In the non-linear structured illumination techniques named saturated pattern excitation microscopy (SPEM) and saturated structured illumination microscopy (SSIM), the ground state (S0) is depleted by saturated excitation (to the S1 state) through an inverse RESOLFT scheme that enables the fluorescence generated by this transition to be recorded on an area-array detector. These techniques are performed using a grid-like array of line-shaped intensity maxima and minima rather than the typical doughnut-shaped phase modulation that is utilized by STED and GSD. The saturated excitation produces narrow line-shaped dark regions in the zero nodes that are surrounded by high levels of fluorescence signal to generate a "negative" imprint of the features being imaged. Thus, in SPEM and SSIM, it is the off state of a fluorescent probe (rather than the on state) that is confined by the phase modulation zero node. The grid lines are rotated several times to generate data for a single image, which is retrieved mathematically during post-acquisition processing.
Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution. Proceedings of the National Academy of Sciences (USA) 102: 13081-13086 (2005). In one of the first papers outlining the concept of saturated structured illumination, Dr. Gustafsson experimentally demonstrates the technique using fluorescently labeled beads and provides a theoretical description of the principles surrounding the methodology.
Saturated patterned excitation microscopy: A concept for optical resolution improvement. Journal of the Optical Society of America A 19: 1599-1609 (2002). Professors Heintzmann, Jovin, and Cremer report the concept of saturated patterned excitation microscopy (SPEM), similar in principle to SSIM but introduced several years earlier. The authors discuss theory, microscope configuration, point-spread function geometry, and resolution.
Saturated patterned excitation microscopy with two-dimensional excitation patterns. Micron 34: 283-291 (2003). Saturated patterned excitation microscopy is also known in the literature as structured illumination, harmonic excitation, and laterally modulated excitation microscopy. This theoretical report focuses on two-dimensional non-linear patterned excitation simulations and the expected resolution improvement.
Widefield fluorescence microscopy with extended resolution. Histochemistry and Cell Biology 130: 807-817 (2008). An excellent review of saturated structured illumination implemented with a technology known as harmonic excitation light microscopy (HELM). The authors discuss theoretical concepts, illumination strategies, image formation, and computational reconstruction of datasets.
Plasmonic structured illumination microscopy. Nano Letters 10: 2531-2536 (2010). Introducing the technique of plasmonic structured illumination microscopy (PSIM), the authors describe combining structured illumination with tunable surface plasmon interference. The authors present two specific instrument designs and conduct simulations to yield up to 4-fold resolution improvement over widefield fluorescence microscopy.
Saturated structured confocal microscopy with theoretically unlimited resolution. Optics Communications 282: 3657-3664 (2009). A theoretical paper describing a technique termed saturated structured confocal microscopy based on a combination of subtraction microscopy with regular and annular excitation beams. The methodology promises to double the resolution compared to that observed in widefield microscopy and could afford resolution in tens of nanometers with saturation.
Breaking the diffraction limit with dynamic saturation optical microscopy. Applied Physics Letters 87: 094105 (2005). In a theoretical research report, Dr. Enderlein proposes a scheme that enables superresolution microscopy by relying on fast temporal measurements of the fluorescence decay after sudden switch-on of the excitation light.
High-resolution confocal microscopy by saturated excitation of fluorescence. Physical Review Letters 99: 228105 (2007). A demonstration of the use of saturated excitation to improve the spatial resolution in laser scanning confocal microscopy (SAX Microscopy). The authors modulate the excitation intensity temporally and detect the harmonic modulation of fluorescence emission.
Beyond the diffraction-limit biological imaging by saturated excitation microscopy. Journal of Biomedical Optics 13: 050507 (2008). Another demonstration of saturated excitation (SAX) confocal microscopy where the population of fluorescent molecules is saturated at the excited state with high excitation intensity to induce strong non-linear responses in the central region of the laser focus.
Optical saturation as a versatile tool to enhance resolution in confocal microscopy. Biophysical Journal 97: 2623-2629 (2009). In a technique similar to SAX microscopy, the authors apply harmonically modulated excitation light to excite the fluorophores using picosecond laser pulses at different intensities resulting in varying levels of saturation.