A Surprising Photoactivity of Blue Fluorescent Protein TagBFP Allows for Super-Resolution Microscopy

A.S Mishin,D.M Chudakov,E.V Zagaynova,N.V Klementieva,K.A Lukyanov,D.A Gorbachev

doi:10.17691/stm2018.10.1.04

Abstract

The aim of this research was to study photobehavior of a popular blue fluorescent protein TagBFP and apply this marker for super-resolution microscopy. Materials and Methods. Photoactivation of TagBFP was examined both in protein solution in vitro and in living cells. Subdiffraction imaging was performed using total internal reflection fluorescence microscopy followed by super-resolution radial fluctuations or single-molecule localization analysis. Results. We show that TagBFP exhibits blinking behavior upon 405 nm light illumination. Moreover, photoactivation to red-emitting state is occurring in the conditions typically used for TagBFP imaging. The red (photoactivated) form of TagBFP possesses spectral properties similar to TagRFP - a close homologue of TagBFP. We show that both blinking and photoactivation of TagBFP can be utilized for super-resolution imaging. We conclude that photoactivation of TagBFP to red-emitting form should be taken into account in the design of multi-channel imaging experiments involving high-power or prolonged UV illumination.

Highlights

Genetically encoded labels — fluorescent proteins of the green fluorescent protein (GFP) family enable realtime visualization of various structures and events in live cells [1]
We have previously reported that TagRFP and several closely related red fluorescent proteins exhibit fast reversible photoconversion between dark and bright states, which could be utilized for super-resolution imaging [8]
We found that TagBFP, to TagRFP, exhibits robust frame-to-frame pixel intensity fluctuations with exposures between 10 to 100 ms and illumination intensities as low as 0.1 W/cm2 of 405 nm light illumination

Summary

Introduction

Genetically encoded labels — fluorescent proteins of the green fluorescent protein (GFP) family enable realtime visualization of various structures and events in live cells [1]. They provide a rich information in multicolor imaging regime that makes it possible to establish a relationship between different cellular targets in space and time. Unlike other parts of the visible spectrum, the palette of widely used blue fluorescent proteins is limited to only a few bright and photostable monomeric proteins, namely Azurite, EBFP2, and TagBFP [2,3,4]. Regular fluorescence microscopy provides the maximum resolution of 200–350 nm that makes many important fine features of a target structure hidden. Some types of these probes are suitable for fast imaging of live cells [7]

Objectives

Methods

Results