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Related Experiment Video

Updated: Jun 15, 2026

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Published on: April 27, 2018

Monomeric red fluorescent proteins with a large Stokes shift.

Kiryl D Piatkevich¹, James Hulit, Oksana M Subach

¹Department of Anatomy and Structural Biology and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.

Proceedings of the National Academy of Sciences of the United States of America

|March 10, 2010

Summary

Researchers developed new red fluorescent proteins for enhanced two-photon microscopy in living animals. These proteins enable multicolor imaging and revealed tumor cell polarization towards blood vessels in breast cancer models.

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Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

Published on: April 25, 2021

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Last Updated: Jun 15, 2026

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Published on: April 27, 2018

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

Published on: April 25, 2021

Area of Science:

Biophysics
Cell Biology
Biochemistry

Background:

Two-photon microscopy is crucial for in vivo cellular imaging.
Current red fluorescent proteins are limited by laser excitation wavelengths.
Need for red fluorescent proteins compatible with common lasers and multicolor imaging.

Purpose of the Study:

Develop novel red fluorescent proteins (LSS-mKate1 and LSS-mKate2) for improved two-photon microscopy.
Characterize their spectral properties, stability, and suitability for multicolor imaging.
Investigate tumor cell behavior in a breast cancer model using these new proteins.

Main Methods:

Development and characterization of two novel red fluorescent proteins (LSS-mKate1, LSS-mKate2).
Assessment of spectral properties, pH stability, photostability, maturation time, and monomeric behavior.
Application in multicolor two-photon microscopy with blue-green fluorophores.
Intravital imaging in a mouse xenograft model of breast cancer.

Main Results:

LSS-mKate1 and LSS-mKate2 exhibit large Stokes shifts with excitation/emission at 463/624 nm and 460/605 nm.
Proteins demonstrate high pH stability, photostability, rapid maturation, and are monomeric.
Absence of green light absorbance and spectral overlap allow multicolor imaging with a single laser.
Breast cancer cells showed polarization towards blood vessels within 40 µm in vivo.

Conclusions:

LSS-mKates are versatile red fluorescent proteins overcoming limitations of existing red fluorophores for two-photon microscopy.
The developed proteins facilitate multicolor imaging and in vivo studies of cellular dynamics.
Demonstrated tumor cell polarization towards vasculature provides insights into cancer cell behavior.