Abstract
A 3D structured light sheet microscope using a four-faceted symmetric pyramid is presented. The sample is illuminated by the resulting four beam interference field. This approach combines advantages of standing wave and structured illumination microscopy. Examples of micrographs of fluorescently labeled Chinese hamster ovary (CHO) cells as well as of the compound eyes of drosophila are shown and the optical sectioning ability of our system is demonstrated. The capabilities and the limitations of the scheme are discussed.
Highlights
Wide-field fluorescence microscopy techniques with axial section capability have recently met a lot of interest, since they combine high sensitivity down to the single molecule detection limit, short image acquisition times, and high 3D spatial resolution
We present a 3D structured light sheet microscope which combines the advantages of standing wave microscopy and structured illumination microscopy
The normalized spatial frequency νɶ =λ/ (∆*NA) is introduced in order to describe the axial response of the system, where λ is the wavelength and ∆ is the spatial periodicity of the projected structure
Summary
Wide-field fluorescence microscopy techniques with axial section capability have recently met a lot of interest, since they combine high sensitivity down to the single molecule detection limit, short image acquisition times, and high 3D spatial resolution. Different from the single light sheets formed by the cylindrical lens, the intensity distribution is uniform inside each antinodes plane and varies rapidly in the axial direction. This leads to an improved axial resolution of up to 50 nm. Otherwise several planes are illuminated simultaneously and their separation is difficult and will give a strong background [12] This problem is not present using structured illumination, which was introduced by Neil [13] to wide field fluorescence microscopy as a means to discriminate against out-of-focus background and to get higher axial resolution [14, 15]. The optical sectioning capability of the method is demonstrated by imaging two different biological samples
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