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 contributionResearch

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.
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

Fingerprint

Nanodiamonds
Nerve Tissue
Lanthanoid Series Elements
In Situ Hybridization
RNA Probes
Spinal Cord
Central Nervous System
RNA
Brain
Photobleaching
Complementary RNA
Hydrogel
Neuralgia
Viral Proteins
Fluorescein
Fluorescent Dyes
Constriction
Coloring Agents
Pain
Control Groups

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
Parker, Lindsay M. ; Packer, Nicolle H. ; Staikopoulos, Vicky ; Hutchinson, Mark R. ; Cordina, Nicole M. ; Sayyadi, Nima. / 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). Progress in Biomedical Optics and Imaging: Clinical and Translational Neurophotonics; Neural Imaging and Sensing; and Optogenetics and Optical Manipulation : proceedings. editor / Steen J. Madsen ; Victor X. D. Yang ; E. Duco Jansen ; Qingming Luo ; Jun Ding ; Anna W. Roe ; Samarendra K. Mohanty ; Nitish V. Thakor. Bellingham, Washington : SPIE, 2016. pp. 969017-1-969017-1 (Proceedings of SPIE).
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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.",
author = "Parker, {Lindsay M.} and Packer, {Nicolle H.} and Vicky Staikopoulos and Hutchinson, {Mark R.} and Cordina, {Nicole M.} and Nima Sayyadi",
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Parker, LM, Packer, NH, Staikopoulos, V, Hutchinson, MR, Cordina, NM & 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 SJ Madsen, VXD Yang, ED Jansen, Q Luo, J Ding, AW Roe, SK Mohanty & NV Thakor (eds), Progress in Biomedical Optics and Imaging: Clinical and Translational Neurophotonics; Neural Imaging and Sensing; and Optogenetics and Optical Manipulation : proceedings. Proceedings of SPIE, vol. 9690, SPIE, Bellingham, Washington, pp. 969017-1-969017-1, Clinical and Translational Neurophotonics; Neural Imaging and Sensing; and Optogenetics and Optical Manipulation (2016), San Francisco, California, United States, 13/02/16. https://doi.org/10.1117/12.2209249

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

Progress in Biomedical Optics and Imaging: Clinical and Translational Neurophotonics; Neural Imaging and Sensing; and Optogenetics and Optical Manipulation : proceedings. ed. / Steen J. Madsen; Victor X. D. Yang; E. Duco Jansen; Qingming Luo; Jun Ding; Anna W. Roe; Samarendra K. Mohanty; Nitish V. Thakor. Bellingham, Washington : SPIE, 2016. p. 969017-1-969017-1 (Proceedings of SPIE; Vol. 9690).

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

TY - CHAP

T1 - 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)

AU - Parker, Lindsay M.

AU - Packer, Nicolle H.

AU - Staikopoulos, Vicky

AU - Hutchinson, Mark R.

AU - Cordina, Nicole M.

AU - Sayyadi, Nima

PY - 2016

Y1 - 2016

N2 - 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.

AB - 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.

U2 - 10.1117/12.2209249

DO - 10.1117/12.2209249

M3 - Other chapter contribution

SN - 9781628419603

T3 - Proceedings of SPIE

SP - 969017-1-969017-1

BT - Progress in Biomedical Optics and Imaging

A2 - Madsen, Steen J.

A2 - Yang, Victor X. D.

A2 - Jansen, E. Duco

A2 - Luo, Qingming

A2 - Ding, Jun

A2 - Roe, Anna W.

A2 - Mohanty, Samarendra K.

A2 - Thakor, Nitish V.

PB - SPIE

CY - Bellingham, Washington

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Parker LM, Packer NH, Staikopoulos V, Hutchinson MR, Cordina NM, Sayyadi N. 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 Madsen SJ, Yang VXD, Jansen ED, Luo Q, Ding J, Roe AW, Mohanty SK, Thakor NV, editors, Progress in Biomedical Optics and Imaging: Clinical and Translational Neurophotonics; Neural Imaging and Sensing; and Optogenetics and Optical Manipulation : proceedings. Bellingham, Washington: SPIE. 2016. p. 969017-1-969017-1. (Proceedings of SPIE). https://doi.org/10.1117/12.2209249