Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein

Jun Chu; Russell D Haynes; Stéphane Y Corbel; Pengpeng Li; Emilio González-González; John S Burg; Niloufar J Ataie; Amy J Lam; Paula J Cranfill; Michelle A Baird; Michael W Davidson; Ho-Leung Ng; K Christopher Garcia; Christopher H Contag; Kang Shen; Helen M Blau; Michael Z Lin

doi:10.1038/nmeth.2888

Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein

Nat Methods. 2014 May;11(5):572-8. doi: 10.1038/nmeth.2888. Epub 2014 Mar 16.

Authors

Jun Chu¹, Russell D Haynes², Stéphane Y Corbel², Pengpeng Li³, Emilio González-González⁴, John S Burg⁵, Niloufar J Ataie⁶, Amy J Lam¹, Paula J Cranfill⁷, Michelle A Baird⁷, Michael W Davidson⁷, Ho-Leung Ng⁸, K Christopher Garcia⁹, Christopher H Contag⁴, Kang Shen¹⁰, Helen M Blau², Michael Z Lin¹¹

Affiliations

¹ 1] Department of Bioengineering, Stanford University, Stanford, California, USA. [2] Department of Pediatrics, Stanford University School of Medicine, Stanford, California, USA.
² 1] Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA. [2] Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, USA.
³ Department of Biological Sciences, Stanford University, Stanford, California, USA.
⁴ 1] Department of Pediatrics, Stanford University School of Medicine, Stanford, California, USA. [2] Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, California, USA.
⁵ 1] Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California, USA. [2] Department of Structural Biology, Stanford University School of Medicine, Stanford, California, USA.
⁶ Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii, USA.
⁷ 1] Department of Biological Science, Florida State University, Tallahassee, Florida, USA. [2] National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida, USA.
⁸ 1] Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii, USA. [2] University of Hawaii Cancer Center, Honolulu, Hawaii, USA.
⁹ 1] Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California, USA. [2] Department of Structural Biology, Stanford University School of Medicine, Stanford, California, USA. [3] Howard Hughes Medical Institute, Stanford University, Stanford, California, USA.
¹⁰ 1] Department of Biological Sciences, Stanford University, Stanford, California, USA. [2] Howard Hughes Medical Institute, Stanford University, Stanford, California, USA.
¹¹ 1] Department of Bioengineering, Stanford University, Stanford, California, USA. [2] Department of Pediatrics, Stanford University School of Medicine, Stanford, California, USA. [3] Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, California, USA.

Abstract

A method for non-invasive visualization of genetically labeled cells in animal disease models with micrometer-level resolution would greatly facilitate development of cell-based therapies. Imaging of fluorescent proteins (FPs) using red excitation light in the 'optical window' above 600 nm is one potential method for visualizing implanted cells. However, previous efforts to engineer FPs with peak excitation beyond 600 nm have resulted in undesirable reductions in brightness. Here we report three new red-excitable monomeric FPs obtained by structure-guided mutagenesis of mNeptune. Two of these, mNeptune2 and mNeptune2.5, demonstrate improved maturation and brighter fluorescence than mNeptune, whereas the third, mCardinal, has a red-shifted excitation spectrum without reduction in brightness. We show that mCardinal can be used to non-invasively and longitudinally visualize the differentiation of myoblasts into myocytes in living mice with high anatomical detail.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cell Differentiation*
Crystallography, X-Ray
Diagnostic Imaging / methods*
Gene Library
HeLa Cells
Hemoglobins / chemistry
Humans
Hydrogen Bonding
Luminescent Proteins / metabolism*
Male
Mice
Mice, Nude
Microscopy, Fluorescence / methods*
Molecular Sequence Data
Muscle Cells / metabolism
Muscle, Skeletal / pathology
Muscles / pathology
Mutagenesis
Myoblasts / metabolism
Myoglobin / chemistry
NIH 3T3 Cells
Red Fluorescent Protein
Regeneration
Stem Cells / cytology

Substances

Hemoglobins
Luminescent Proteins
Myoglobin

Associated data

GENBANK/KJ131552
GENBANK/KJ131553
GENBANK/KJ131554
PDB/4OJ0
PDB/4OQW

Abstract

Publication types

MeSH terms

Substances

Associated data

Grants and funding