Figure 1. Principle and proof of... | Rockefeller University Press

Figure 1.

$Figure 1. Principle and proof of inducible speckle imaging. (a) A single transverse mode laser beam is scattered by a diffuser before the microscope objective, creating a pattern composed of speckles with a characteristic size (correlation length) at the diffraction limit. This high-contrast pattern is generated at all planes: at, before, and after the objective’s focal plane. The beam induces a heterogeneous photoswitch pattern across the sample. (b) A PSF volume comprises a large number of potentially tagged slots. In FSM, slots are randomly occupied, leading to faint density fluctuations at the PSF scale. Increased fluctuations can be induced by narrowing the PSF or, conversely, by clustering fluorescent molecules, as does ISI, to emulate PSF-sized slots. (c) Comparison between simulated and observed fluorescence pattern structure after an ISI pulse on a uniform fluorescent layer (concanavalin A–conjugated Alexa Fluor 488) at different bleaching strengths (γ). Bars, 2 µm. (d) ISI on GFP–α-tubulin in a fixed Drosophila S2 mitotic cell. To produce a roughly uniform fluorophore pool, microtubules were depolymerized using colchicine. Bars: 5 µm; (inset) 1 µm.$

Principle and proof of inducible speckle imaging. (a) A single transverse mode laser beam is scattered by a diffuser before the microscope objective, creating a pattern composed of speckles with a characteristic size (correlation length) at the diffraction limit. This high-contrast pattern is generated at all planes: at, before, and after the objective’s focal plane. The beam induces a heterogeneous photoswitch pattern across the sample. (b) A PSF volume comprises a large number of potentially tagged slots. In FSM, slots are randomly occupied, leading to faint density fluctuations at the PSF scale. Increased fluctuations can be induced by narrowing the PSF or, conversely, by clustering fluorescent molecules, as does ISI, to emulate PSF-sized slots. (c) Comparison between simulated and observed fluorescence pattern structure after an ISI pulse on a uniform fluorescent layer (concanavalin A–conjugated Alexa Fluor 488) at different bleaching strengths (γ). Bars, 2 µm. (d) ISI on GFP–α-tubulin in a fixed Drosophila S2 mitotic cell. To produce a roughly uniform fluorophore pool, microtubules were depolymerized using colchicine. Bars: 5 µm; (inset) 1 µm.