than the usual Gaussian excitation scheme. These results open the way to SHG-based histological diagnoses.
Project description:We combined instant structured illumination microscopy (iSIM) with total internal reflection fluorescence microscopy (TIRFM) in an approach referred to as instant TIRF-SIM, thereby improving the lateral spatial resolution of TIRFM to 115 ± 13 nm without compromising speed, and enabling imaging frame rates up to 100 Hz over hundreds of time points. We applied instant TIRF-SIM to multiple live samples and achieved rapid, high-contrast super-resolution imaging close to the coverslip surface.