Adaptive movement compensation for in vivo imaging of fast cellular dynamics within a moving tissue.

Ethics Statement

All experimental procedures were performed in accordance with guidelines from the Canadian Council on Animal Care and the animal protection committee of Laval University (approval 2008019-3).

Multimodal video-rate imaging system

The video rate microscope has been designed for in vivo imaging. The main requirements of such system are: i) the speed of imaging has to be faster than any characteristic movement of the specimen, ii) multimodality to allow for simultaneous acquisition and separation of different signals based on their wavelengths. Typically, an acquisition speed of 30 frames per second while maintaining a relatively high resolution (500×500 scanned points for a 250 µm field of view) must be achieved. The imaging platform has been described in details elsewhere [18] . Briefly, the heart of the system is the fast laser scanning mechanism and its timing circuitry. The approach for the scanning platform is based on the original design of Rajadhyaksha and Webb [20] . It consists in a galvanometer mounted mirror and a spinning polygonal mirror that produce a unidirectional raster scan pattern. The horizontal (or fast) axis is scanned by the spinning polygonal mirror (DT-36-290-025, Lincoln Laser) at a speed of 480 revolutions per second yielding a line scanning rate of approximately 18 kHz. The vertical (or slow) axis scan is performed by the galvanometer-based optical scanner (6240H, Cambridge Technology) coupled to a 15 mm clear aperture silver coated mirror. To obtain the raster scan, the galvanometer mounted mirror is synchronously scanned vertically at 30 Hz. Movement compensation device The device is a highly modified ATF-4 sensor from WDI Inc ( Fig. S1 ). It emits a hemicircular light beam at 785 nm, and an integrated line CCD sensor operating at 7 kHz is used to monitor the position of the reflected beam. An internal processor uses a signal processing algorithm involving a rolling window average (down to 60 Hz) and digital filtering coupled with a peak fitting procedure and linear interpolation to determine the best current position of the imaging plane of interest. The predicted Z position for each data point is generated by subtracting the system self motion from the data points before averaging. The correction is then computed based on the best linear fit to extrapolate to current position. The signal is sent to the nanopositioner for correction.

Image acquisition and real-time

X/Y stabilization processing Light excitation is provided by a femtosecond Ti-Sapphire laser (Maitai HP, Spectra-Physics, Irvine, CA), operating at 80 MHz with a pulse duration of about 140 fs. The excitation beam is focused onto the sample via a 40× water immersion objective (0.85°NA, Olympus Plan Apochromat). The average power delivered to the samples is typically around 30 mW. An ultrasteep beamsplitter at 45°(LPD01-785RS-25, Semrock, Rochester, NY) is used to combine the laser light used for imaging (λ = 800 nm) with the one used for the movement compensation (λ = 785 nm). The emitted fluorescence is first separated from excitation with a primary dichroic filter (FF735-Di01, Semrock). Then signals arising from the Ca 2+ dye OGB1 and the structural dye Calcein are separated by a secondary dichroic filter (FF568-Di01, Semrock), filtered respectively with emission filters 525/50 (FF01-525/50, Semrock) and 620/52 (FF568-Di01, Semrock) and finally focussed on two photomultiplier tubes (R3896, Hamamatsu). The analog signal from each detector and the synchronization signals, VSYNC and HSYNC, are sent to a video acquisition board (Snapper-24, Active Silicon). A typical pixel clock, internally generated by the frame grabber board, of 10 MHz is used. This corresponds to 100 ns of pixel dwell time. The software used for acquisition, written in-house, allows for real-time XY movement correction that enables in-plane stabilization with a real-time visual feedback to the user [18] . The real-time XY correction is based on the cross-correlation between two successive frames that gives an instantaneous displacement vector used to correct the position of the first frame with respect to the second one.

