Around-the-objective total internal reflection fluorescence microscopy

Abstract

Total internal reflection fluorescence (TIRF) microscopy uses the evanescent field on the aqueous side of a glass/aqueous interface to selectively illuminate fluorophores within approximately 100 nm of the interface. Applications of the method include epi-illumination TIRF, where the exciting light is refracted by the microscope objective to impinge on the interface at incidence angles beyond critical angle, and prism-based TIRF, where exciting light propagates to the interface externally to the microscope optics. The former has higher background autofluorescence from the glass elements of the objective where the exciting beam is focused, and the latter does not collect near-field emission from the fluorescent sample. Around-the-objective TIRF, developed here, creates the evanescent field by conditioning the exciting laser beam to propagate through the submillimeter gap created by the oil immersion high numerical aperture objective and the glass coverslip. The approach eliminates background light due to the admission of the laser excitation to the microscopic optics while collecting near-field emission from the dipoles excited by the approximately 50 nm deep evanescent field.

Fig. 1

Schematic aoTIRF diagrams for (a) upright and (b) inverted microscope configurations. Dashed lines show the beam path through the prisms to the glass/aqueous interface where the exciting beam totally internally reflects. The inverted configuration has two other total internal reflections at glass/air interfaces. Coverslips and Littrow prisms are optically coupled by immersion oil. The upright microscope configuration also shows the beam conditioning elements consisting of a beam waist reducer from 10× and 20× microscope objectives and a low-aperture focusing lens. Beam conditioning is needed in both upright and inverted microscope configurations but is shown only for the upright.

Fig. 2

Detailed schematic aoTIRF upright microscope configuration. Symbols defining distances or angles are used in the text. Distance C is ~0.15 mm for a #1 coverslip. The approach-limiting edge requires θ₂ ≤ 3 deg and is the strictest constraint on laser beam shape. The upright configuration works with both small and large base Littrow prisms. Part of the objective is shown with the incident laser beam path. The exiting laser beam pathway is a mirror image of the incident path with reflection through the objective symmetry axis.

Fig. 3

Detailed schematic aoTIRF inverted microscope configuration. Symbols defining distances or angles used in Fig. 2 are equivalently defined here. Numeric distances are given in millimeters. Distance B is ~0.21 mm for a #2 coverslip. Oil is confined by the objective, the lower edge of the #1 coverslip, the short edges of the #2 coverslips, and the oil retainers. The inverted configuration requires the large base Littrow prisms. The exiting laser beam pathway is a mirror image of the incident path with reflection through the objective symmetry axis.

Fig. 4

Two views of the exciting laser beam intensity profile (rippled surface) and (FOV plane for the microscope objective near the focal plane for the exciting light. The x_p and y_p axes lie in the focal plane of the exciting light with z_p in the direction of light propagation and y_p in the incidence plane at the glass/aqueous interface. The vertical axis is intensity in arbitrary units for the intensity profile and micrometers for the (a) y_p or (b) x_p spatial dimensions.

Fig. 5

The exciting laser beam cross section for various z_p values, where the z_p axis is parallel to the direction of light propagation, and the 1/e² intensity level. The beam full width at z_p = 3.8 mm must be <250 μm for the exciting light to propagate past the approach-limiting edge without producing excessive light scattering.

Fig. 6

Close up photographs of the (a) upright and (b) inverted aoTIRF configurations. The slightly red pencil of light inside the prisms is the 488 nm laser beam exciting autofluorescence.

Fig. 7

The BFP microscopy emission pathway optical setup where OP is the object plane, OBJ is the objective, BFP is the back focal plane, TL is the tube lens with focal length f _TL, RL is the chromatic and spherical aberrations corrected removable lens, and CCD is the image plane for the camera.

Fig. 8

(a) Sequential images of 100 nm diameter fluorescent spheres under aoTIRF illumination in the upright microscope configuration. The images are 20 ms exposures of the CCD camera to light but are separated in time by 59 ms. (b) Sequential images of 100 nm diameter fluorescent spheres under aoTIRF illumination in the inverted microscope configuration. Exposure time was 100 ms on the EMCCD camera and frames are separated in time by 135 ms. (c) Sequential images of 40 nm diameter spheres diffusing in bulk solution under epi-illumination and observed in the upright microscope configuration. The images are 10 ms exposures of the CCD camera to light but are separated in time by 80 ms. Scale bars are shown for each panel.

Fig. 9

The BFP image from a 100 nm diameter sphere emitting fluorescence excited by aoTIRF illumination (top half) and the pattern computed from a single emitting dipole lying 50 nm above the interface and pointing horizontally along the x-axis direction (bottom half). The arrow at the bottom of the figure designates the bright ring due to critical angle emission.

Fig. 10

Fluorescence images of cardiac papillary muscle fibers. Fluorescence is detected from GFP tagged myosin regulatory light chain specifically replacing the native regulatory light chain on myosin cross bridges. Images in (a) and (b) compare the same sample but under epi-illumination and aoTIRF illumination. (c) is from another cardiac papillary fiber under oTIRF. All images were taken with the inverted microscope and the scale bar applies to all panels.