Abstract
Azimuthal beam scanning eliminates the uneven excitation field arising from laser interference in through-objective total internal reflection fluorescence (TIRF) microscopy. The same principle can be applied to scanning angle interference microscopy (SAIM), where precision control of the scanned laser beam presents unique technical challenges for the builders of custom azimuthal scanning microscopes. Accurate synchronization between the instrument computer, beam scanning system and excitation source is required to collect high quality data and minimize sample damage in SAIM acquisitions. Drawing inspiration from open-source prototyping systems, like the Arduino microcontroller boards, we developed a new instrument control platform to be affordable, easily programmed, and broadly useful, but with integrated, precision analog circuitry and optimized firmware routines tailored to advanced microscopy. We show how the integration of waveform generation, multiplexed analog outputs, and native hardware triggers into a single central hub provides a versatile platform for performing fast circle-scanning acquisitions, including azimuthal scanning SAIM and multiangle TIRF. We also demonstrate how the low communication latency of our hardware platform can reduce image intensity and reconstruction artifacts arising from synchronization errors produced by software control. Our complete platform, including hardware design, firmware, API, and software, is available online for community-based development and collaboration.
Highlights
Modern imaging applications typically require the cooperative action of several independent devices, such as stages, filters, excitation modulators, and many others
We demonstrate the advantages of hardware control in a custom azimuthal beam scanning microscope by characterization of the effects of excitation quality and timing accuracy in a pair of complementary axial localization techniques: scanning angle interference microscopy (SAIM)[5,6] and multiangle total internal reflection fluorescence microscopy (MA-TIRF)[7]
Given the high component and trace density required while considering cost and availability, we opted for a 4-layer printed circuit board (PCB) design with minimum 6 mil trace width and separation
Summary
Modern imaging applications typically require the cooperative action of several independent devices, such as stages, filters, excitation modulators, and many others. The latency introduced by serial communications, the operating system (OS), application overhead and suboptimal API implementations can have a significant effect on synchronization accuracy Because of these limitations, the time taken to update the experimental parameters across multiple peripherals can be much greater than the individual devices’ response times, limiting the effective acquisition speed and increasing the risk of synchronization errors that could cause artifacts in the collected data. The time taken to update the experimental parameters across multiple peripherals can be much greater than the individual devices’ response times, limiting the effective acquisition speed and increasing the risk of synchronization errors that could cause artifacts in the collected data To overcome these challenges, hardware controllers centralize device control within a single unit, typically using digital triggers or analog voltages to step peripheral devices through a series of predefined states. We demonstrate the advantages of hardware control in a custom azimuthal beam scanning microscope by characterization of the effects of excitation quality and timing accuracy in a pair of complementary axial localization techniques: scanning angle interference microscopy (SAIM)[5,6] and multiangle total internal reflection fluorescence microscopy (MA-TIRF)[7]
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