dye hmsir maleimide  (GORYO Chemical)

Journal: Nature

Article Title: Overlapping nuclear import and export paths unveiled by two-colour MINFLUX

doi: 10.1038/s41586-025-08738-0

Figure Lengend Snippet: a,b , The time between two successive MINFLUX localizations for Nb GFP -HMSiR in datasets 1 and 2. The insets are EFO ( e mission f requency at o ffset) histograms. The EFO is a measure of the emission intensity. With a higher average EFO, the localization is less likely to fail since sufficient photons are collected within the dwell time of the localization. Longer localization times occur when the dwell time needs to be extended to collect enough photons. These EFO histograms reveal a significantly higher EFO peak within dataset 2 compared to dataset 1. This difference explains why the average localization time of dataset 1 was longer than dataset 2. We note that the different distributions of HMSiR localization times for the two datasets should have no material effect on the NPC reconstructions since the data were collected over 15–20 min and the NPC scaffolds have been shown to be stable over this time period . Further explanation and discussion of the EFO can be found in the and Extended Data Fig. . c,d , The time between two successive MINFLUX localizations for Imp α-JF549 in datasets 1 and 2. See Supplementary Tables and for acquisition parameters.

Article Snippet: The spontaneously blinking dye HMSiR maleimide (SaraFluor 650B-maleimide; A209-01, Goryo Chemical) was attached to the C-terminal cysteine on the anti-GFP nanobody LaG-9(S151C) by incubating with a 15-fold molar excess at room temperature for 15 min to yield Nb GFP –HMSiR.

Techniques:

Journal: Nature

Article Title: Overlapping nuclear import and export paths unveiled by two-colour MINFLUX

doi: 10.1038/s41586-025-08738-0

Figure Lengend Snippet: a , Equal distribution of 32 NUP96 molecules between the cytoplasmic (maroon) and nucleoplasmic (orange) rings of human NPCs. Adapted from the electron microscopy density map EMD-2444 (refs. , , Springer Nature, and ref. , Cell Press). b – e , MINFLUX imaging of NPCs in permeabilized U2OS cells containing NUP96–mEGFP. The confocal image of eGFP fluorescence identifies the outline of a cell nucleus and a gold bead (100 nm) used for image stabilization (lower left corner, b ). A section of the bottom of the nucleus in b ( c ), and 3D MINFLUX imaging of NPCs ( d , e ) are also shown. Anti-GFP nanobodies (Nb GFP ) modified with the HMSiR blinking dye were used to visualize the NPCs within the region shown in c via the stochastic blinking of the dye. In d , the curvature of the nuclear envelope is apparent from the layers defined by the cytoplasmic and nucleoplasmic rings of the NPCs (see a ). In e , all NPCs identified by confocal imaging in c were detected. Coloration shows the z scale. During data collection, the cytoplasm was on the bottom, but images throughout this article were flipped to place the cytoplasm on top for consistency with convention. f , MINFLUX images of single NPCs (more examples in Extended Data Fig. ). g , h , Composite 2D histogram images of an averaged NPC obtained by aligning individual pores on the basis of their centroids and rotated on the basis of their expected eightfold rotational symmetry (see and Extended Data Fig. ; 37 cells, 541 NPCs, n = 82,331 localizations). The scale is percent of maximum. i , Localization precision determined from centroid deviations within HMSiR ‘trajectories’ (20 points or more per trajectory, 37 cells, 269 clusters; n = 32,184 localizations; σ x = 6.5 ± 0.1 nm (black), σ y = 7.0 ± 0.1 nm (red) and σ z = 4.2 ± 0.1 nm (blue)). The values σ x /σ y = 0.93 and σ x /σ z = 1.55 were assumed throughout this article. j , Jump step histogram analysis of localization precision. The predicted distribution assuming the localization precision values determined in i (blue curve) fits the experimental data (black) poorly, thus indicating that the method in i overestimates the localization precision. A simulation model assuming diffusional drift (red; n = 96,000 jump steps, 25 localizations per trajectory; σ x = 4.1 nm = 0.93σ y = 1.55σ z ; D x , D y and D z = 0.00072, 0.00083 and 0.0003 µm 2 s −1 , respectively) agrees with the data and yields the same centroid deviations as determined in i (see Extended Data Fig. ). See and Extended Data Fig. for a description of the analytical approach and a fit with no diffusional drift.

Article Snippet: The spontaneously blinking dye HMSiR maleimide (SaraFluor 650B-maleimide; A209-01, Goryo Chemical) was attached to the C-terminal cysteine on the anti-GFP nanobody LaG-9(S151C) by incubating with a 15-fold molar excess at room temperature for 15 min to yield Nb GFP –HMSiR.

