Multiphoton Photochemistry of Red Fluorescent Proteins in Solution and Live Cells

Abstract

Genetically encoded fluorescent proteins (FPs), and biosensors based on them, provide new insights into how living cells and tissues function. Ultimately, the goal of the bioimaging community is to use these probes deep in tissues and even in entire organisms, and this will require two-photon laser scanning microscopy (TPLSM), with its greater tissue penetration, lower autofluorescence background, and minimum photodamage in the out-of-focus volume. However, the extremely high instantaneous light intensities of femtosecond pulses in the focal volume dramatically increase the probability of further stepwise resonant photon absorption, leading to highly excited, ionizable and reactive states, often resulting in fast bleaching of fluorescent proteins in TPLSM. Here, we show that the femtosecond multiphoton excitation of red FPs (DsRed2 and mFruits), both in solution and live cells, results in a chain of consecutive, partially reversible reactions, with individual rates driven by a high-order (3–5 photon) absorption. The first step of this process corresponds to a three- (DsRed2) or four-photon (mFruits) induced fast isomerization of the chromophore, yielding intermediate fluorescent forms, which then subsequently transform into nonfluorescent products. Our experimental data and model calculations are consistent with a mechanism in which ultrafast electron transfer from the chromophore to a neighboring positively charged amino acid residue triggers the first step of multiphoton chromophore transformations in DsRed2 and mFruits, consisting of decarboxylation of a nearby deprotonated glutamic acid residue.

Transgenic quail to dynamically image amniote embryogenesis

“…S6). We found that the Tg(PGK1.H2B-chFP) cells are best excited from 1040-1160 nm (data not shown), which agrees with previous reports (Drobizhev et al, 2011(Drobizhev et al, , 2014Vadakkan et al, 2009). Unfortunately, high z-resolution image acquisition requires largely overlapping zstacks and therefore results in longer laser exposure.…”

Hidden photoinduced reactivity of the blue fluorescent protein mKalama1

“…S23). 67 The transient absorption at 430 nm is also consistent with the spectral window for anionic tyrosine-based chromophores in teal FPs (l ex/em = 453/488 nm), 68 blue-shifted relative to GFPs. The BFP proteins, including mKalama1 variants, have successfully been used in two-laser imaging experiments where dark anionic species are optically depleted.…”

Two-photon absorption spectra of fluorescent isomorphic DNA base analogs

“…fluorescent proteins without disrupting biological function. Under typical 2PEF microscope sample illumination conditions, the quantum efficiency of photobleaching of L-trp, φ~0.07-6.5% [9], is comparable or even lower than in fluorescent proteins (φ~0.9%) [29]. Therefore, even though the maximum peak σ 2PA of IXP may lack behind relative to some other types of probes, we estimate that the upper most FPS may still exceed what is typical for fluorescent proteins [13].…”