lambert instruments lifa flim system  (Lambert Instruments BV)

Journal: bioRxiv

Article Title: hpGRISZ: a high-performance fluorescent biosensor for in vivo imaging of synaptic zinc dynamics

doi: 10.1101/2025.10.06.680726

Figure Lengend Snippet: (A) Targeting of hpGRISZ to the surface of HEK293T cells. Top: Schematic of hpGRISZ-TM construct, including murine Igκ signal peptide and PDGFRβ transmembrane (TM) domain. Middle: Representative brightfield and fluorescence images of HEK293T cells co-transfected with hpGRISZ-TM and R-GECO1 (used here as a Zn 2+ -unresponsive cytosolic marker). Scale bar: 10 μm. Bottom: Fluorescence intensity profiles of hpGRISZ-TM (green) and R-GECO1 (magenta) along the indicated lines in the middle panel. (B–D) Time-lapse imaging of cells exposed to Zn 2+ (100 μM) and EDTA (250 μM). B: Representative fluorescence images. Scale bar: 10 μm. C: Normalized fluorescence intensity traces over time. D: Quantification of maximal normalized fluorescence change (mean ± SD; **** P < 0.0001, two-tailed unpaired t-test). (E) Dose-dependent fluorescence response of hpGRISZ at the HEK293T cell surface ( K d = 4.4 ± 0.3 μM; mean ± SEM from 77 cells). (F) Time-lapse fluorescence images (left) and intensity trace (right) of hpGRISZ expressed at the SH-SY5Y cell surface in response to Zn 2+ and EDTA (mean ± SEM from 27 cells). Scale bar: 10 μm. (G) Dose-dependent fluorescence lifetime response of hpGRISZ at the HEK293T cell surface. Left : Pseudo-color lifetime images. Right: Corresponding dose-response curve ( K d = 0.82 ± 0.13 μM; mean ± SEM from 34 cells). Scale bar: 10 μm.

Article Snippet: Fluorescence lifetime imaging was performed with a Lambert LIFA-Toggle frequency-domain FLIM system using a 474-nm LED, 480/40 nm excitation and 527/30 nm emission filters, 1 s exposure, 40 MHz modulation, and 12 phase steps.

Techniques: Construct, Fluorescence, Transfection, Marker, Imaging, Two Tailed Test

Journal: Nature Cell Biology

Article Title: Dynamic regulation of integrin β1 phosphorylation supports invasion of breast cancer cells

doi: 10.1038/s41556-025-01663-4

Figure Lengend Snippet: a , A schematic representation of the possible conformations for the ITGB1 intramolecular FRET biosensor (Illusia), where the mTurquoise2–YPet FRET pair is separated by the PTB from Dok1, a linker and the cytoplasmic domain (aa772–798) from ITGB1 (including the two NPxY motifs). Illusia is recruited to the membrane through an acylation substrate sequence derived from Lyn kinase. Ex, excitation; Em, emission. b , Representative FLIM images (left) and quantification of apparent FRET efficiency (right) of MM231 cells stably expressing Illusia after Dox-inducible overexpression of either Src(WT), kinase-dead Src(K295R) or constitutively active Src(Y527F)/Src(E378G) ( n = 60 cells in all conditions with the exception of 70 for Src(Y527F) (−)Dox and 65 for Src(Y527F) (+)Dox pooled from three biological replicates; one-way analysis of variance (ANOVA) with a Šidák correction for multiple comparisons). Scale bars, 10 μm. c , Representative western blot of MM231 cells after Dox-inducible Src(WT), Src(K295R), Src(Y527F) or Src(E378G) overexpression. d , Densitometry analysis of western blots from c ( n = 4 biological replicates; one-sample two-tailed t -test against the normalized control values for each cell line without Dox). The data are mean ± s.e.m. e , A scheme of an ELISA for pY. f , An ELISA for changes in ITGB1 phosphorylation using recombinant ITGB1 peptide and Src kinase in the absence or presence of ATP and the Src inhibitor Sara (1 μM; n = 3 biological replicates; triplicate wells/replicates; one-way ANOVA with a Šidák correction for multiple comparisons). Unphosphorylated ITGB1 and phosphorylated ITGB1 p(Y783) peptides were included as negative and positive controls, respectively. The data are the mean ± s.e.m. g , Representative FLIM–FRET images (left) and quantification (right) of MM231 and TIF cells with stable expression of Illusia and Sara treatment (1 μM) for 24 h (MM231s, n = 129 (DMSO) and 125 (Sara) cells pooled from five biological replicates; TIFs, n = 74 (DMSO) and 75 (Sara) cells pooled from three biological replicates; unpaired two-tailed Student’s t -test with a Welch’s correction). Scale bars, 10 μm. The boxplots represent the median and IQR. The whiskers extend to the minimum and maximum values. The grey areas on the boxplots highlight the IQR of the control conditions. NS, not significant. * P < 0.05, *** P < 0.001. .

