Fluorescent nanodiamond and lanthanide labelled in situ hybridization for the identification of RNA transcripts in fixed and clarity-cleared central nervous system tissues (conference presentation)

Lindsay M. Parker, Nicolle H. Packer, Vicky Staikopoulos, Mark R. Hutchinson, Nicole M. Cordina, Nima Sayyadi

Research output: Chapter in Book/Report/Conference proceedingOther chapter contribution

Abstract

Despite significant advancement in the methodology used to conjugate, incorporate and visualize fluorescent molecules at the cellular and tissue levels, biomedical imaging predominantly relies on the limitations of established fluorescent molecules such as fluorescein, cyanine and AlexaFluor dyes or genetic incorporation of fluorescent proteins by viral or other means. These fluorescent dyes and conjugates are highly susceptible to photobleaching and compete with cellular autofluorescence, making biomedical imaging unreliable, difficult and time consuming in many cases. In addition, some proteins have low copy numbers and/or poor antibody recognition, further making detection and imaging difficult. We are developing better methods for imaging central nervous system neuroinflammatory markers using targeted mRNA transcripts labelled with fluorescent nanodiamonds or lanthanide chelates. These tags have increased signal and photostability and can also discriminate against tissue/cell autofluorescence. Brains and spinal cords from BALB/c mice with a chronic constriction model of neuropathic pain (neuroinflammation group) or that have undergone sham surgeries (control group) were collected. A subset of brains and spinal cords were perfused and fixed with paraformaldehyde (n=3 sham and n=3 pain groups) prior to sectioning and in situ hybridization using nanodiamond or lanthanide chelate conjugated complementary RNA probes. Another subset of brains and spinal cords from the same cohort of animals were perfused and processed for CLARITY hydrogel based clearing prior to in situ hybridization with the same probes. We will present our findings on the photostability, sensitivity and discrimination from background tissue autofluorescence of our novel RNA probes, compared to traditional fluorophore tags.
Original languageEnglish
Title of host publicationProgress in Biomedical Optics and Imaging
Subtitle of host publicationClinical and Translational Neurophotonics; Neural Imaging and Sensing; and Optogenetics and Optical Manipulation : proceedings
EditorsSteen J. Madsen, Victor X. D. Yang, E. Duco Jansen, Qingming Luo, Jun Ding, Anna W. Roe, Samarendra K. Mohanty, Nitish V. Thakor
Place of PublicationBellingham, Washington
PublisherSPIE
Pages969017-1-969017-1
Number of pages1
ISBN (Print)9781628419603
DOIs
Publication statusPublished - 2016
EventClinical and Translational Neurophotonics; Neural Imaging and Sensing; and Optogenetics and Optical Manipulation (2016) - San Francisco, California, United States
Duration: 13 Feb 201616 Feb 2016

Publication series

NameProceedings of SPIE
PublisherSPIE
Volume9690
ISSN (Print)1605-7422

Conference

ConferenceClinical and Translational Neurophotonics; Neural Imaging and Sensing; and Optogenetics and Optical Manipulation (2016)
CitySan Francisco, California, United States
Period13/02/1616/02/16

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  • Cite this

    Parker, L. M., Packer, N. H., Staikopoulos, V., Hutchinson, M. R., Cordina, N. M., & Sayyadi, N. (2016). Fluorescent nanodiamond and lanthanide labelled in situ hybridization for the identification of RNA transcripts in fixed and clarity-cleared central nervous system tissues (conference presentation). In S. J. Madsen, V. X. D. Yang, E. D. Jansen, Q. Luo, J. Ding, A. W. Roe, S. K. Mohanty, ... N. V. Thakor (Eds.), Progress in Biomedical Optics and Imaging: Clinical and Translational Neurophotonics; Neural Imaging and Sensing; and Optogenetics and Optical Manipulation : proceedings (pp. 969017-1-969017-1). (Proceedings of SPIE; Vol. 9690). Bellingham, Washington: SPIE. https://doi.org/10.1117/12.2209249