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The depth of anaesthesia was monitored by hind paw retraction in response to toe pinch, and by the rate of heart beats anaesthesia level was increased when mice retracted their hind paw in response to toe pinch. 2 J (right) were conducted under ketamine/xylazine (100 mg kg −1/10 mg kg −1, respectively) anaesthesia. 2 J (right) were conducted under isoflurane (1.5–2% vol./vol. All experiments except for those shown in Fig. The head of the animal was immobilized by attaching the head plate to a custom machined stage mounted on the microscope table. Two-photon excitation was achieved using a titanium:sapphire laser (Chameleon Ultra II, Coherent) tuned to 900 nm and attenuated, so that 15 mW or less average power entered the brain.
#Medfilt2 matlab 2012 software
2003) software and custom scripts written in MATLAB 7 (see TrImager below) on Windows XP Professional (Microsoft). The microscope was controlled by a personal computer equipped with an Intel Core i7 CPU 950 is part of the technical description 3.07 GHz and 3 GB of RAM running ScanImage (v3.6) ( Pologruto et al. Animals with this configuration were imaged 1–2 days after surgery.įluorescence images were collected using a Movable Objective Microscope (MOM) (Sutter Instrument) with a Zeiss ×20, 1.0 NA or an Olympus ×40, 0.8 NA objective. For PoRTS window experiments, a head plate was attached using a similar procedure, and then a 2 mm × 2 mm region of skull was thinned to a thickness of approximately 15 μm and processed further, as described ( Drew et al. Imaging was initiated at least 2 weeks after surgery. A No.1 cover glass was placed on the dura mater and the edges sealed with dental cement (Caulk Division, Dentsply International Grip Cement). A 2 mm × 2 mm area of skull in the centre of the opening was removed using a dental drill (Osada XL-230) fitted with a conical drill bit (Meisinger HM23SR007) under forced air cooling. After drying, an 8 mm wide aluminum head plate with a 4.5 mm diameter circular opening centred 1.5 mm posterior from bregma and 1.5 mm left of midline was attached to the skull using dental cement (C&B Metabond Parkell Bio-Materials Div.). The periostium was then shaved off and approximately 3 mm of muscle surrounding the exposed skull was covered with a thin layer of cyanoacrylate cement. The scalp was incised, resected, and a mixture of lidocaine (lignocaine) HCl and adrenaline (epinephrine) (Hospira 10 mg ml −1 and 10 μg ml −1, respectively) were applied topically to the exposed skull. and petroleum jelly was applied to the eyes. As soon as animals were unconscious, dexamethasone (Vedco 0.1 mg per mouse) was injected i.p. injection of ketamine (100 mg kg −1) and xylazine (10 mg kg −1).
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Here, we show that the impact of brain motion on images collected through in vivo two-photon microscopy can be substantially reduced by synchronizing image acquisition to the cardiac cycle.įor craniotomies, mice were anaesthetized by i.p. Nevertheless, these post hoc in-frame correction approaches rely on statistical assumptions that are difficult to validate, and data lost during image acquisition cannot be recovered. The impact of these frequent movements can be reduced using line-by-line correction algorithms based on the Lucas–Kanade framework or hidden Markov models ( Dombeck et al. However, it is more difficult to compensate motion artifacts that occur during the acquisition of individual image frames (in-frame motion artifacts).
#Medfilt2 matlab 2012 registration
Post hoc application of whole-frame registration algorithms based on cross-correlation ( Rosenfeld & Kak, 1982) is effective at reducing the impact of both translational movements of the brain and thermal drift arising from instrumentation. 2010), leaving the challenge of brain motion unresolved. Although it is possible to reduce brain movement in the open skull configuration by pressing a glass coverslip or other transparent material against the surface of the brain, such manipulations are not possible when imaging is combined with drug application, or when imaging is performed using less traumatic thinned skull preparations ( Dombeck et al. In vivo imaging of fluorescent structures in the brain requires removal or thinning of the overlying skull. The complex motions produced by these vital functions reduce image stability, compromise the resolving power of the microscope, and limit our ability to study both physiological responses and pathological changes in the intact CNS. However, rapid movements of the brain due to beating of the heart and breathing present an inherent challenge for in vivo studies. To accurately follow cellular dynamics, such as Ca 2+ changes in subcellular compartments, the growth of dendritic spines, and organelle movements, it is essential to maximize spatial resolution. In vivo two-photon imaging has become an indispensable approach for monitoring changes in the morphology and physiological activity of cells in the central nervous system ( Kerr & Denk, 2008).