title: Pixel timing correction in time-lapsed calcium imaging using point scanning microscopy.
abstract: In point scanning imaging, data are acquired by sequentially scanning each pixel of a predetermined area. This way of scanning leads to time delays between pixels, especially for lower scanning speed or large scanned areas. Therefore, experiments are often performed at lower framerates in order to ensure a sufficient signal-to-noise ratio, even though framerates above 30 frames per second are technically feasible. For these framerates, we suggest that it becomes crucial to correct the time delay between image pixels prior to analyses. In this paper, we apply temporal interpolation (or pixel timing correction) for calcium imaging in two-photon microscopy as an example of fluorescence imaging. We present and compare three interpolation methods (linear, Lanczos and cubic B-spline). We test these methods on a simulated network of coupled bursting neurons at different framerates. In this network, we introduce a time delay to simulate a scanning by point scanning microscopy. We also assess these methods on actual microscopic calcium imaging movies recorded at usual framerates. Our numerical results suggest that point scanning microscopy imaging introduces statistically significant time delays between image pixels at low frequency. However, we demonstrate that pixel timing correction compensates for these time delays, regardless of the used interpolation method.
authors.affiliation: Department of Physiology, Hyogo College of Medicine, Nishinomiya, Japan. Electronic address: dboiroux@hotmail.com.
authors.forename: Dimitri
authors.initials: D
authors.lastname: Boiroux
authors.affiliation: Department of Physiology, Hyogo College of Medicine, Nishinomiya, Japan.
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Oke
authors.affiliation: The Institute of Statistical Mathematics, Tokyo, Japan.
authors.forename: Fumikazu
authors.initials: F
authors.lastname: Miwakeichi
authors.affiliation: Department of Physiology, Hyogo College of Medicine, Nishinomiya, Japan. Electronic address: yoku@hyo-med.ac.jp.
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Oku
mesh_terms: D000465:Algorithms; D000818:Animals; D000831:Animals, Newborn; D001933:Brain Stem / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D003952:Diagnostic Imaging; D005453:Fluorescence; D007091:Image Processing, Computer-Assisted; D066298:In Vitro Techniques; D008564:Membrane Potentials / Q000502:physiology; D008959:Models, Neurological; D009415:Nerve Net / Q000502:physiology; D009474:Neurons / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D059629:Signal-To-Noise Ratio; D013997:Time Factors
chemical_list: D002118:Calcium
keywords: Calcium imaging; Computational neuroscience; Point scanning; Time delay correction