Abstract:

Stimulated emission depletion (STED) microscopy usually employs a scanning excitation beam that is superimposed by a donut-shaped STED beam for keeping the fluorophores at the periphery of the excitation spot dark. Here, we introduce a simple birefringent device that produces a donut-shaped focal spot with suitable polarization for STED, while leaving the excitation spot virtually intact. The device instantly converts a scanning (confocal) microscope with a co-aligned STED beam into a full-blown STED microscope. The donut can be adapted to reveal, through the resulting fluorescence image, the orientation of fluorophores in the sample, thus directly providing subdiffraction resolution images of molecular orientation.

©2010 Optical Society of America

1. Introduction

Fluorescence microscopy is one of the most extensively used tools for the structural and functional investigation of the interior of cells. Its popularity has steadily grown despite the fact that it notoriously fails to image structures smaller than about half the wavelength of light (~200 nm). While electron, X-ray, and scanning probe microscopy offer a substantially better resolution, they all fall short in imaging intact or even living cells in three dimensions (3D).

The invention of Stimulated Emission Depletion Microscopy (STED) in 1994 highlighted the then unexpected fact that the diffraction resolution barrier can be effectively overcome in a microscope that uses regular lenses and focused visible light [fusion_builder_container hundred_percent=”yes” overflow=”visible”][fusion_builder_row][fusion_builder_column type=”1_1″ background_position=”left top” background_color=”” border_size=”” border_color=”” border_style=”solid” spacing=”yes” background_image=”” background_repeat=”no-repeat” padding=”” margin_top=”0px” margin_bottom=”0px” class=”” id=”” animation_type=”” animation_speed=”0.3″ animation_direction=”left” hide_on_mobile=”no” center_content=”no” min_height=”none”][1,2]. Other subdiffraction resolution techniques, such as PALM, STORM and structured illumination have since emerged as well [3–5]. STED microscopy currently provides nanometer scale resolution [6–8] in biological and non-biological samples, while retaining most of the advantages of far-field optical operation, such as the ability to non-invasively image cells in 3D [9].

While the principles of scanning STED microscopy do not rest on those of the confocal microscope, STED can be implemented in a scanning confocal microscope to great effect. To this end, one overlaps the focused excitation beam of a scanning (confocal) microscope with a donut-shaped STED beam [10,11], whose role is to keep fluorophores dark even when they are exposed to excitation photons. The fluorophores remain dark, because the wavelength and the intensity of the STED beam are adjusted such as to instantly de-excite potentially excited fluorophores by stimulated emission. Consequently, fluorophores subject to a STED beam of intensity I >3 s I are practically confined to the ground state and hence switched off. This is a consequence of the fact that the normalized probability of the molecule to spend time in the excited state follows ~ exp(− I Is ) , with Is being a characteristic of the molecule. Any molecule subject to s I >> I is deprived of its ability to fluoresce, because the fluorescent state is disallowed by the presence of the STED beam. Since I increases from the center of the donut on outwards to the donut crest, the probability for a molecule to be off is highest at the donut crest. Molecules located at the donut center remain fluorescent. At a certain distance from the center where I >3 s I , practically all molecules (95%) will be off.

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