Article Snippet: Frequency-domain FLIM–FRET was performed using a LIFA fast frequency-domain FLIM system (Lambert Instruments) attached to an inverted microscope (Zeiss AXIO Observer.D1) with sinusoidally modulated (40 MHz) epi-illumination (1 W for 405 nm or 3 W for 470 nm) from a temperature-stabilized multi-light-emitting diode (LED) system (Lambert Instruments) and a ×63/1.15 objective (Zeiss, Objective LD C-Apochromat ×63/1.15 W Corr M27).

Techniques: Membrane, Sequencing, Derivative Assay, Stable Transfection, Expressing, Over Expression, Western Blot, Two Tailed Test, Control, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Recombinant

Journal: Nature Cell Biology

Article Title: Dynamic regulation of integrin β1 phosphorylation supports invasion of breast cancer cells

doi: 10.1038/s41556-025-01663-4

Figure Lengend Snippet: a , Representative western blots of MM231 and MCF10A cells expressing either Venus or Dok1 (n = 4 biological replicates). b , A schematic of the intermolecular FRET approach between Dok1 and ITGB1 (WT or YYFF). c , Representative FLIM-FRET images (left) and quantification of apparent FRET efficiency (right) from MM231 shβ1 cells reexpressing mRuby2-tagged ITGB1(WT or YYFF) and transfected with Dok1-Clover (n = 60 cells in each condition pooled from three biological replicates; significance assessed using an unpaired two-tailed Student’s t -test with a Welch’s correction; *** p < 0.001). Scale bars, 10 μm. d , Representative FLIM-FRET images (left) and quantification of apparent FRET efficiency (right) from MM231 shβ1 cells reexpressing mRuby2-tagged ITGB1(WT or YYFF), and transfected with GFP-F 0 F 3 talin head domain fragment (n = 62 cells in each condition pooled from three biological replicates; significance assessed using an unpaired two-tailed Student’s t -test with a Welch’s correction; *** p < 0.001). Scale bars, 20 μm. e & f , Apparent FRET efficiencies ( e ) and representative images ( f ) of MM231 cells stably expressing Illusia and Dox-inducible ABL2(WT) or kinase-dead ABL2(K281M) ± Dox treatment. Parental cells treated with Dox are used as an additional control (n = 60 cells in each condition pooled from three biological replicates; significance assessed using a one-way ANOVA with a Šidák correction for multiple comparisons; NS, not significant, *** p < 0.001). Scale bars, 10 μm. g & h , Representative western blot ( g ) and densitometry ( h ) analysis of ITGB1 phosphorylation levels in MM231 cells with Dox-induced ABL2(WT) or ABL2(K281M) overexpression (n = 5 biological replicates; significance assessed using a one-sample two-tailed t -test against the normalised control values for each cell line without Dox; NS, not significant, * p < 0.05). Data are mean ± SEM. i & j , Representative western blot ( i ) and densitometry ( j ) of MM231 cells treated with saracatenib (Sara) for 0, 2, 24, 48 and 72 h (1 μM; n = 5 biological replicates; significance assessed using a one-sample two-tailed t -test against the normalised DMSO control). Source data and exact p-values are provided in the statistical source data file. Boxplots represent median and interquartile range. Whiskers extend to min and max values. Grey areas on boxplots highlight the interquartile range of the control conditions.

Article Snippet: Frequency-domain FLIM–FRET was performed using a LIFA fast frequency-domain FLIM system (Lambert Instruments) attached to an inverted microscope (Zeiss AXIO Observer.D1) with sinusoidally modulated (40 MHz) epi-illumination (1 W for 405 nm or 3 W for 470 nm) from a temperature-stabilized multi-light-emitting diode (LED) system (Lambert Instruments) and a ×63/1.15 objective (Zeiss, Objective LD C-Apochromat ×63/1.15 W Corr M27).

