Abstract
Conventional standing-wave (SW) fluorescence microscopy uses a single wavelength to excite fluorescence from the specimen, which is normally placed in contact with a first surface reflector. The resulting excitation SW creates a pattern of illumination with anti-nodal maxima at multiple evenly-spaced planes perpendicular to the optical axis of the microscope. These maxima are approximately 90 nm thick and spaced 180 nm apart. Where the planes intersect fluorescent structures, emission occurs, but between the planes are non-illuminated regions which are not sampled for fluorescence. We evaluate a multi-excitation-wavelength SW fluorescence microscopy (which we call TartanSW) as a method for increasing the density of sampling by using SWs with different axial periodicities, to resolve more of the overall cell structure. The TartanSW method increased the sampling density from 50 to 98% over seven anti-nodal planes, with no notable change in axial or lateral resolution compared to single-excitation-wavelength SW microscopy. We demonstrate the method with images of the membrane and cytoskeleton of living and fixed cells.
Highlights
Confocal laser scanning microscopy (CLSM)[1] and multi-photon laser scanning microscopy[2] are widely used for 3D cell imaging
Total internal reflection fluorescence (TIRF) microscopy can improve the axial resolution to 70 nm, overcoming the diffraction limit, but this technique is limited to 2D imaging of the basal cell m embrane[17] as is the comparable method supercritical illumination m icroscopy[18]
We evaluated the TartanSW method using live and fixed cells, labelling the plasma membrane and actin network of mammalian cells with fluorescent probes or common photoproteins, and we explored whether colour-ordering in TartanSW could reduce or remove the ambiguity of cell shape that occurs in conventional SW microscopy
Summary
Confocal laser scanning microscopy (CLSM)[1] and multi-photon laser scanning microscopy[2] are widely used for 3D cell imaging. Molecules, and the study of actin polymerisation areas in dendritic cells[12], as well as imaging the two layers of actin at the leading edge of the c ell[13] These super-resolution techniques are, hampered by limited probe availability[14]. Total internal reflection fluorescence (TIRF) microscopy can improve the axial resolution to 70 nm, overcoming the diffraction limit, but this technique is limited to 2D imaging of the basal cell m embrane[17] as is the comparable method supercritical illumination m icroscopy[18]. An improvement in both lateral and axial resolution of up to a factor of 2 can be achieved by the use of Structured Illumination Microscopy (SIM)[19,20], which is an intrinsically slow and computationally intensive method, but has been made faster by optomechanical design improvements
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