Techniques: Electron Microscopy, Imaging, Fluorescence, Modification

Journal: Nature

Article Title: Overlapping nuclear import and export paths unveiled by two-colour MINFLUX

doi: 10.1038/s41586-025-08738-0

Figure Lengend Snippet: a , MINFLUX images of single NPCs. Images are 2D histograms of localizations for individual NPCs from permeabilized U2OS cells containing NUP96-mEGFP labeled with Nb GFP -HMSiR. b , Scatter plot of HMSiR localizations. Only high-density circular clusters were analyzed further ( squares ); deformed or incomplete clusters were rejected. c , Double-circle fitting. High quality localization clusters were fit to a double-circle model , reflecting the double-ring structure of the NPC. d , Rotation phase angle. The angles in the xy plane of the individual localizations in c relative to the centroid of the double-circle fit were binned (0–45°; assumes an eightfold periodicity), normalized, and fit to y = 1/9 + (1/20.6)*sin(8( x -ϕ)), as described previously . The 1/9 term reflects the average frequency expected for the 9 bins (5° each), and the sine scaling factor is a reasonable average based on simulations. Note that improving the chi-square of the fit by allowing for an adjustable scaling factor does not change the estimated phase angle due to the orthogonality of frequency and angle. e , 2D histogram of aligned NPC scaffolds. Localization clusters were rotated by a phase angle, as determined in d , and aligned based on their centroids, as determined in c (37 cells, 541 NPCs, N = 82,331 localizations). This is the same image as in Fig. . f , Angular distribution of rotationally corrected localizations. The angle distribution for the individual localizations in e was fit to y = 1/180 + c sin(8( x -ϕ)), where c and ϕ are fit parameters, and the 1/180 term reflects the average frequency expected for the 180 bins (2° each). g , The number of HMSiR localizations obtained per NPC scaffold. As few as ten localizations were used earlier to identify an NPC scaffold , but here more than 10-fold more localizations were obtained, on average.

Article Snippet: The spontaneously blinking dye HMSiR maleimide (SaraFluor 650B-maleimide; A209-01, Goryo Chemical) was attached to the C-terminal cysteine on the anti-GFP nanobody LaG-9(S151C) by incubating with a 15-fold molar excess at room temperature for 15 min to yield Nb GFP –HMSiR.

Techniques: Labeling

Journal: Nature

Article Title: Overlapping nuclear import and export paths unveiled by two-colour MINFLUX

doi: 10.1038/s41586-025-08738-0

Figure Lengend Snippet: a , Averaged structure for NPCs from uncorrected MINFLUX measurements (37 cells, 541 NPCs, 82,331 HMSiR localizations). b,c , The z ( b ) and radial ( c ) distributions for the data in a . The distance between the two peaks in b yields the ring separation (76.8 ± 0.8 nm), and the peak in c is considered the radius of the pore (51.1 ± 1.4 nm). The data/results for a-c are summarized in Supplementary Table (row 8). d , Averaged structure for NPCs from astigmatism measurements of the pore scaffolds used for the alignment of mEosEM data in Fig. and Extended Data Fig. (129 cells, 1453 NPCs, 17,234 HMSiR localizations). e,f , The z ( e ) and radial ( f ) distributions for the data in d . The data/results for d-f are summarized in Supplementary Table (rows 2–5). g , Averaged structure for NPCs from corrected MINFLUX measurements. The data in a were corrected by multiplying all z values by 0.67. This factor was determined as indicated in Supplementary Table (note ‘e’). The images on the left are shown in Fig. . h,i , The z ( h ) and radial ( i ) distributions for the data in g . Note that the radial distribution for the corrected MINFLUX data ( i ) is identical to c . The data/results for g-i are summarized in Supplementary Table (row 9).

Article Snippet: The spontaneously blinking dye HMSiR maleimide (SaraFluor 650B-maleimide; A209-01, Goryo Chemical) was attached to the C-terminal cysteine on the anti-GFP nanobody LaG-9(S151C) by incubating with a 15-fold molar excess at room temperature for 15 min to yield Nb GFP –HMSiR.