Techniques: Western Blot, Expressing, Transfection, Two Tailed Test, Stable Transfection, Control, Phospho-proteomics, Over Expression

Journal: Nature Cell Biology

Article Title: Dynamic regulation of integrin β1 phosphorylation supports invasion of breast cancer cells

doi: 10.1038/s41556-025-01663-4

Figure Lengend Snippet: a , A schematic of phosphorylation-dependent Dok1 recruitment to ITGB1. b , c , Representative western blots ( b ) and densitometry ( c ) of MM231 (left) or MCF10A (right) cells treated with the broad-spectrum PTP inhibitor sodium orthovanadate ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\rm{VO}}}_{4}^{3-}$$\end{document} VO 4 3 − ; 100 μM, 2 h; n = 4 biological replicates; one-sample two-tailed t -test against the normalized control value without \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\rm{VO}}}_{4}^{3-}$$\end{document} VO 4 3 − ). The data are the mean ± s.e.m. d , A representative western blot (left) of MM231 ITGB1(WT or YYFF) cells treated for 2 h with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\rm{VO}}}_{4}^{3-}$$\end{document} VO 4 3 − (100 μM) and densitometry analysis (right) of MM231 ITGB1(WT) cells ( n = 5 biological replicates; one-sample two-tailed t -test against the normalized control value without \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\rm{VO}}}_{4}^{3-}$$\end{document} VO 4 3 − ). The data are the mean ± s.e.m. e , Representative FLIM images (left) and quantification of apparent FRET efficiency (right) after \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\rm{VO}}}_{4}^{3-}$$\end{document} VO 4 3 − treatment (100 μM, 2 h) of MM231 cells stably expressing Illusia(WT) or a non-phosphorylatable mutant reporter Illusia(YYFF) ( n = 88 (Illusia(WT) ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$-{{\rm{VO}}}_{4}^{3-}$$\end{document} − VO 4 3 − )), 88 (Illusia(WT) ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${+{\rm{VO}}}_{4}^{3-}$$\end{document} + VO 4 3 − )), 93 (Illusia(YYFF) ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${-{\rm{VO}}}_{4}^{3-}$$\end{document} − VO 4 3 − )) and 86 (Illusia(YYFF) ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${+{\rm{VO}}}_{4}^{3-}$$\end{document} + VO 4 3 − )) cells pooled from four biological replicates; one-way analysis of variance with a Šidák correction for multiple comparisons). n.s., not significant. Scale bars, 10 μm. The boxplot represents the median and IQR. The whiskers extend to the minimum and maximum values. The grey area on the boxplot highlights the IQR of the control condition. NS, not significant, * P < 0.05, *** P < 0.001.

Article Snippet: Frequency-domain FLIM–FRET was performed using a LIFA fast frequency-domain FLIM system (Lambert Instruments) attached to an inverted microscope (Zeiss AXIO Observer.D1) with sinusoidally modulated (40 MHz) epi-illumination (1 W for 405 nm or 3 W for 470 nm) from a temperature-stabilized multi-light-emitting diode (LED) system (Lambert Instruments) and a ×63/1.15 objective (Zeiss, Objective LD C-Apochromat ×63/1.15 W Corr M27).

Techniques: Phospho-proteomics, Western Blot, Two Tailed Test, Control, Stable Transfection, Expressing, Mutagenesis

Journal: Nature Cell Biology

Article Title: Dynamic regulation of integrin β1 phosphorylation supports invasion of breast cancer cells

