Simultaneous voltage and calcium imaging and optogenetic stimulation with high sensitivity and a wide field of view

Abstract

Transmembrane voltage and intracellular calcium concentration are coupled parameters essential to the function of neurons, cardiomyocytes, and other excitable cells. Here we introduce the Firefly-HR microscope for simultaneous optogenetic stimulation and voltage and calcium imaging with fluorescent proteins using three spectrally distinct visible color bands. Firefly-HR combines patterned stimulation, near-total internal reflection laser excitation through a prism located between the sample and a water-immersion objective, and concurrent imaging of three color channels. The microscope has efficient light collection, low fluorescent background, and a large field of view (0.24 x 1.2 mm @ 1000 frames/sec). We characterize optical crosstalk and demonstrate capabilities with three applications: (1) probing synaptically connected neuronal microcircuits, (2) examining the coupling between neuronal action potentials and calcium influx, and (3) studying the pharmacology of paced human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) via simultaneous recordings of voltage, calcium, and contraction.

Fig. 1

Firefly-HR design. (A) The proteins involved in the measurement system: Membrane bound, light-gated ion channel CheRiff is opened by 470 nm light. Cytosolic calcium sensor jRGECO1a is excited by 560 nm light. Membrane bound voltage sensor QuasAr is excited by 638 nm light. (B) An optical diagram of the microscope. There are four key light sources: 1) Patterned blue light reflected off the digital micromirror device (DMD) for e.g. targeted stimulation of individual neurons. 2-3) 638 & 560 nm lasers for at near-total internal reflection (TIR) for fluorescence excitation of QuasAr and jRGECO1a. 4) Green light in trans-illumination for measuring cardiac contractions. Two synchronized cameras record the three light sources simultaneously. The top camera is designated as a brightfield collector, while the bottom camera is designated as a fluorescent collector. (C) An expanded view of prism. The water-immersion objective is optically coupled to the bottom of the prism with water, and the prism is optically coupled to the glass-bottomed dish with immersion oil. This geometry enables TIR or near TIR illumination over a large field of view, does not rout intense lasers through the objective, and maintains the high numerical aperture of 1.0. “1” and “2”, which are reproduced in (E), are the objective access and laser access apertures, respectively. (D) The prism design. (E) The design of the prism plate that prevents oil and water from mixing or obscuring the laser path. (F) Spectra. JRGECO1A spectrum is mApple, and filters are from Semrock. Optopatch component spectra from [11].

Fig. 2

Optical characterization. Images in the QuasAr fluorescence channel of a pristine, empty cyclic olefin copolymer (COC) substrate illuminated by a 638 nm laser routed (A) through the prism at near-TIR and (B) through the objective in traditional Epi illumination. For both light paths, the 638 nm laser was apertured to illuminate exactly the same area, and the laser power in both paths was tuned to match illumination intensities. Counts in the Epi sample were scaled so that the median of measured bead intensities in a separate sample (after background subtraction) matched between Epi and near-TIR illumination. In near-TIR illumination, there is a small amount of background from the immersion oil and substrate. In Epi illumination, there is a much larger amount of out-of-focus autofluorescence from glass in the objective, still present when the sample is removed entirely. (C) The histogram of pixel intensities after background subtraction from the rectangles shown in A and B show 13x lower autofluorescence in the near-TIR illumination geometry. (D) The light collection efficiency of the 2 fluorescence imaging pathways [QuasAr left, GFP right]. A relatively homogeneous fluorescent orange paper sample was illuminated by an oversized, homogeneous LED source from above. Images were captured at many locations within the paper and averaged to minimize effects of sample inhomogeneity. The lineout reveals some light loss from the objective where the colors are still colinear and some light loss in the dual-view path after the colors have been separated. (E) A dualview image of 1 um Tetraspeck beads emitting multiple fluorescent colors is captured on the bottom camera (of Fig. 1(B)). The image spans the full camera width and shows the 1.2 mm wide FOV for both the QuasAr channel (left) and the GFP channel (right). The dichroic reflecting green light to the first camera was removed. Each half of the image has been independently background subtracted and contrasted. Expanded views of indicated sub-regions in (E) for red (F - H) and green (I - K) fluorescence channels. Expanded images from each fluorescence channel are shown with the same brightness and contrast values.

