A compact multiphoton 3D imaging system for recording fast neuronal activity

Abstract

We constructed a simple and compact imaging system designed specifically for the recording of fast neuronal activity in a 3D volume. The system uses an Yb:KYW femtosecond laser we designed for use with acousto-optic deflection. An integrated two-axis acousto-optic deflector, driven by digitally synthesized signals, can target locations in three dimensions. Data acquisition and the control of scanning are performed by a LeCroy digital oscilloscope. The total cost of construction was one order of magnitude lower than that of a typical Ti:sapphire system. The entire imaging apparatus, including the laser, fits comfortably onto a small rig for electrophysiology. Despite the low cost and simplicity, the convergence of several new technologies allowed us to achieve the following capabilities: i) full-frame acquisition at video rates suitable for patch clamping; ii) random access in under ten microseconds with dwelling ability in the nominal focal plane; iii) three-dimensional random access with the ability to perform fast volume sweeps at kilohertz rates; and iv) fluorescence lifetime imaging. We demonstrate the ability to record action potentials with high temporal resolution using intracellularly loaded potentiometric dye di-2-ANEPEQ. Our design proffers easy integration with electrophysiology and promises a more widespread adoption of functional two-photon imaging as a tool for the study of neuronal activity. The software and firmware we developed is available for download at http://neurospy.org/ under an open source license.

Figure 1. The diagram of our simple imaging system.

A LeCroy WaveRunner 64xi oscilloscope acts as both the scan controller and the data acquisition system. Radio-frequency signals generated by the AD9959 Direct Digital Synthesis chip, which is controlled by the oscilloscope via USB, are amplified and injected into a compact two-axis acousto-optic deflector. Long laser pulses have narrow spectral bandwidth and so obviate the need for dispersion compensation. The use of mirrors M1 and M2 instead of lenses allows for a very compact microscope. Mirror M3 steers fluorescence collected by the condenser to the photomultiplier (PMT). A fast photodiode (PD) collects transmitted excitation light that was deflected to large angles to provide oblique contrast for observing cells in non-fluorescent tissue. A very fast photodiode (FastPD) reports laser pulse timing for fluorescence lifetime measurements.

Figure 2. Design and performance of the Yb:KYW laser.

a) Diagram of the laser cavity. Pump light is collimated and focused by lenses L1–4 to a 51 µm × 85 µm waist in air; pump beam is tilted relative to the lasing beam to keep reflections from reentering the pump diode and destabilizing it. Beamsplitter (BS) has high transmissivity at 981 nm and high reflectivity at >1010 nm. Lasing medium is a d = 1.2 mm Brewster-cut 10%-at. Yb:KYW crystal. M1,M4 = −200 mm, M2,M3 = −100 mm are cavity mirrors with standard λ/4 dielectric coating. SF14 – uncoated isosceles prisms. OC – output coupler. b) Low pump beam quality makes the laser prone to multimode operation, which is easily observed by a photodiode as the circulation of pulse energy between lobes of higher-order modes on subsequent passes. c) Restricting the cavity to single-mode operation results in even pulsing at 113 MHz with 300 mW of average power. d) Output spectrum is 3.7 nm wide and centered near 1033 nm, indicating sech² transform-limited pulse width of 310 fs. e) Spectrum of the transmitted pump light. The narrow Yb:KYW absorption peak near 981 nm is readily visible; the pump wavelength must be temperature-tuned to overlap it for maximum output power and stability. f) Output power variability. With the off-axis pumping arrangement the laser is capable of very quiet operation.

Figure 3. Imaging performance and resolution.

a) Transmitted light image of a neuron in acute rat brain slice. Video-rate acquisition permits patch-clamping without the use of a separate camera. b) Fluorescence image of a cortical L2/3 pyramidal cell filled with the fluorescent dye FM 4–64. Inset: zoom onto the 10×10 µm area outlined in the image showing dendritic spines. c,d) x–y and z–y projections of a 100 µm deep stack of one quadrant of the basal dendritic tree of a cortical L2/3 pyramidal cell filled with the voltage-sensitive dye di-2-ANEPEQ. Thin processes at 100 µm depth can be observed easily. The axial resolution is worse than the objective's limit because we deliberately underilluminate the back aperture. All images were acquired with a 60×0.9NA water-immersion objective and have 100×100 µm field of view.

Figure 4. Fast digital control of acousto-optic deflection enables three-dimensional scanning with no moving parts.

Direct Digital Synthesis permits a) precise control of radio-frequency signals at microsecond timescales, as well as b) accurate and exactly repeatable sweeping between preset frequencies. The nominal aperture of the deflector we use is approximately the size of the entire trace in a,b). c) A frequency sweep makes an acousto-optic deflector act as a lens, so the output beam can be made to converge or diverge depending on the direction of the sweep. d) The lensing induced in this manner translates into axial displacement of the focus from the nominal focal plane of the objective. e) Large volumes can be scanned rapidly through the use of bidirectional line sweeps. By underilluminating the back-aperture of an objective the axial point-spread function (PSF) can be extended to match the excursion from the nominal focal plane caused by the frequency sweep, so a sweep in one direction can be made to excite a large number of sparsely labeled neurons in a thick slab of tissue to either side of the nominal focal plane. The images show two focal planes, displaced by 40 µm in z, of EYFP-labeled neurons in mouse olfactory bulb taken simultaneously without moving the objective.

Figure 5. Point-dwelling ability and photon counting permit optical recording of fast events with good signal-to-noise ratio.

a) The measured probability of detecting a photomultiplier pulse as a function of the oscilloscope sampling rate for a Hamamatsu 7422-40 module biased to 1000V at 2.5mV discrimination threshold. With 25 MS of sample memory and 250 MS/s sampling rate (arrow), photons can be counted for 100 ms per episode with 80% detection probability. b) Electrical and unfiltered optical (10 kHz) traces of an action potential in a rat cortical pyramidal neuron loaded with the potentiometric dye di-2-ANEPEQ. The traces are averages of four recordings.

Figure 6. Fluorescence lifetime imaging.

a) The high temporal resolution of oscilloscope data acquisition enables the measurement of temporal delay between an exciting laser pulse and a fluorescence photon. A histogram b) of relative arrival times measured in this manner can be fit to measure the fluorescence lifetime of fluorophores at the scanned location. By performing this measurement at every pixel (or voxel) a fluorescence lifetime image is constructed. c) Mouse hippocampal tissue loaded extracellularly with the dye FM 4–64. d) Fluorescence lifetime image clearly reveals Thy1.2-EYFP neurons (orange) within the background staining (green). Video-rate imaging of transmitted light or of fluorescence intensity can be used for guided patching of neurons that are first identified by their fluorescence lifetime.