doi: 10.1038/s41556-025-01663-4

Figure Lengend Snippet: a & b , Representative immunoprecipitation (IP) of phosphorylated ITGB1 in MM231 ( a ) and TIF ( b ) cells using anti-pY beads after \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\rm{VO}}}_{4}^{3-}$$\end{document} VO 4 3 − treatment (100 mM, 2 h; n = 3 biological replicates). c , Representative western blot and densitometry analysis of ITGB1 phosphorylation levels in TIF cells after \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\rm{VO}}}_{4}^{3-}$$\end{document} VO 4 3 − treatment (100 mM, 2 h; n = 4 biological replicates; significance assessed using a one-sample two-tailed t -test against the normalised control value without \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\rm{VO}}}_{4}^{3-}$$\end{document} VO 4 3 − ; ** p < 0.01). Data are mean ± SEM. d , Representative images (left) and quantification of apparent FRET efficiency (right) of VO 4 3- (100 mM, 2 h) treated TIF cells stably expressing Illusia (WT), or a non-phosphorylatable mutant (YYFF) (n = 60 cells in each condition pooled from three biological replicates; significance assessed using a one-way ANOVA with a Šidák correction for multiple comparisons; NS, not significant, *** p < 0.001). Scale bars, 10 μm. e , Representative FLIM-FRET images (left) and apparent FRET efficiencies (right) from HEK293 cells transfected with different Illusia variants (WT, Y783F, Y795F, YYFF and YYEE; n = 127 [WT \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(-){\rm{VO}}}_{4}^{3-}$$\end{document} ( − ) VO 4 3 − ], 152 [WT \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(+){\rm{VO}}}_{4}^{3-}$$\end{document} ( + ) VO 4 3 − ], 113 [Y783F \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(-){\rm{VO}}}_{4}^{3-}$$\end{document} ( − ) VO 4 3 − ], 120 [Y783F \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(+){\rm{VO}}}_{4}^{3-}$$\end{document} ( + ) VO 4 3 − ], 109 [Y795F \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(-){\rm{VO}}}_{4}^{3-}$$\end{document} ( − ) VO 4 3 − ], 117 [Y795F \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(+){\rm{VO}}}_{4}^{3-}$$\end{document} ( + ) VO 4 3 − ], 115 [YYFF \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(-){\rm{VO}}}_{4}^{3-}$$\end{document} ( − ) VO 4 3 − ], 121 [YYFF \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(+){\rm{VO}}}_{4}^{3-}$$\end{document} ( + ) VO 4 3 − ], 106 [YYEE \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(-){\rm{VO}}}_{4}^{3-}$$\end{document} ( − ) VO 4 3 − ], and 109 [YYEE \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(+){\rm{VO}}}_{4}^{3-}$$\end{document} ( + ) VO 4 3 − ] cells pooled from four biological replicates; significance assessed using one-way ANOVA with a Šidák correction for multiple comparisons; NS, not significant, ***p < 0.001). Scale bars, 20 μm. f & g , Representative western blots of MM231 and TIF cells stably expressing Illusia and treated with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\rm{VO}}}_{4}^{3-}$$\end{document} VO 4 3 − ( f ; 100 mM, 2 h; n = 3 biological replicates) or Sara for 24 h ( g ; 1 μM; n = 4 biological replicates). h , Representative GFP-trap IP of mT2 or Illusia from MM231 cells stably expressing the reported constructs (n = 3 biological replicates). Source data and exact p-values are provided in the statistical source data file. Boxplots represent median and interquartile range. Whiskers extend to min and max values. Grey areas on boxplots highlight the interquartile range of the control conditions.

Article Snippet: Frequency-domain FLIM–FRET was performed using a LIFA fast frequency-domain FLIM system (Lambert Instruments) attached to an inverted microscope (Zeiss AXIO Observer.D1) with sinusoidally modulated (40 MHz) epi-illumination (1 W for 405 nm or 3 W for 470 nm) from a temperature-stabilized multi-light-emitting diode (LED) system (Lambert Instruments) and a ×63/1.15 objective (Zeiss, Objective LD C-Apochromat ×63/1.15 W Corr M27).

Techniques: Immunoprecipitation, Western Blot, Phospho-proteomics, Two Tailed Test, Control, Stable Transfection, Expressing, Mutagenesis, Transfection, Construct

Journal: Nature Cell Biology

Article Title: Dynamic regulation of integrin β1 phosphorylation supports invasion of breast cancer cells

doi: 10.1038/s41556-025-01663-4

Figure Lengend Snippet: a , A waterfall plot from an RNAi sensitized emission (SE)-FRET screen of MM231 cells stably expressing Illusia to assess changes after KD of 96 out of the 108 PTPs in the human genome, using three siRNAs/target (A, B and C) ( n = 3; one-way analysis of variance (ANOVA) with a Tukey correction for multiple comparisons after normalizing replicates into robust z -scores). PTPs that significantly ( P < 0.01) increased phosphorylation upon KD are indicated with red text and circles (15 hits), while those that significantly ( P < 0.01) decreased phosphorylation upon KD are indicated with blue text and circles (15 top hits). The data are the mean ± s.e.m. b , c , Representative FLIM–FRET images (left) and quantification (right) of MM231 and TIF cells with stable Illusia expression and transfected with siRNAs (A, B or C) against either PTPN11 (Shp2) ( b ) or PTPN12 (PTP-PEST) ( c ) (MM231 PTPN11 silencing, n = 100 (siNTC), 98 (siRNA A), 100 (siRNA B) and 100 (siRNA C); PTPN12 silencing, n = 99 (siNTC), 99 (siRNA A), 98 (siRNA B) and 97 (siRNA C) cells pooled from three biological replicates; TIF PTPN11 silencing, n = 75 (siNTC), 76 (siRNA A), 76 (siRNA B) and 78 (siRNA C); PTPN12 silencing, n = 74 (siNTC), 74 (siRNA A), 77 (siRNA B) and 74 (siRNA C) cells pooled from four biological replicates; one-way ANOVA with a Dunnett correction for multiple comparisons). n.s., not significant. Scale bars, 20 μm. The boxplots represent the median and IQR. The whiskers extend to the minimum and maximum values. The grey areas on the boxplots highlight the IQR of the control conditions. siNTC, non-targeting control siRNA. d , e , Densitometry from western blots of PTPN11 (representative western blot) ( d ) (Extended Data Fig. ) and PTPN12 (representative western blot) ( e ) (Extended Data Fig. ) siRNA KD in MM231 ( n = 5 biological replicates) and TIF ( n = 5 (Shp2) or 6 (PTP-PEST) biological replicates) cells. The data are the mean ± s.e.m. * P < 0.05, *** P < 0.001.

