An open-hardware platform for optogenetics and photobiology

Abstract

In optogenetics, researchers use light and genetically encoded photoreceptors to control biological processes with unmatched precision. However, outside of neuroscience, the impact of optogenetics has been limited by a lack of user-friendly, flexible, accessible hardware. Here, we engineer the Light Plate Apparatus (LPA), a device that can deliver two independent 310 to 1550 nm light signals to each well of a 24-well plate with intensity control over three orders of magnitude and millisecond resolution. Signals are programmed using an intuitive web tool named Iris. All components can be purchased for under $400 and the device can be assembled and calibrated by a non-expert in one day. We use the LPA to precisely control gene expression from blue, green, and red light responsive optogenetic tools in bacteria, yeast, and mammalian cells and simplify the entrainment of cyanobacterial circadian rhythm. The LPA dramatically reduces the entry barrier to optogenetics and photobiology experiments.

Figure 1. The LPA.

(a) Top-down view of an LPA lacking the 24-well plate and plate lid. In the configuration shown, the device contains a 535 nm LED in each top position and a variable wavelength LED (364–947 nm) in each bottom position. The SD card (blue), circuit board (purple), reset button (red) and power cord (red/black wires) are visible. (b) Fully assembled LPA. (c) Schematic cross-section showing the light path from the LEDs to the culture plate wells. (1) mounting plate, (2) LED spacer, (3) plate adapter, (4) plate lid, (5) incubator platform, (6) PCB, (7) LED atop LED socket, (8) gasket, (9) 24 well plate, (10) wing nut assembled with mounting bolt. (d) Spectra of 22 LEDs (Supplementary Table S5) used in this study measured and calibrated using a spectrophotometer and probe adapter (Supplementary Figs S7 and S20, and Supplementary Methods).

Figure 2. Iris.

(a) Steady State Mode. Constant light functions are specified by entering the desired greyscale intensity (0–4095) of up to all 48 LEDs in the Input Panel spreadsheet (left). The download button is shown at the bottom of the Input Panel. In Plate View mode (shown), the Simulation Panel (right) displays a schematic visualization of the output intensity of all 48 LEDs. The top and bottom LEDs are visualized as red and green, respectively, regardless of the actual LEDs used. (b) Advanced Mode. Constant and dynamic light functions are specified in the Input Panel. In the latter case, Iris automatically runs a staggered-start algorithm (Supplementary Fig. 19). In Well View mode (shown), the Simulation Panel (right) visualizes the output of the top and bottom LEDs in a given well over the duration of the experiment. The Simulation Panel also plays movies of specified light functions.

Figure 3. Benchmarking the LPA against E. coli CcaS-CcaR.

(a) CcaS-CcaR system. (b) CcaS-CcaR 533 nm light intensity versus sfGFP transfer functions in the presence of increasing 678 nm light. (c) Kinetic response to an increase in 533 nm light from 0.00 to 17.88 μmol m⁻² s⁻¹ and simultaneous decrease in 678 nm light from 12.79 μmol m⁻² s⁻¹ to 0.00 μmol m⁻² s⁻¹ (green dots), or decrease in 533 nm light from 17.88 to 0.00 μmol m⁻² s⁻¹ and simultaneous increase in 678 nm from 0.00 μmol m⁻² s⁻¹ to 12.79 μmol m⁻² s⁻¹ (red dots). Black lines represent best fits (Supplementary Table S9 and S10) of our previous CcaS-CcaR mathematical model to these data. Gray envelopes represent 95% confidence intervals. (d) Biological Function Generation. Reference waveform (black line), pre-computed 533 nm light intensity time course (green dashed lines), experimental sfGFP levels (green dots). Constant 12.79 μmol m⁻² s⁻¹ 678 nm was applied. 533 nm intensity values are mapped to sfGFP units through the transfer function in panel b. RMSE between experimental data and reference over three days is shown. Error bars represent the SEM of three experiments over three days.

Figure 4. Using the LPA to characterize CcaS-CcaR forward and reverse action spectra.

For the FAS (hollow circles), bacteria were exposed to 0.40 μmol m⁻² s⁻¹ photons from a variable wavelength bottom LED (Fig. 1a,c). For the RAS (black circles), bacteria were exposed to 0.40 μmol m⁻² s⁻¹ from the maximally activating 533 nm LED in the top position and 3.21 μmol m⁻² s⁻¹ photons from the variable wavelength bottom LED. Error bars represent the SEM of three experiments over a single day.

Figure 5. Validating the LPA with yeast by characterizing S. cerevisiae CRY2-CIB1 Y2H.

(a) CRY2-CIB1 Y2H system. (b) 467 nm intensity transfer function. The black line represents the best fit to a Hill function. (c) Step activation and de-activation kinetics. Cells were either preconditioned for 4 h in the dark and switched to 88.8 μmol m⁻² s⁻¹ 467 nm (blue dots) or preconditioned in 88.8 μmol m⁻² s⁻¹ 467 nm for 10 h and switched to dark (black dots) at time zero. Black lines represent best fits (Supplementary Tables S9 and S11) to a kinetic model (Methods). (d) FAS. Experiments were performed as in Fig. 4, but with 88.8 μmol m⁻² s⁻¹ photon flux. Grey envelopes represent 95% confidence interval. Error bars represent the SEM of mCherry levels from three experiments over three days (b–c) or a single day (d).

Figure 6. Validating the LPA with mammalian cells by spatial patterning of transgene delivery with PHYB/VNP-PIF6.

(a) PHYB/VNP-PIF6 system. (b) HeLa cells expressing PhyB(908)-NLS were treated with 15μM phycocyanobilin and VNP-PIF6 (1,000 viruses per cell) and exposed through a photomask (top row and Supplementary Fig. S18) to 2.0 μmol m⁻² s⁻¹ 733 nm light for 30 min followed by 1.0 μmol m⁻² s⁻¹ 733 nm light and variable 647 nm light intensities (shown in μmol m⁻² s⁻¹ in upper left of cell fluorescence images) for 60 min. Image corresponding to 647 nm intensity of 1.75 μmol m⁻² s⁻¹ was acquired with photomultiplier setting of 80 while all others were acquired with photomultiplier setting of 60. (c) Contrast ratio of cell fluorescence patterns from images in panel b (bottom row). Symbols represent the average ratio of pixel intensity in three light-exposed regions to an unexposed region, while error bars represent the SEM.

Figure 7. Validating the LPA with cyanobacteria by entraining circadian rhythm in S. elongatus.

(a) Schematic representation of entrainment protocol. Cells grown under constant light were placed in the LPA and entrained with three light-dark cycles, with dark periods starting at different times in order to shift the phases of each well. The cells were then transferred to a plate reader for measurement of luminescence under constant light. (b) Luminescence oscillation for cultures entrained starting at different times. Cultures were entrained starting at zero (red), four (orange), eight (yellow), 12 (green), 16 (blue) and 20 (purple) hours. Fits of raw data were normalized to equalize peak height, and the normalized fits were averaged across three replicates except for the cultures in yellow for which only two replicates could be obtained. Bars indicate SEM. (c) Phase of cultures entrained starting at different times. Colors are the same as in (b). Dots indicate cosine acrophase (phase of peak) of the replicates. Lines indicate the best fit for the expected locations of acrophase for perfect entrainment.