Techniques:

Journal: Nature

Article Title: Overlapping nuclear import and export paths unveiled by two-colour MINFLUX

doi: 10.1038/s41586-025-08738-0

Figure Lengend Snippet: a-f , Simulated jump and R 2 / t histograms illustrating the effects of particle movement, time step duration, and precision. Conditions are indicated in the various panels. For a-d , isotropic diffusion ( D x = D y = D z ) was assumed; the value D = 0.055 µm 2 /s corresponds to the value obtained for species 3 of the best-fit for the localizations of the JF549 dye on Imp α (see Fig. ). The range of t values in a and b approximate the first ten values from the Imp α-JF549 time step histograms (Extended Data Fig. ). The precision (σ) values used in c-d correspond to the range used to simulate fits to experimental data. e-f show the effect of t for an immobilized particle. Jump step histograms are unaffected by t ( e ) since the measured jump step is determined entirely by the precision of the two localizations needed; in contrast, larger t values promote a sharp peak near zero in the R 2 / t histogram ( f ), which is the key observation that promoted the inclusion of species 1 for the fit in Fig. . g-i , Analysis of HMSiR localizations. g , The blue and red simulated fits and the black experimental data curve are identical datasets and parameters for those of the same color in Fig. . The green curve corresponds to simulations where σ x = 4.45 nm = 0.93σ y = 1.55σ z with no diffusional drift; thus, the centroid (molecular position) is constant throughout the trajectory and jump steps in x , y , and z are distributed according to the localization precision values, which do not agree with the localization precisions as estimated from experimental centroid deviations (Fig. ). So, the green fit cannot be considered consistent with the data despite the agreement with the experimental jump step and R 2 / t [see h ] histograms. The green and red curves substantially overlap and are therefore difficult to distinguish. h , R 2 / t histograms corresponding to the curves in g . i , To determine the effect of diffusional motion on the estimated precision, centroid deviations were calculated from the data for the red curve in g (same curve as in Fig. ) yielding estimated precision values of σ x = 6.5 nm, σ y = 7.0 nm, and σ z = 4.2 nm, which match those from the experimental centroid-based precision calculation (Fig. ). Note that the D x , D y , and D z values were adjusted to reproduce the experimental centroid deviations while keeping σ x = 0.93σ y = 1.55σ z . Due to particle movement during the acquisition of repeated localizations, the precision estimated from centroid deviations therefore overestimates the localization precision (simulation input: σ x = 4.1 nm, σ y = 4.4 nm, and σ z = 2.6). j-l , Analysis of Imp α-JF549 localizations. j,k , The black curves (experimental data) are identical to those in Fig. . The orange curves have parameters identical to the red curves in Fig. , except that particle rotation around a centroid with r = 6 nm was included for species 2, and the precision for this species was reduced to σ x = 7.2 nm = 0.93σ y = 1.55σ z . This approximates slow rotation within a confined region for a transport complex with the dye on the surface. A two species model did not allow for simultaneous good fits to both the jump step and the R 2 / t histograms. Two examples are provided. The blue curves correspond to simulations where σ x = 7.8 nm = 0.93σ y = 1.55σ z with 60% stuck particles and 40% of particles have D = 0.07 µm 2 /s. The green curves correspond to simulations with 58% stuck particles (σ x = 7.1 nm = 0.93σ y = 1.55σ z ) and 42% of particles have D = 0.065 µm 2 /s (σ x = 7.8 nm = 0.93σ y = 1.55σ z ). Note that for the jump step histograms the orange and blue curves largely overlap, and for the R 2 / t histograms the orange and green curves almost exactly overlap. l , A plot of R 2 vs ∆ t for Imp α-JF549 localizations supports the interpretation that the transiting particles are ‘stuck’ most of the time since the displacements do not follow an expected < R 2 > = 6 Dt profile for a diffusing particle. The red line is the expected slope (6 D ) for D = 0.055 µm 2 /s. For all simulations, the number of jump distances was N = 96,000 with 25 localizations per trajectory.

Article Snippet: The spontaneously blinking dye HMSiR maleimide (SaraFluor 650B-maleimide; A209-01, Goryo Chemical) was attached to the C-terminal cysteine on the anti-GFP nanobody LaG-9(S151C) by incubating with a 15-fold molar excess at room temperature for 15 min to yield Nb GFP –HMSiR.

Techniques: Diffusion-based Assay

Journal: Nature

Article Title: Overlapping nuclear import and export paths unveiled by two-colour MINFLUX

doi: 10.1038/s41586-025-08738-0

Figure Lengend Snippet: a , Schematic of the concurrent import and export of Imp α labelled with JF549. The NPC structure was adapted from refs. , , AAAS. b , Target coordinate pattern. The MINFLUX 3D donut was scanned in a seven-point octahedral pattern (black dots) for the Imp α–JF549 tracking algorithm, yielding successive localizations (gold stars). The NPC structure was adapted from ref. , Springer Nature, ref. , AAAS and the RCSB Protein Data Bank . c , d , Unfiltered two-colour MINFLUX localization data obtained in the presence of transport mix. NPC localizations (blue; Nb GFP –HMSiR; see Fig. ) were collected for 20 min, and these were followed by tracking localizations (coloured z scale, Imp α–JF549) collected for 20 min. Views from the cytoplasm ( c ; xy ) and the side ( d ; xz ) for two different cells are shown. e , Tracks (magenta) that satisfied MINFLUX filtering criteria (see and Extended Data Fig. ) overlaid onto four NPC scaffolds (blue).