Article Snippet: Frequency-domain FLIM–FRET was performed using a LIFA fast frequency-domain FLIM system (Lambert Instruments) attached to an inverted microscope (Zeiss AXIO Observer.D1) with sinusoidally modulated (40 MHz) epi-illumination (1 W for 405 nm or 3 W for 470 nm) from a temperature-stabilized multi-light-emitting diode (LED) system (Lambert Instruments) and a ×63/1.15 objective (Zeiss, Objective LD C-Apochromat ×63/1.15 W Corr M27).

Techniques: Stable Transfection, Expressing, Phospho-proteomics, Transfection, Control, Western Blot

Journal: Nature Cell Biology

Article Title: Dynamic regulation of integrin β1 phosphorylation supports invasion of breast cancer cells

doi: 10.1038/s41556-025-01663-4

Figure Lengend Snippet: a , b , A malachite green assay for free phosphate release after incubation of phosphorylated/non-phosphorylated ITGB1 peptides with recombinant Shp2 ( n = 5 independent replicates, each performed in triplicate) ( a ) or PTP-PEST ( n = 4 independent replicates, each performed in triplicate) ( b ). The significance was assessed using a Kruskal–Wallis test with a Dunn’s correction for multiple comparisons. The data are presented as the mean ± s.e.m. c , d , Schematics of FRET experiments (left) using mRuby2-tagged ITGB1 and Clover-tagged PTPs. Representative FLIM–FRET images (right) and quantification of apparent FRET efficiency of MM231 cells with stable expression of either ITGB1(WT)–mRuby2 or ITGB1(YYFF)–mRuby2 transfected with either Shp2–Clover ( c ) or PTP-PEST-Clover ( d ) and treated with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\rm{VO}}}_{4}^{3-}$$\end{document} VO 4 3 − (100 μM, 2 h) (for c , n = 73 (ITGB1(WT) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(-){\rm{VO}}}_{4}^{3-}$$\end{document} ( − ) VO 4 3 − ), 62 (ITGB1(WT) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(+){\rm{VO}}}_{4}^{3-}$$\end{document} ( + ) VO 4 3 − ), 64 (ITGB1(YYFF) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(-){\rm{VO}}}_{4}^{3-}$$\end{document} ( − ) VO 4 3 − ) and 67 (ITBG1(YYFF) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(+){\rm{VO}}}_{4}^{3-}$$\end{document} ( + ) VO 4 3 − ) cells pooled from three biological replicates; for d , n = 75 (ITGB1(WT) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(-){\rm{VO}}}_{4}^{3-}$$\end{document} ( − ) VO 4 3 − ), 58 (ITGB1(WT) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(+){\rm{VO}}}_{4}^{3-}$$\end{document} ( + ) VO 4 3 − ), 58 (ITGB1(YYFF) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(-){\rm{VO}}}_{4}^{3-}$$\end{document} ( − ) VO 4 3 − ) and 65 (ITBG1(YYFF) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(+){\rm{VO}}}_{4}^{3-}$$\end{document} ( + ) VO 4 3 − ) cells pooled from three biological replicates; one-way analysis of variance with a Tukey correction for multiple comparisons). Scale bars, 20 μm. The boxplots represent the median and IQR. The whiskers extend to the minimum and maximum values. The grey areas highlight the IQR of the control conditions. NS, not significant. * P < 0.05, ** P < 0.01, *** P < 0.001. .

