Mechanistic investigation of mEos4b reveals a strategy to reduce track interruptions in sptPALM

Abstract

Green-to-red photoconvertible fluorescent proteins repeatedly enter dark states, causing interrupted tracks in single-particle-tracking localization microscopy (sptPALM). We identified a long-lived dark state in photoconverted mEos4b that results from isomerization of the chromophore and efficiently absorbs cyan light. Addition of weak 488-nm light swiftly reverts this dark state to the fluorescent state. This strategy largely eliminates slow blinking and enables the recording of significantly longer tracks in sptPALM with minimum effort. Main textFluorescent proteins (FPs) and in particular green-to-red photoconvertible fluorescent proteins (PCFPs) have become indispensable tools for advanced imaging such as singlemolecule localization microscopy (SMLM) or single-particle tracking photoactivated localization microscopy (sptPALM). Both techniques are however limited by blinking, a process in which the fluorophores stochastically enter reversible dark states. PCFPs display blinking on multiple timescales, arising from different underlying photochemical processes. 1 Fluorescence intermittencies shorter than the typical exposure times used in these imaging methodologies (~tens of milliseconds), such as caused by intersystem crossing to the triplet state, reduce the apparent brightness of the label. Intermittencies longer than the exposure time, in contrast, can cause severe complications such as multiple counting of target molecules in quantitative SMLM and interruptions of single-molecule tracks in sptPALM. 2 However, the mechanistic origin of long-lived dark states in PCFPs has remained unclear and there is presently no strategy to eliminate these. We set out to investigate the nature of long-lived fluorescence intermittencies in photoconverted (red) mEos4b, 3 one of the latest probes in a series of highly popular greento-red PCFPs. Individual molecules of mEos4b were immobilized in a polyacrylamide (PAA) matrix, converted to the red emissive state using 405-nm illumination, and the fluorescence emission visualized in time using a sensitive widefield microscope. The single-molecule fluorescence traces (Fig. 1A) displayed reversible and long-lived intermittencies, which we identified as the blinking giving rise to interpretation difficulties in SMLM and sptPALM. Histograms of the intermittency duration (Supplementary Fig. 1) revealed the presence of at least two dark states, as previously reported in mEos2 or Dendra2 4,5 (Supplementary Note 1). While the shorter-lived dark state was insensitive to the intensity of the employed 561-nm illumination (Supplementary Figure 2), the rate at which the longer-lived dark state returned to the emissive state increased with the illumination intensity, reaching a saturation regime at ~0.8 s -1 above a power density of ~ 1.5 kW/cm² (Fig. 1B), indicating a sensitivity to light.

Dual Illumination Enhances Transformation of an Engineered Green‐to‐Red Photoconvertible Fluorescent Protein

“…An efficient transition between the red states is highly beneficial for SR imaging as it allows for enhanced optical control and simplifies the experimental methodology by using distinct imaging and photoswitching wavelengths . Recently, during single‐particle‐tracking localization microscopy (sptPALM) with 561 nm light irradiation on mEos4b, a long‐lived dark state that leads to blinking and the track interruptions was shown to be effectively suppressed by the addition of weak 488 nm light . The dark state species was identified as Red off that adopts a bent trans conformation and is highly dynamic, reminiscent of the red species of LEA that can undergo photochromism in this work.…”

“…140 min, the sequential condition unequivocally produces a reduced red/green ratio owing to the aforementioned GA′ species, which apparently has reduced capabilities to photoconvert or photoswitch. The pretreatment of 505 nm light without 400 nm light also increases the likelihood of producing more photobleached species or some long‐lived dark states, ultimately resulting in the worst red/green contrast and lowest photoconversion yield among all tested conditions (e.g., the green trace is below the black trace after ca. 140 min in Figure b).…”

“…Moreover, dual illumination effectively reduces near‐UV irradiation and may therefore benefit bioimaging. One can increase the irradiation power by using four 400‐nm LEDs, but since 405 nm near‐UV light has been widely known to be phototoxic for live‐cell microscopy using PcFPs, this could lead to more detrimental photobleaching and phototoxicity on the bioimaging targets and cellular components in general before molecular counting can occur …”

SOFIevaluator: a strategy for the quantitative quality assessment of SOFI data

“…The SOFI images obtained with these labels are equally good under the conditions that we tested here. An advantage of these labels is that they show photoswitching behavior (which is moderate, though present in mEos and its derived proteins [40]), the dynamics of which can be tuned by modifying 488-or 405-nm light intensities, thus modifying the blinking behavior to match the SOFI algorithm best, yielding high quality SOFI images.…”

Integrated structure-function dataset reveals key mechanisms underlying photochromic fluorescent proteins

“…For example, it is known that photochromism typically involves a chromophore cis/trans isomerization [15] and (de-)protonation [2,16,17], and requires an environment capable of both allowing this isomerization and of stabilizing the resulting states. [12,14,18] However, achieving a more quantitative understanding is complicated by the fact that most studies investigate one or only a few mutants, possibly focusing on only a few spectroscopic parameters, and do so for different scaffolds arising from all over the diverse FP family.…”