Fig. 3

Synaptic microcircuit. Neurons were collected from an E18 rat hippocampus and cultured for 14 days. (A) QuasAr fluorescence image of excitatory cells E1 and E2 connected in a microcircuit with inhibitory cell I. (B) The digital micromirror device (DMD) is used to stimulate single cell individually, indicated by the blue rectangle, while recording from the other 2. The left of each panel shows inferred connectivity for excitatory (red) and inhibitory (green) synaptic coupling. (C) A diagram of microcircuit connectivity determined from the functional recordings in B. (D) – (H) Frames from the video where E2 is stimulated (blue square), at times indicated by black arrows in B. (D) Before cell E2 fires. (E) The stimulated cell E2 fires, which triggers (F) cell I to fire. (G) & (H) Cell I induces inhibitory post synaptic potentials in cells E1 and E2.

Fig. 4

Optical Crosstalk. (A) The average fluorescence from 19 FOV’s of jRGECO1a-expressing neurons with (gold) and without (gray) CheRiff expression. Blue light photoconverts jRGECO1a into a brighter fluorescent state as shown in the blue stimulated portions of the gray trace. 560nm laser intensity: 80 mW/cm². 488nm illumination intensities: 10 – 100 mW/cm². At higher blue stimulation intensities, the optical crosstalk can exceed the true calcium signal, where the true calcium signal is calculated by the difference between the yellow trace and the gray trace. Direct blue-light induced fluorescence, captured with the 560nm laser off, is negligible. (B) 560 nm laser-induced stimulation of the CheRiff. The average firing rate from at least 780 neurons/condition is shown before any intervention (gray) and after illuminating the cells with different intensities of yellow light in response to a slow ramped blue stimulus. The highest 560 nm intensity of 406 mW/cm² is the largest we use in any experiment. There may be a slight change in the blue light intensity to induce the first action potential at the stronger yellow illumination intensities, but it does not rise to statistical significance and typically one can ignore this effect.

Fig. 5

Simultaneous voltage and calcium imaging in neurons. (A) QuasAr fluorescence from an example field of view. (B) jRGECO1a fluorescence from the same field, imaged on the other half of the camera. (C) A color overlay after image registration. (D) The pixel weights used to calculate the time traces for 5 neurons in this FOV. (E) Synchronized voltage and calcium recordings from the cells labeled in D. The blue light stimulus of CheRiff is shown below. (F) A raster plot from 47 FOVs in 3 dishes with no pharmacological modulation. Each row in 1 cell and each point is one action potential. (G) The calcium traces from the same cells. (H) The average spike rate for all cells treated with 30 μM 4-AP (green), 10 μM isradipine (purple), or vehicle control (blue). (I) The corresponding average calcium time traces. The voltage action potential waveform averaged over all spikes during (J) spontaneous recording and (K) all epochs excluding the comb. (L) – (N) For each cell, the integrated calcium signal is plotted against the total number of spikes. Although the change is not statistically significant, the changes in slope suggest that the calcium flux per action potential may change. (O) The number of spiking neurons per FOV, demonstrating throughput.

Fig. 6

Tri-functional recordings in human iPS-derived cardiomyocytes. (A) Voltage and (B) calcium fluorescent recordings were captured on one camera, and (C) motion was captured on a second, synchronized camera. The optogenetic stimulus was delivered from above with a 470 nm LED offset to stimulate left of the FOV (not shown in Fig. 1(B)). The action potential propagated from left to right with a velocity of ~20 μm/ms. (D) Multiple beats from the regions in A are aligned in time to the optogenetic stimulus. The good overlap shows the highly reproducible conduction velocity. (E) Simultaneous voltage, calcium, and motion recordings. Motion was calculated as the root-mean-square deviation from the average baseline image. As an action potential enters the FOV, the voltage deflection precedes the calcium transient, which precedes motion, as expected. (Fi - Fxii) Pharmacology in the tri-view system. Each FOV was measured three times: before drug application (blue), 1 min after drug application (purple), and 10 min. after drug application (magenta). Columns show functional readouts and rows correspond to different drugs. After application of a vehicle control (0.1% DMSO), voltage and calcium changed minimally upon re-imaging while motion showed an unusual, temporary change. Calcium channel blocker nifedipine (0.1 μM) drastically changed AP and CT shapes after 1 min treatment and eliminated firing part way through the recording. Motion was stopped before electrical activity ceased. β-adrenergic receptor agonist isoproterenol (0.1 μM) increased conduction velocity and contraction strength after 10 min. It also increased spontaneous beat rate (data not shown). para-Nitroblebbistatin (20 μM), a photostable myosin II inhibitor [30], had relatively small effects on AP’s and CT’s but drastically reduced, and then eliminated, contraction.