Article Snippet: Frequency-domain FLIM–FRET was performed using a LIFA fast frequency-domain FLIM system (Lambert Instruments) attached to an inverted microscope (Zeiss AXIO Observer.D1) with sinusoidally modulated (40 MHz) epi-illumination (1 W for 405 nm or 3 W for 470 nm) from a temperature-stabilized multi-light-emitting diode (LED) system (Lambert Instruments) and a ×63/1.15 objective (Zeiss, Objective LD C-Apochromat ×63/1.15 W Corr M27).

Techniques: Malachite Green Assay, Incubation, Recombinant, Expressing, Transfection, Control

Journal: Nature Cell Biology

Article Title: Dynamic regulation of integrin β1 phosphorylation supports invasion of breast cancer cells

doi: 10.1038/s41556-025-01663-4

Figure Lengend Snippet: a , Schematic of the FRET experiment (left), with representative FLIM-FRET images (middle) and quantification of apparent FRET efficiency (right) of MM231 cells with stable expression of either ITGB1(WT)-mRuby2 or ITGB1(YYFF)-mRuby2 transfected with Clover and treated with VO 4 3- (n = 65 [ITGB1(WT) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(-){\rm{VO}}}_{4}^{3-}$$\end{document} ( − ) VO 4 3 − ], 70 [ITGB1(WT) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(+){\rm{VO}}}_{4}^{3-}$$\end{document} ( + ) VO 4 3 − ], 72 [ITGB1(YYFF) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(-){\rm{VO}}}_{4}^{3-}$$\end{document} ( − ) VO 4 3 − ], and 66 [ITGB1(YYFF) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(+){\rm{VO}}}_{4}^{3-}$$\end{document} ( + ) VO 4 3 − ]) cells pooled from three biological replicates. Scale bars, 20 μm. b , Representative FLIM-FRET images (left) and quantification of apparent FRET efficiency (right) in MM231 cells with stable Illusia expression and constitutive overexpression of PTPN11 (Shp2, WT) or a phosphatase-dead mutant (Shp2, Mut; PTPN11(D425A, C459S)) (n = 97 (mScarlet), 96 (WT) and 96 (Mut) cells pooled from four biological replicates; significance assessed using a one-way ANOVA with a Tukey correction for multiple comparisons; NS, not significant, *** p < 0.001). Scale bars, 20 μm. c , Representative western blot (left) and densitometry (right) of MM231 cells with stable Illusia expression and constitutive overexpression of PTPN11 (Shp2, WT) or a phosphatase-dead mutant (Shp2, Mut; PTPN11(D425A, C459S); n = 8 biological replicates; significance assessed using a one-sample two-tailed t -test against the normalised control value; * p < 0.05, ***p < 0.001; NS, not significant). d-e , Representative FLIM-FRET images ( d ) and quantification of apparent FRET efficiency ( e ) of MM231 cells with stable Illusia expression treated overnight with Dox to induce overexpression of PTP-PEST WT or a phosphatase-dead mutant (Mut, PTPN12(D199A, C231S) (n = 100 cells in each condition pooled from four biological replicates; significance assessed using a one-way ANOVA with a Šidák correction for multiple comparisons; NS, not significant, *** p < 0.001). Scale bars, 20 μm. f , Western blot (left) and densitometry (right) from parallel data in ( d ; n = 4 biological replicates; significance assessed using a one-sample two-tailed t -test against the normalised control value; * p < 0.05; NS, not significant). g & h , Representative FLIM-FRET images (left) and quantification of apparent FRET efficiency (right) of MM231 ( g ) and TIF ( h ) cells with stable Illusia expression and treated with SHP099 for 2 h (100 nM) (MM231, n = 96 (DMSO) and 95 (SHP099) | TIFs, n = 100 (DMSO) and 99 (SHP099) cells pooled from four biological replicates; significance assessed using an unpaired two-tailed Student’s t -test with a Welch’s correction (NS, not significant, *** p < 0.001). Scale bars, 20 μm. i , Representative western blots (left) and densitometry (right) of MM231 and TIF cells with stable Illusia expression and treated with SHP099 for 2 h (100 nM; n = 6 biological replicates; significance assessed using a one-sample two-tailed t -test against the normalised control value; * p < 0.05). Data are mean ± SEM. Source data and exact p-values are provided in the statistical source data file. Boxplots represent median and interquartile range. Whiskers extend to min and max values. Grey areas on boxplots highlight the interquartile range of the control conditions.

