Journal: Biophysical Journal

Article Title: Calibrating Evanescent-Wave Penetration Depths for Biological TIRF Microscopy

doi: 10.1016/j.bpj.2019.07.048

Figure Lengend Snippet: Fundamentals of TIRF excitation. (A) A prismless (objective-type) TIRF is shown (left). A laser beam is focused to an eccentric position in the back-focal plane (BFP) (dashed line) of a high-NA objective, generating a collimated beam impinging at an oblique angle θ at the dielectric interface (solid gray, n3 > n1). For θ exceeding the critical angle, θc = asin(n1/n3), the beam is totally reflected at the interface and an inhomogeneous surface wave is generated in the rare medium (n1) (middle). This evanescent wave (EW) propagates along the surface (the Pointing vector, S, is oriented in +x direction for a beam impinging from the left, arrow), and its intensity decays exponentially with axial (+z) distance from the reflecting interface (right) with a length constant (“penetration depth”) δ of the order of 100 nm. (B) Dependence of δ on θ, for λ = 488 nm, n1 = 1.35, and for different substrate indices n3 is shown. The higher n3, the smaller the critical angle θc and the better the optical sectioning. For a typical borosilicate glass/cell interface and an NA of 1.45, the maximally attainable angle θΝΑ limits the penetration depth to 73 nm (dashed). In this angle range, δ depends steeply on θ, demanding high precision and accuracy when adjusting θ. (C) Dependence of θc and δ∞ on substrate index, n3, is shown. The asymptotes of the critical angle θc and limiting penetration depth δ∞ for grazing incidence (θ → 90°), respectively, decrease monotonously with n3. The solid line indicates n3 = 1.52 (BK-7), as earlier. To see this figure in color, go online.

Article Snippet: The penetration depth was measured on a commercial multiangle objective-type TIRF setup (Visitron Systems, Puchheim, Germany) built around an I × 83 inverted microscope equipped with an autofocus module and a UAPON100XOTIRF (NA 1.49 objective) (all Olympus, Tokyo, Japan).

Techniques: Generated, Plasmid Preparation

Journal: Biophysical Journal

Article Title: Calibrating Evanescent-Wave Penetration Depths for Biological TIRF Microscopy

doi: 10.1016/j.bpj.2019.07.048

Figure Lengend Snippet: Quantitative uses of TIRF. (A) Variable-angle TIRF (VA-TIRF) is shown. Smaller θ translates into larger illumination depths δ(θ) (block arrow) allowing a topographic reconstruction of axial fluorophore profiles from a multi-θ stack. (B) Multi-λ excitation is shown. δ scales linearly with λ (block arrows). Toggling between different excitation wavelengths alters the excited volume (gray arrowheads, top). Simultaneously adjusting θ between different-color acquisitions maintains a constant excitation volume, permitting quantitative colocalization or FRET studies at or near the basal plasma membrane (bottom). (C) TIR-FRAP, a variant of fluorescence recovery after photobleaching (FRAP), sequentially images, bleaches, and reimages a sample with EW excitation. An intense pulse of evanescent light (flash) selectively bleaches the surface-proximal fluorophores. Unbleached molecules from deeper sample regions repopulate the bleached volume. TIRF-FRAP allows studying the average mobility and mobile fraction of near-membrane fluorescent species (inset). Here, the penetration depth is constant, and the illumination intensity is modulated between imaging and bleaching episodes. To see this figure in color, go online.

Article Snippet: The penetration depth was measured on a commercial multiangle objective-type TIRF setup (Visitron Systems, Puchheim, Germany) built around an I × 83 inverted microscope equipped with an autofocus module and a UAPON100XOTIRF (NA 1.49 objective) (all Olympus, Tokyo, Japan).

