title: Image registration and averaging of low laser power two-photon fluorescence images of mouse retina.
abstract: Two-photon fluorescence microscopy (TPM) is now being used routinely to image live cells for extended periods deep within tissues, including the retina and other structures within the eye . However, very low laser power is a requirement to obtain TPM images of the retina safely. Unfortunately, a reduction in laser power also reduces the signal-to-noise ratio of collected images, making it difficult to visualize structural details. Here, image registration and averaging methods applied to TPM images of the eye in living animals (without the need for auxiliary hardware) demonstrate the structural information obtained with laser power down to 1 mW. Image registration provided between 1.4% and 13.0% improvement in image quality compared to averaging images without registrations when using a high-fluorescence template, and between 0.2% and 12.0% when employing the average of collected images as the template. Also, a diminishing return on image quality when more images were used to obtain the averaged image is shown. This work provides a foundation for obtaining informative TPM images with laser powers of 1 mW, compared to previous levels for imaging mice ranging between 6.3 mW [Palczewska G., Nat Med.20, 785 (2014) Sharma R., Biomed. Opt. Express4, 1285 (2013)].
authors.affiliation: Department of Pharmacology, Cleveland Center for Membrane and Structural Biology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA; nsa36@case.edu.
authors.forename: Nathan S
authors.initials: NS
authors.lastname: Alexander
authors.affiliation: Polgenix Inc., 11000 Cedar Ave, Cleveland, Ohio 44106, USA.
authors.forename: Grazyna
authors.initials: G
authors.lastname: Palczewska
authors.affiliation: Faculty of Physics, Astronomy and Informatics, Institute of Physics, Nicolaus Copernicus University, 87-100 Torun, Poland.
authors.forename: Patrycjusz
authors.initials: P
authors.lastname: Stremplewski
authors.affiliation: Faculty of Physics, Astronomy and Informatics, Institute of Physics, Nicolaus Copernicus University, 87-100 Torun, Poland.
authors.forename: Maciej
authors.initials: M
authors.lastname: Wojtkowski
authors.affiliation: Department of Pharmacology, Cleveland Center for Membrane and Structural Biology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA.
authors.forename: Timothy S
authors.initials: TS
authors.lastname: Kern
authors.affiliation: Department of Pharmacology, Cleveland Center for Membrane and Structural Biology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA; Polgenix Inc., 11000 Cedar Ave, Cleveland, Ohio 44106, USA; kxp65@case.edu.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Palczewski
keywords: (100.2960) Image analysis; (170.2520) Fluorescence microscopy; (170.3880) Medical and biological imaging