Article Snippet: Frequency-domain FLIM–FRET was performed using a LIFA fast frequency-domain FLIM system (Lambert Instruments) attached to an inverted microscope (Zeiss AXIO Observer.D1) with sinusoidally modulated (40 MHz) epi-illumination (1 W for 405 nm or 3 W for 470 nm) from a temperature-stabilized multi-light-emitting diode (LED) system (Lambert Instruments) and a ×63/1.15 objective (Zeiss, Objective LD C-Apochromat ×63/1.15 W Corr M27).

Techniques: Expressing, Transfection, Over Expression, Mutagenesis, Western Blot, Two Tailed Test, Control

Journal: Nature Cell Biology

Article Title: Dynamic regulation of integrin β1 phosphorylation supports invasion of breast cancer cells

doi: 10.1038/s41556-025-01663-4

Figure Lengend Snippet: a , b , Representative FLIM images ( a , left) and apparent FRET efficiencies ( a , right) and cell area ( b ) of MM231 cells stably expressing Illusia and seeded on either glass or hydrogels (60, 2 or 0.5 kPa) (for a , n = 110 (glass), 117 (60 kPa), 116 (2 kPa) and 113 (0.5 kPa) cells pooled from four biological replicates; for b , n = 121 (glass), 137 (60 kPa), 157 (2 kPa) and 165 (0.5 kPa) cells pooled from four biological replicates; one-way analysis of variance (ANOVA) with a Tukey correction for multiple comparisons). Scale bars, 10 μm. c , Representative images (left) of MM231 ITGB1(WT or YYFF) cells invading into 3D fibroblast-contracted collagen I treated with DMSO or SHP099 (100 nM). Pan-cytokeratin (PanCK) staining was used to mark cancer cells and exclude fibroblasts from the analysis. Quantification of invasion beyond 100 μm (right), normalized to the total number of cells/region, or proliferation, normalizing the number of Ki67-positive nuclei to the total number of cells/region ( n = 24 regions per cell line pooled from four biological replicates; one-way ANOVA with a Tukey correction for multiple comparisons). Scale bars, 100 μm. d , A scheme for the basement membrane invasion assay. e , Left: representative images of MM231 and MM468 cells invading into the basement membrane matrix for 4 or 5 days, respectively, in the presence of SHP099 (100 nM) or Sara (1 μM) (collagen I is labelled with HaloTag-CNA35 (magenta), fibroblasts by mScarlet expression (red) and all nuclei stained with DAPI (cyan); the cancer cells are apparent by nuclei staining alone (cyan-positive, mScarlet-negative cells)). Quantification of basement membrane invasion (MM231 cells, n = 31 (from 11 basement membranes; DMSO), 24 (from 9 basement membranes; Sara) and 23 (from 8 basement membranes; SHP099) regions pooled from three biological replicates (right); MM468 cells, n = 31 (from 12 basement membranes; DMSO), 32 (from 12 basement membranes; Sara) and 27 (from 9 basement membranes; SHP099) regions pooled from four biological replicates; one-way ANOVA with a Dunnett’s correction for multiple comparisons). Scale bars, 50 μm. f , Illusia-expressing MM231s (green) embedded in collagen I (magenta) and treated with SHP099 (100 nM) or Sara (1 μM) for 24 h (cell size, n = 119 (DMSO), 93 (Sara) and 85 (SHP099); solidity, n = 122 (DMSO), 91 (Sara) and 94 (SHP099) cells from four biological replicates; one-way ANOVA with a Dunnett’s correction). The boxplots represent the median and IQR. The whiskers extend to the minimum and maximum values. The grey areas highlight the IQR of the control conditions. NS, not significant. * P < 0.05, *** P < 0.001. .

Article Snippet: Frequency-domain FLIM–FRET was performed using a LIFA fast frequency-domain FLIM system (Lambert Instruments) attached to an inverted microscope (Zeiss AXIO Observer.D1) with sinusoidally modulated (40 MHz) epi-illumination (1 W for 405 nm or 3 W for 470 nm) from a temperature-stabilized multi-light-emitting diode (LED) system (Lambert Instruments) and a ×63/1.15 objective (Zeiss, Objective LD C-Apochromat ×63/1.15 W Corr M27).

