Microfluidics-based selection of red-fluorescent proteins with decreased rates of photobleaching

Abstract

Fluorescent proteins offer exceptional labeling specificity in living cells and organisms. Unfortunately, their photophysical properties remain far from ideal for long-term imaging of low-abundance cellular constituents, in large part because of their poor photostability. Despite widespread engineering efforts, improving the photostability of fluorescent proteins remains challenging due to lack of appropriate high-throughput selection methods. Here, we use molecular dynamics guided mutagenesis in conjunction with a recently developed microfluidic-based platform, which sorts cells based on their fluorescence photostability, to identify red fluorescent proteins with decreased photobleaching from a HeLa cell-based library. The identified mutant, named Kriek, has 2.5- and 4-fold higher photostability than its progenitor, mCherry, under widefield and confocal illumination, respectively. Furthermore, the results provide insight into mechanisms for enhancing photostability and their connections with other photophysical processes, thereby providing direction for ongoing development of fluorescent proteins with improved single-molecule and low-copy imaging capabilities.

Figure 1

Beta-strand 7/10 dynamics, microfluidic selection strategy, and photostability population shift. (A) Crystal structure (PDB 2H5Q) of mCherry, highlighting the targeted region between β-strands 7 and 10 ^, , including residues targeted for mutagenesis, indicated in red (V16, M66, W143, I161, and Q163). (B) Cells expressing a single genomically integrated copy of a mutant FP are hydrodynamically focused to a velocity of ~ 6-8 mm/s and subjected to four illumination events . Each illumination event is ~ 2 ms in duration, and it takes ~ 8 ms for the cell to travel to the next beam, thereby providing sufficient time to allow recovery from reversible dark-states ^-. Photobleaching is quantitated by the quotient of the fluorescence intensity from the fourth and first excitation events. Cells exhibiting improved photostability (e.g., higher photobleaching ratio) are deflected to a separate output channel by applying an optical force with a 1064 nm focused laser. (C) Histogram of the photobleaching ratio (Beam 4 / Beam 1) normalized to the photostability of mCherry for the library before, and (D) after being subjected to two rounds of sorting. Ratios at higher values represent cells expressing mutant FPs with improved photostability.

Figure 2

Spectral and Photobleaching Properties of Isolated Mutants. S2c and Kriek are the same FP variant (mCherry W143I I161M Q163V). (A) Absorption spectra of Sort2 mutants normalized at λabs ~ 230 nm and (B) λabs ~ 590 nm. (C) Widefield (λex = 520-560 nm) and (D) laser-scanning confocal (λex = 561 nm) photobleaching decays for Kriek (Black) and mCherry (Red).

Figure 3

Comparative Molecular Dynamics of Kriek and mCherry. (A) Interstrand dynamics were quantitated by measuring the time-dependent distance between the α-carbons on A145 (β-7) and K198 (β-10), highlighted in red. (B) 100 ns time-trajectory of interstrand dynamics for Kriek and (C) mCherry. (D) Alternative ³O₂ pathway, and ’gatekeeper’ residues, Q64, and F99, highlighted in red.

Figure 4

Kriek Oxygen Dependent Photobleaching and Fluorescence Lifetime. (A) Photobleaching of Kriek expressing E. coli before (aerobic) and after (anaerobic) environmental purging with N2. Purging of O2 significantly decreased the rate of photobleaching. The initial non-exponential phase of photobleaching for Kriek is attributed to fluorescent protein kindling. (B) Fluorescence lifetime measurement by time-correlated single-photon counting. Kriek has a lifetime of 0.87 ns, whereas mCherry has a lifetime of 1.64 ns.