Techniques: Blocking Assay, Clinical Proteomics, Membrane, Variant Assay, Fluorescence, Imaging

Journal: Biophysical Journal

Article Title: Calibrating Evanescent-Wave Penetration Depths for Biological TIRF Microscopy

doi: 10.1016/j.bpj.2019.07.048

Figure Lengend Snippet: Intensity-based techniques for calibrating axial intensity decays. (A) Raisin-cake subdiffraction fluorescent microspheres are embedded in an index-matched (RI ≈ n1) agarose gel. (B) Oblique-fluorescent-layer sample, consisting of a fluorophore-coated coverslip and a spacer (of height d, typically another cover glass), is shown. In a variant, (C), the surface of a long-f lens of known curvature radius is coated with fluorescent beads and positioned on the interface. Again, the known axial fluorophore profile is used to probe the EW decay. In the “infinitely” thick (d ≫> λ) dye layer approach (D), a homogenous fluorophore solution is used to measure the cumulative fluorescence at a given penetration depth, δ(θ). Upon multi-θ sweeps, depending on how far the EW reaches in the fluorescent solution, the intensity changes in a predictable manner. (E) In a dilute dye solution, the intensity fluctuations resulting from the diffusion of single molecules through the EW-excited volume allow measuring the penetration depth through TIR-FCS. (F) A point emitter attached to the tip of an atomic force microscope (data not shown) or attached to DNA samples the EW in a single point. To see this figure in color, go online.

Article Snippet: The penetration depth was measured on a commercial multiangle objective-type TIRF setup (Visitron Systems, Puchheim, Germany) built around an I × 83 inverted microscope equipped with an autofocus module and a UAPON100XOTIRF (NA 1.49 objective) (all Olympus, Tokyo, Japan).

Techniques: Agarose Gel Electrophoresis, Variant Assay, Fluorescence, Diffusion-based Assay, Microscopy

Journal: Biophysical Journal

Article Title: Calibrating Evanescent-Wave Penetration Depths for Biological TIRF Microscopy

doi: 10.1016/j.bpj.2019.07.048

Figure Lengend Snippet: Determining the effective penetration depth with a raisin-cake test sample. (A) The experimental workflow is shown. Acquisition of a z-stack of images in epifluorescence (EPI) (θ = 0°) is shown (left). EPI image taken at z0, localizing the bottom layer of beads on the coverslip, is shown (middle). Corresponding TIRF image at z0 and different beam angles, θ, is shown (right). (B) Each bead was localized from its axial intensity profile by fitting a Gaussian distribution (black line) with the average fluorescence, F(z), measured in a 3 × 3 pixel region of interest (+). Example bead at z = 38 nm, with F(z) well described by a Gaussian distribution is shown (left). The top images correspond to planes identified by arrows. An example of a distorted profile discarded from analysis, z = 44 nm, is shown (right). (C) The intrinsic fluorescence (i.e., bead intensity measured from the peak of the Gaussian distribution as in (B) for each bead) versus its in-focus position z is shown. (D) The bead fluorescence F(EPI)(z; z0) when focusing at the lowest bead layer at z0, upon EPI illumination, as a function of previously measured bead position z and after normalization for its respective intrinsic fluorescence as in (C) is shown. The observed axial intensity decay (exponential fit, Dz = 481 ± 166 nm) is the result of the increasing defocus for surface-distant beads and the objective’s finite depth of field and distance-dependent collection efficiency. (D) The same information in (C) but for TIRF excitation (●) and fit of a double-exponential decay (line) with the measured fluorescence F(TIRF)(z; z0) from all beads are shown. Color codes for different θ are shown. The long-range component (Dz) was identical to that measured for out-of-focus beads upon EPI excitation, (D), confirming our interpretation of spurious nonevanescent excitation. The short-range component δ(θ) was taken as the effective EW the penetration depth and was, respectively, 349 ± 118, 139 ± 20, 109 ± 16, and 91 ± 13 nm for θ = 65.0, 67.5, 70.0, and 72.5°. Note the surface-enhancement by a factor of ∼4 of F(TIRF)(z0) versus F(EPI)(z0), as expected from theory. Depending on θ, the fractional amplitude of the nonevanescent long-range excitation component varied between 13 and 15.5% (see main text). The inset shows double-exponential fits on a log scale and 95% confidence interval of the fit (thin lines and shaded in the respective color code). In all cases, the measured short-range component δ(θ) was larger than predicted by theory. To see this figure in color, go online.

Article Snippet: The penetration depth was measured on a commercial multiangle objective-type TIRF setup (Visitron Systems, Puchheim, Germany) built around an I × 83 inverted microscope equipped with an autofocus module and a UAPON100XOTIRF (NA 1.49 objective) (all Olympus, Tokyo, Japan).

Techniques: Fluorescence