DEVELOPMENT OF A STANDING-WAVE FLUORESCENCE MICROSCOPE WITH HIGH NODAL PLANE FLATNESS

This article reports about the development and application of a standi ng-wave fluorescence microscope (SWFM) with high nodal plane flatness. As opposed to the uniform excitation field in conventional fluorescen ce microscopes an SWFM uses a standing-wave pattern of laser light, Th is pattern consists of alternating planar nodes and antinodes, By shif ting it along the axis of the microscope a set of different fluorescen t structures can be distinguished, Their axial separation may just be a fraction of a wavelength so that an SWFM allows distinction of struc tures which would appear axially unresolved in a conventional or confo cal fluorescence microscope, An SWFM is most powerful when the axial e xtension of the specimen is comparable to the wavelength of light, Oth erwise several planes are illuminated simultaneously and their separat ion is hardly feasible, The objective of this work was to develop a ne w SWFM instrument which allows standing-wave fluorescence microscopy w ith controlled high nodal plane flatness, Earlier SWFMs did not allow such a controlled flatness, which impeded image interpretation and pro cessing, Another design goal was to build a compact, easy-to-use instr ument to foster a more widespread use of this new technique. The instr ument developed uses a green-emitting helium-neon laser as the light s ource, a piezoelectric movable beamsplitter to generate two mutually c oherent laser beams of variable relative phase and two single-mode fib res to transmit these beams to the microscope, Each beam is passed on to the specimen by a planoconvex lens and an objective lens, The only reflective surface whose residual curvature could cause wavefront defo rmations is a dichroic beamsplitter, Nodal plane flatness is controlle d via interference fringes by a procedure which is similar to the inte rferometric test of optical surfaces, The performance of the instrumen t was tested using dried and fluorescently labelled cardiac muscle cel ls of rats, The SWFM enabled the distinction of layers of stress fibre s whose axial separation was just a fraction of a wavelength, Layers a t such a small distance would lie completely within the depth-of-field of a conventional or confocal fluorescence microscope and could there fore not be distinguished by these two methods, To obtain futher infor mation from the SWFM images it would be advantageous to use the images as input-data to image processing algorithms such as conceived by Kri shnamurthi et al, (Proc. SPIE, 2655, 1996, 18-25). To minimize specime n-caused nodal plane distortion, the specimen should be embedded in a medium of closely matched refractive index. The proper match of the re fractive indices could be checked via the method presented here for th e measurement of nodal plane flatness. For this purpose the fluorescen t layer of latex beads would simply be replaced by the specimen, A com bination of the developed SWFM with a specimen embedded in a medium of matched refractive index and further image processing would exploit t he full potential of standing-wave fluorescence microscopy.