Measurement of the movement compensation accuracy

To test the performances of the system, we imposed a series of movements to a sample (either highly reflective or highly diffusive) mounted on a micromanipulator (MPC-385, Sutter) and monitored simultaneously the movement of the objective and the one of the manipulator. For the former, the piezo controller (E-665, PI) brings a real-time readout of the exact position of the nanopositioner onto which the objective is mounted. For the latter, a glass pipette with a thin tip (∼1 µm) was also fixed to the micromanipulator and imaged at video-rate with a CCD camera for which the pixel size is known. This ensures an independent calibration of the micromanipulator movement. Animal surgery for in vivo experiments and ex-vivo spinal cord preparation Adult male Wistar rats (250–300 g body weight) were deeply anaesthetized with 4 vol % isoflurane applied through an anaesthesia mask or with urethane (1.5 g/kg i.p.). For experiments conducted under isoflurane, anesthesia was maintained using 1.5 vol % isoflurane. Core temperature was kept at 37.5±2°C with a feedback-controlled heating blanket. The level of anesthesia and the cardiopulmonary health were continuously monitored with a Mouse Ox non-invasive sensor clip (Starr Life Science, Oakmont, PA). Note that movement compensation was achieved with the movement compensation device in both conditions of anaesthesia, even though complete immobilisation of the spinal cord appears difficult to achieve under urethane anaesthesia [11] . The lumbar segments L4–L6 were exposed by laminectomy. A portable stereotaxic frame with two adjustable clamps for vertebral fixation, rostral and caudal to the exposed section, was custom made to fit on a micromanipulator (MP-125, Sutter Instrument, Novato, CA), under the microscope (see above). An agar pool was formed around the exposed spinal segments and filled with artificial cerebrospinal fluid (ACSF). The dura and pia maters were incised, and the lamina I of the dorsal horn of the spinal cord was directly accessed by gently lifting the roots. After the imaging experiment, animals were decapitated under deep anaesthesia. For experiment on the tissue target, a piece of spinal cord was isolated and fixed overnight in 4% PFA, rinsed several times in 0.1 M phosphate buffer. In vivo dye loading of spinal cord neurons We adapted the bulk-loading method of fluorescent indicator previously published by Garaschuk et al. [21] . Briefly, a 2 mM stock of the Ca 2+ dye Oregon Green BAPTA-1 AM (OGB1 AM, Invitrogen, Eugene, OG) in DMSO +20% pluronic (made fresh for each experiment) and a 1.3 mM stock of the structural dye Cell Trace Calcein Red Orange (Calcein, Invitrogen) were respectively diluted 10 and 13 fold in a HEPES-buffered solution composed of the following (in mM): 126 NaCl, 10 HEPES, 2.5 KCl, 2 MgCl2, 2 CaCl2, and 10 glucose (pH 7.4). 200 nl of the mixture was pressure ejected into the superficial laminae (100 µm in depth) of the exposed spinal cord via a glass pipette (resistance around 300 kΩ) connected to a nanoinjector (Micro 4, WPI) at the rate of 50 nl/min. After 30 min of de-esterification of the AM dye, the animal was placed under the video-rate two-photon microscope for imaging. In vivo electrical stimulations of the sciatic nerve for Ca 2+ imaging of spinal cord neurons For this experiment, animals were paralysed with pancuronium bromide and ventilated (Inspira asv, Harvard Apparatus). The left sciatic nerve was exposed during the surgery and a double silver hook electrode was implanted to allow for electrical stimulations of this nerve (15 pulses of 1 ms-duration, 5 mA at 0.1 Hz synchronized with imaging sequence). Data analysis for Ca 2+ transient dynamics Analysis was performed using custom macros in Image J (National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gov/ij ). Regions of interest (ROIs) were manually defined to closely approximate the outline of the structure of interest (neural cell bodies). The fluorescence intensity time courses were measured for both dyes (OGB1 and Calcein, respectively green and red traces Fig. 3 B ), and expressed as ΔF/F = (F−F basal )/F basal . F is the fluorescence at any time point, and F basal the baseline fluorescence acquired at the very beginning of an experiment, before any stimulation. ΔF/F for a given frame is calculated on the mean of all pixels in a ROI. ΔF/F calculated for Calcein can be subtracted to ΔF/F calculated for OGB1 to improve the SNR ( Fig. 3 B , blue traces). To evaluate the kinetics of individual Ca 2+ responses, the τ rise and the τ decay were extracted from the fit of two exponential functions (one for the rise, one for the decay) to the average of the 15 responses of Cell 1.

