High performance, LED powered, waveguide based total internal reflection microscopy

Abstract

Total internal reflection fluorescence (TIRF) microscopy is a rapidly expanding optical technique with excellent surface sensitivity and limited background fluorescence. Commercially available TIRF systems are either objective based that employ expensive special high numerical aperture (NA) objectives or prism based that restrict integrating other modalities of investigation for structure-function analysis. Both techniques result in uneven illumination of the field of view and require training and experience in optics. Here we describe a novel, inexpensive, LED powered, waveguide based TIRF system that could be used as an add-on module to any standard fluorescence microscope even with low NA objectives. This system requires no alignment, illuminates the entire field evenly, and allows switching between epifluorescence/TIRF/bright field modes without adjustments or objective replacements. The simple design allows integration with other imaging systems, including atomic force microscopy (AFM), for probing complex biological systems at their native nanoscale regimes.

Figure 1

(A) Schematic of the LED based waveguide TIRF microscopy system and its principle of operation. Light from LED sources scatter diffusely at the waveguide surface, part of the light is trapped in the waveguide and undergoes refraction at all angles. For angles less than critical angle ‘θ' (θ_c being 48°, for the combination of 1.78 and 1.33 RIs), light refracts out into the other side of the cover glass and is absorbed by the black rubber. For angles greater than 48° (θ_i), light undergoes total internal reflection within the cover glass and generates an evanescent field on either side of the cover glass interface. Scattered light or direct light from the source is prevented from reaching the sample by the inner cylindrical tube. (B) Schematic of the overall system design. A high index waveguide (SF 11 cover glass, R.I. 1.76, ø 25 mm) sandwiched between thin black rubber sheets is mounted in the center of two concentrically arranged steel cylinders. The outer cylinder (ø 60 mm) machined to have optical access from the bottom for high magnification objectives. Six high power LED's mounted on a copper tube are placed inside the cylinder.

Figure 2. TIRF imaging of cells with both low and high power objectives.

Methanol fixed N2A cells transfected with Cx43-YFP were imaged with low power objectives (10x–40x, A–F) under both TIRF and epi-illumination modes. The TIRF image shows the distribution of Cx43 fluorescence at the margin of the cells while rejecting the background from cytoplasmic Cx43 that is evident in epi-illumination images. Live cell imaging of human mesenchymal stem cells transfected with paxillin-GFP was imaged with a 60x oil immersion objective (G,H). The TIRF panel shows the focal adhesion sites selectively while rejecting the background fluorescence from the cytoplasmic pool. 100x epi-illumination imaging of Cx43-YFP in methanol fixed cells (I) shows the Cx43 distribution across the entire thickness of the cell, while TIRF illumination (J) selectively shows Cx43 distributed at the periphery of the cells. Scale bar: 100 μm.

Figure 3. Demonstration of image contrast through comparative imaging of fluorescent beads against a background of Lucifer yellow dye under epi-illumination (A) and TIRF (B) modes.

Rejection of background fluorescence in the TIRF image results in better image contrast compared to the epi-illumination. Corresponding intensity line plots (inset) across a narrow rectangular region of interest in the images shows the contribution of background fluorescence in epi-illumination, resulting in the upward shift of the baseline reducing the contrast. Scale bar is 20 μm.

Figure 4. Measurement of evanescent field depth using AFM.

A fluorescent bead immobilized on the tip of a cantilever (k = 200 N/m) was moved away from the surface at defined intervals using a high precision AFM scanner. Using force curve motion of the scanner, and halting the movement for 5 seconds both at the top and bottom of the force curve movement, fluorescence images were collected at different positions of z-travel of the AFM scanner using different Z scan sizes in the force curves. An 80 nm trigger was used to determine the exact point at which tip-surface separation occurred. Fluorescence intensities were normalized with respect to the surface fluorescence. The plot shows the characteristic exponential decay of the evanescent field.

Figure 5. Simultaneous combined AFM and TIRF imaging of cells.

Simultaneous AFM and TIRF imaging was carried out on methanol fixed N2A cells transfected with Cx43-YFP. (A) TIRF image of a cell before it was force dissected using the AFM tip. The white and red rectangular boxes indicate region of interest (ROI) zoomed in for AFM imaging. (B, C) TIRF and AFM images respectively of the ROI before force dissection, (D, E) are corresponding images after force dissection. (F, G) The height of the neuronal process to be ~200 nm (red arrows) which is within the evanescence field of excitation; cell bodies that are >200 nm, are saturated in AFM images, are not registered in TIRF images (blue arrows).