Simple structured illumination microscope setup with high acquisition speed by using a spatial light modulator

Abstract

Abstract:We describe a two-beam interference structured illumination fluorescence microscope. The novelty of the presented system lies in its simplicity. A programmable electro-optical spatial light modulator in an intermediate image plane enables precise and rapid control of the excitation pattern in the specimen. The contrast of the projected light pattern is strongly influenced by the polarization state of the light entering the high NA objective. To achieve high contrast, we use a segmented polarizer. Furthermore, a mask with six holes blocks unwanted components in the spatial frequency spectrum of the illumination grating. Both these passive components serve their purpose in a simpler and almost as efficient way as active components. We demonstrate a lateral resolution of 114.2 ± 9.5 nm at a frame rate of 7.6 fps per reconstructed 2D slice.

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

“…The first in vivo images obtained with TIRF-SIM were reported in 2009 16 achieving frame rates of 11 Hz to visualize tubulin and kinesin dynamics, and two color TIRF-SIM systems have been presented 17,18 . Most recently, a guide for the construction and use of a single color two-beam SIM system was presented featuring frame-rates of up to 18 Hz 19,20 .…”

Laser Illumination in Live Cell Microscopy: Scattering and Structured Illumination

“…Two laser beams (e.g. the first diffraction orders of an optical grating or a spectral light modulator (SLM) upon blocking of k = 0 [11]) create an interference pattern at the entrance plane FD of the microscope. They are further focused at the edges of the microscope aperture, as depicted in Fig.…”

Method of lateral image reconstruction in structured illumination microscopy with super resolution

“…One main drawback of SIM is the time consuming process (normally 0.1 to 30 s) necessary to reconstruct a super-resolution image. 9,[17][18][19] Nonlinear SIM operates with the same process of linear SIM, but produces higher resolution by generating the harmonic spatial frequencies of the illumination pattern through nonlinear response of°u orescence yield rate. 20 Considering the simplest case, the nonlinear function fðIÞ of illumination intensity I can be described by the Taylor series fðIðxÞÞ ¼ a 1 IðxÞ þ a 2 IðxÞ 2 þ a 3 IðxÞ 3 þ Á Á Á and the normalized structured illumination intensity IðxÞ ¼ 1 þ cosð2k 0 xÞ.…”

“…Although the principle of SIM is straightforward, the image reconstruction algorithm in the post-process is complex. [8][9][10] The reconstruction programming details in SIM have not been publicly released up to date, making it di±cult to conduct the experiment. To give a comprehensive insight and guide the programming, in this work we analyze the whole process of lateral image reconstruction in SIM, including pattern generation, spectra encoding, parameter estimation, spectra decoding, image reconstruction and the analysis of artifacts sources.…”