Article Snippet: The spontaneously blinking dye HMSiR maleimide (SaraFluor 650B-maleimide; A209-01, Goryo Chemical) was attached to the C-terminal cysteine on the anti-GFP nanobody LaG-9(S151C) by incubating with a 15-fold molar excess at room temperature for 15 min to yield Nb GFP –HMSiR.

Techniques:

Journal: Nature

Article Title: Overlapping nuclear import and export paths unveiled by two-colour MINFLUX

doi: 10.1038/s41586-025-08738-0

Figure Lengend Snippet: A description of the MINFLUX parameters reported in this figure and what they mean are described in the . a , The c enter f requency r atio (CFR) values obtained for the HMSiR channel (EX = 642 nm). The CFR is a measure of localization quality. The primary reason for a high CFR in these measurements is the contribution from a second fluorophore, which leads to inaccurate localization values. Thus, an upper bound cutoff of 0.8 was used for HMSiR localizations during acquisition (see ). b , The e ffective f requency at o ffset (EFO) for the HMSiR channel. Background, i.e., low-level emission from the permeabilized cells, was partially eliminated during acquisition of HMSiR dataset 1 by using an EFO > 25 kHz so as not to spend acquisition time on weak signals. A lower threshold of 15 kHz was used during the iteration sequence (Supplementary Table ), which still captures background (peak at ~20 kHz), but the 25 kHz threshold during pattern repeats (25 photons/ms) eliminated most of these weak signals. Post-acquisition, a trajectory length of ≥ 5 localizations reduced background contributions further. Also post-acquisition, an upper threshold of 60 kHz was used to eliminate localizations contaminated by on-switching of a second fluorophore. Due to the higher EFO values for HMSiR dataset 2 (see Extended Data Fig. , inset ), an EFO range of 50–100 kHz was used for this dataset. c,d , Detector channel ratio (DCR) for 561 nm excitation in the absence ( c ) and presence ( d ) of Imp α-JF549. Some background fluorescence in the JF549 channel was detectable within permeabilized U2OS NUP96-mEGFP cells that had been treated with Nb GFP -HMSiR but without addition of ‘transport mix’ (see ). This background signal ( c ) was identified and filtered out based on its DCR, which was generally higher than that of JF549 fluorescence. DCR is defined as the emission frequency in detector 1 divided by the emission frequencies measured for detector 1 + detector 2. Here, detector 1 = 650–685 nm and detector 2 = 580–630 nm. For cells without the transport mix ( c ), the DCR was mostly > 0.5 (40 min acquisition), whereas in the presence of the transport mix ( d ), most DCR values were <0.5, with the values above 0.5 likely reflecting the background. Thus, the data filtration criterion for tracking Imp α-JF549 was DCR < 0.5. e , CFR for the JF549 channel (EX = 561 nm). The data filtration criterion for JF549 was CFR < 0.8 and implemented post-acquisition. Unlike for HMSiR localizations where the CFR check during imaging was set to a low value to select for high quality localizations at the time of acquisition, for tracking Imp α-JF549 the CFR ratio was set to a large cut-off ( > 2.0) to avoid rejecting tracks that were temporarily interrupted. f , EFO for the JF549 channel (EX = 561 nm). This EFO distribution has peaks at ~37, 42, and 83 kHz. The distribution underlying the first peak represents background noise, and the latter two peaks result from the photon emission streams generated by one or two JF549 dyes. Imp α has four reactive cysteine residues and, while the protein was under-labeled with dye, some dual labeling could not be avoided. Since trafficking behavior was not expected to be influenced by the number of dyes on Imp α, the data filtration criterion was an EFO of 40–150 kHz. Of the 225 tracks used in the analysis, 19 had an EFO > 80 kHz (two JF549 dyes). Note that the clear third peak observed here is not present in b , indicating that simultaneous detection of more than one HMSiR dye was infrequent.

Article Snippet: The spontaneously blinking dye HMSiR maleimide (SaraFluor 650B-maleimide; A209-01, Goryo Chemical) was attached to the C-terminal cysteine on the anti-GFP nanobody LaG-9(S151C) by incubating with a 15-fold molar excess at room temperature for 15 min to yield Nb GFP –HMSiR.

Techniques: Sequencing, Fluorescence, Filtration, Imaging, Generated, Labeling