Abstract
Combining fluorescence and transmitted light sources for microscopy is an invaluable method in cellular neuroscience to probe the molecular and cellular mechanisms of cells. This approach enables the targeted recording from fluorescent reporter protein expressing neurons or glial cells in brain slices and fluorescence-assisted electrophysiological recordings from subcellular structures. However, the existing tools to mix multiple light sources in one-photon microscopy are limited. Here, we present the development of several microcontroller devices that provide temporal and intensity control of light emitting diodes (LEDs) for computer controlled microscopy illumination. We interfaced one microcontroller with μManager for rapid and dynamic overlay of transmitted and fluorescent images. Moreover, on the basis of this illumination system we implemented an electronic circuit to combine two pulsed LED light sources for fast (up to 1 kHz) ratiometric calcium (Ca2+) imaging. This microcontroller enabled the calibration of intracellular Ca2+ concentration and furthermore the combination of Ca2+ imaging with optogenetic activation. The devices are based on affordable components and open-source hardware and software. Integration into existing bright-field microscope systems will take ∼1 day. The microcontroller based LED imaging substantially advances conventional illumination methods by limiting light exposure and adding versatility and speed.
Highlights
The continuous improvement of equipment and methodologies to visualize cells and their fine processes is critical to the advancement of neuroscience research
We present three microcontroller devices that are used for controlling light emitting diodes (LEDs) to provide epifluorescence and transmitted light illumination for microscopes
We describe the development of three microcontroller-based systems for brain slice illumination during patch-clamp recordings and epifluorescence imaging in combination with optogenetic activation
Summary
The continuous improvement of equipment and methodologies to visualize cells and their fine processes is critical to the advancement of neuroscience research. The application of long infrared (IR) wavelengths for visualization of cells in brain tissue reduces light scattering and in combination. Loading unlabeled neurons with chemical fluorescent dyes through the patch pipette further facilitated visualization of processes for targeted subcellular recording (Stuart et al, 1993). The widespread use of IR differential interference contrast (DIC), to improve the contrast of in vitro brain slices, prohibits the simultaneous use of transmitted and fluorescence light and requires switching of the analyzer and fluorescence cube. Alternatives for DIC are either the simpler oblique contrast or the Dodt gradient contrast illumination (Dodt et al, 1999) both enabling fluorescence illumination without switching components in the light path
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