Supporting Information Figure S1 Principle of half-aperture method of distance to focus measurement using a hemicircular beam. The transverse shift (X in ) in the detector image plane of the centre of gravity (COG) of the bell shaped beam profile from the initial position measured at best focal plane (X make0 ) is in first approximation proportional to the distance from focus measured along the optical axis (X in ). The measured distance to focus is used as a measurement error signal in a closed loop control system which attempts to minimize the error. The closed loop control is a modified variant of the industry standard PID control with only K D (differential) and K P (proportional) gains used. Modifications were made to address the problem of measurement method that proved to be susceptible to the local variation of tissue density at the point of focus of the ATF4 illumination laser. The biological tissue is reflecting only small portions of the incident light and has a tendency to scatter. This phenomenon is causing the light measured by the detector to be received from a substantially larger surface area (as compared to the diffraction limited spot at focus) and from a region of substantial depth. This, in turn, results not only in a broader beam profile, but also makes the COG position dependent on a local tissue structure. Because in live animal experiments the cyclical movement has both Z and X components, the observed position includes a significant error that, if left not compensated, leads to vibrations. To increase the signal-to-noise ratio, the ATF4 control loop is optimized using different strategies: first, using a dynamically adapting laser power; second, using a signal processing algorithm involving a limiter and rolling window average of N Avg samples and digital filtering coupled with a peak fitting procedure to determine the best current position of the imaging plane of interest. However, low pass filtering of N Avg samples introduces poles in the closed loop transfer function leading to instability, which, in turn, requires lowering the overall proportional gain K P . To offset this effect, the predicted position Z Abs Pred for each data point is generated by decoupling the system self motion from the measured position before averaging. The correction is then computed in a linear predictor based on the best linear fit and is extrapolated to a current position. The difference in linearized correction and current position serves as a differential component of the control loop and is sent to the output with the gain K D . The ATF4 control loop takes advantage of large signal oversampling, since measurements were available at 1 kHz rate, while the highest frequency component in the signal was estimated at 15 Hz. It should also be noted that to correctly provide the Z Abs Pred position, the system needs to have N DS delay identified, which corresponds to the difference in time between induced motion and observed results. The limiter prevents signal excursions that are too rapid and could not result from biological movements. The rolling window average signal Z Abs Avg is used as reference to center the limiter acceptance band. Because of the use of a piezo actuator, the absolute Z position Z Abs Out (rather than Z delta correction) was necessary as a control output. (TIF) Click here for additional data file. Table S1 Residual movement amplitude (µm) with movement compensation. Residual movement amplitude (in µm), not compensated by the device, over a range of calibrated movement in terms of amplitude and frequencies applied to two types of targets: one highly reflective and homogeneous (a mirror) and one highly diffusive and heterogeneous (a tissue). Data are means ± SEM of 3 experiments per condition, 10 measurements per experiment. There were no significant differences in mean residual movement amplitudes obtained with the mirror versus tissue targets (two-way ANOVA; P >0.05). (DOC) Click here for additional data file. Video S1 Movie illustrating movement compensation efficiency under in vivo conditions. Complete sequence from experiment shown in Fig. 2A . The breathing of the living animal under anesthesia induces a large amplitude movement, resulting in spinal neurons appearing and disappearing cyclically. When the movement compensation is ON, the imaging plane is locked and stable over time. The raw images were corrected for background noise. Scale bar, 10 µm. (AVI) Click here for additional data file.