Fig 1.
Optical scheme of the spinning spot TIRF illuminator.
(A) Optical schematic. Laser light from a single-mode fiber optic (FO) is collimated by a parabolic mirror beam expander (PM) and directed onto a pair of orthogonal galvanometer-driven mirrors (GM: only a single mirror is shown for clarity). Reflected light is relayed through a scan lens (SL: formed by a 10x eyepiece) and a tube lens (TL: formed by a 50 mm f 1.8 camera lens) which focus the laser light to a diffraction-limited spot at the back focal plane (BFP) of the microscope objective (obj). A dichroic mirror placed between the tube lens and objective (not shown) reflects laser light to the objective, while allowing emitted fluorescence to pass through to the imaging camera. IP = image plane; cIP = conjugate image planes; cBFP = conjugate back focal plane. (B) Photograph of the spinning spot TIRF illuminator, constructed from Thor Labs optical cage components. (C, D) Photographs taken through the microscope ocular, illustrating the alignment procedure using a rhodamine film/fluorescein solution test specimen. (C) Images taken with a Bertrand lens present so as to view the back focal plane of the objective lens. The left panel shows the narrow green circle traced by the spinning lase spot when the radius is set below the critical angle for total internal reflection. The right panel was captured after increasing the scan radius to achieve TIRF excitation. The appearance of the orange ring arises from selective fluorescence excitation of the film of rhodamine within the evanescent field. (D) Corresponding appearance of the fluorescein solution/rhodamine film test specimen viewed conventionally (i.e. without the Bertrand lens) with the radius of the scan circle below (left) and above (right) that required for TIRF excitation. The white box in the right panel outlines the approximate imaging field of the EMCCD camera.
Fig 2.
Improved field uniformity with shadowless TIRF excitation.
(A, B) Images (500x300 pixel, 40 x 24 μm) of rhodamine film on a coverglass. The image in A was captured using stationary-spot (conventional) TIRF excitation at 488 nm; that in B shows the same specimen but imaged by spinning-spot TIRF excitation. Dirt specks and fluorescent beads are readily discerned in B, but are almost completely masked by irregularities in the excitation field in A. The grey-scale levels in each panel were adjusted to equalize black levels and the brightest regions (excluding the fluorescent beads in B). (C) Measurements of fluorescence intensity along horizontal lines drawn across the center of the images in A (irregular black trace) and B (grey trace). (D) Stability of fluorescence excitation over time. The plot shows mean fluorescence intensity within a 100 x 100 pixel region in the center of the field, measured from 600 image frames acquired at 5 ms intervals in spinning-spot TIRF mode. (E-G) Representative images showing COS-7 cells expressing tubulin tagged by EGFP, captured using wide-field excitation (E), by stationary-spot TIRF (F) and spinning-spot TIRF (G). Images were captured with 488 nm excitation during 2s exposures using an 18 Mpixel Canon EOS M camera and eyepiece adapter.
Fig 3.
Imaging local Ca2+ puffs by shadowless TIRF.
(A) Averaged image (mean of 100 frames; 51 x 38 μm) of resting fluorescence in SH-SY5Y cells loaded with Cal520. (B) Representative examples of individual image frames captured at different times after photorelease of IP3, illustrating puffs arising at three different locations in the cells. (C) Maximum intensity profile across 5000 image frames acquired following photorelease of IP3 showing sites of puff activity in the cells. (D) Traces show fluorescence ratio measurements (ΔF/F0) derived from small (~1x1 μm) regions of interest centered on the puff sites shown in (B). (E) Selected examples of events shown on an expanded timescale to illustrate temporal resolution of stepwise transitions during puffs.
Fig 4.
Interleaved stationary- and spinning-spot TIRF imaging of Ca2+ puffs.
Alternate 5ms exposures were made in each mode, and image panels and traces were derived after de-interlacing the image sequence. Image panels acquired in stationary spot (left) and spinning spot (right) modes show (A), resting fluorescence (F0) averaged from 100 frames before photorelease of IP3; (B), maximum intensity projections of fluorescence (F) during 900 frames after photoreleasing IP3 to evoke puff activity; (C), maximum intensity projections of fluorescence ratio changes (ΔF/F0) during the same 900 frames. Image size is 40 x 40 μm. (D, E) Traces showing fluorescence measured, respectively, from the regions of interest marked as 1,2 in the panels in (B). In each case, the black trace was obtained in spinning spot mode, and the red trace in stationary spot mode. (F) Traces as in (E), after normalizing as fluorescence ratio (ΔF/F0) measurements.
Fig 5.
Interleaved imaging of Ca2+ puffs in TIRF and in wide-field modes.
(A) Fluorescence recordings from two puff sites, where Ca2+ release originated close to the plasma membrane (top) and deeper into the cell (bottom). Each record shows fluorescence monitored in TIRF mode (black) and WF mode (red). Alternate 5ms exposures were made in each mode, and fluorescence measurements were made from 3x3 pixel (~1 x 1 μm) regions of interest centered identically over the puff site on the two de-interlaced image sequences. (B) Distribution of puff amplitude ratios as measured in TIRF and WF modes. A ratio >1 indicates that a puff gave a larger signal in TIRF than in WF mode; and vice versa for ratios <1. Data are from 189 events at sites that gave ≥4 puffs, in 14 SH-SY5Y cells. (C) Puff amplitude ratios (TIRF/WF) segregated by sites which showed mean ratios >1.1 (red) and <0.9 (black). (D) Examples of ‘superficial’ puffs imaged in interleaved TIRF (black) and WF (red) modes, shown on an expanded timescale to illustrate the improved kinetic resolution of TIRF imaging.
Fig 6.
Multi-mode imaging of STIM/Orai relocalization.
Images in each panel show EGFP-tagged Orai visualized by shadowless TIRF excitation at 488 nm (top left); mCherry-tagged STIM visualized by TIRF excitation at 532 nm (top right); mCherry-tagged STIM visualized by skimming- plane (SP) excitation at 532 nm (bottom left); and a pseudocolored overlay (Orai in green, TIRF-STIM in red, SP-STIM in blue) of these three images. Images were acquired before (A) and about 3 min after (B) addition of thapsigargin to deplete ER Ca2+ stores and induce relocalization and clustering of Orai and STIM. Each image (81 x 61 μm) is an average of two successive exposures; brightness and contrast settings were individually adjusted for each mode, but are consistent between control and thapsigargin conditions. Traces in (C) and (D) show line-scans of EGFP-Orai (red traces) and mCherry-STIM (blue traces) fluorescence in TIRF mode obtained before from 20-frame averages captured before (C) and after (D) thapsigargin treatment.