Techniques: Stable Transfection, Expressing, Staining, Membrane, Invasion Assay, Control

Journal: Nature Cell Biology

Article Title: Dynamic regulation of integrin β1 phosphorylation supports invasion of breast cancer cells

doi: 10.1038/s41556-025-01663-4

Figure Lengend Snippet: a , Representative BiFC-FLIM-FRET images (left) and quantification of apparent FRET efficiency (right) from MM231 cells transfected with Venus or ITGB1-V1/ Dok1-V2 and either mScarlet-tagged Annexin A6 or VPS35 (n = 61 for each Annexin A6 condition, 58 (VPS35, Venus) and 60 (VPS35, BiFC) cells pooled from three biological replicates; significance assessed using an unpaired two-tailed Student’s t -test with a Welch’s correction; *** p < 0.001). Scale bars, 20 μm. b , Schematic of an invadopodium degrading the ECM. c , Representative images (left) and quantification (right) of MM231 ITGB1(WT or YYFF) cells with doxycycline(Dox)-inducible Src(E378G), treated (+/-)Dox overnight and then seeded on fluorescent gelatin (green) for 6 h (white, SiR-Actin stain; blue, DAPI nuclear stain; n = 30 [ITGB1 (WT) (-)Dox], 41 [ITGB1 (WT) ( + )Dox], 34 [ITGB1(YYFF) (-)Dox] and 41 [ITGB1(YYFF) ( + )Dox] fields of view pooled from three biological replicates; significance assessed using a one-way ANOVA with a Šidák correction for multiple comparisons; NS, not significant, *** p < 0.001). Scale bars, 20 μm. Source data and exact p-values are provided in the statistical source data file. Boxplots represent median and interquartile range. Whiskers extend to min and max values. Grey areas on boxplots highlight the interquartile range of the control conditions.

Article Snippet: Frequency-domain FLIM–FRET was performed using a LIFA fast frequency-domain FLIM system (Lambert Instruments) attached to an inverted microscope (Zeiss AXIO Observer.D1) with sinusoidally modulated (40 MHz) epi-illumination (1 W for 405 nm or 3 W for 470 nm) from a temperature-stabilized multi-light-emitting diode (LED) system (Lambert Instruments) and a ×63/1.15 objective (Zeiss, Objective LD C-Apochromat ×63/1.15 W Corr M27).

Techniques: Transfection, Two Tailed Test, Staining, Control

Journal: Nature Cell Biology

Article Title: Dynamic regulation of integrin β1 phosphorylation supports invasion of breast cancer cells

doi: 10.1038/s41556-025-01663-4

Figure Lengend Snippet: a , A schematic of the interaction between ITGB1-V1 and a V2-tagged adaptor or regulator, highlighting the resulting V1/V2 (Venus) protein complex as the restored epitope for BiCAP (top) or donor for BiFC–FLIM–FRET (bottom). The Venus tag alone was used as a control for the BiCAP and BiFC–FLIM–FRET experiments. b , A representative BiCAP immunoblot after HEK293T cell transfection with either Venus or ITGB1-V1/Dok1-V2 ( n = 3 biological replicates). c , Representative BiCAP immunoblots from MM231 and HEK293T cells where annexin A6 (AnxA6), VPS35 and Cofilin coimmunoprecipitate with the Dok1/ITGB1 complex ( n = 3 biological replicates). d , Representative BiFC–FLIM–FRET images (left) and quantification of apparent FRET efficiency (right) from MM231 cells transfected with RFP-tagged Cofilin mutants WT, S3A and S3E (Venus, n = 73 (WT), 72 (S3A) and 64 (S3E); BiFC, n = 92 (WT), 64 (S3A) and 64 (S3E) cells from three biological replicates; one-way analysis of variance (ANOVA) with a Tukey correction for multiple comparisons). Scale bars, 20 μm. e , Representative images (left) and quantification of gelatin degradation (right) by MM231 ITGB1(WT or YYFF) cells with Dox-inducible Src(E378G) expression. The MM231 cells were transfected with siRNAs against Dok1 (siDok1_1 and siDok1_2) or a NTC siRNA and treated with Dox for 24 h before being seeded on fluorescent gelatin (green) for 6 h (actin labelled with SiR-actin (white), nuclei with DAPI (blue)) (ITGB1(WT), n = 36 (NTC), 38 (siDok1_1) and 36 (siDok1_2); ITGB1(YYFF), n = 38 (NTC), 34 (siDok1_1) and 37 (siDok1_2) fields of view pooled from three biological replicates; one-way ANOVA with a Šidák correction for multiple comparisons). Scale bars, 20 μm; insets, 10 μm. The boxplots represent the median and IQR. The whiskers extend to the minimum and maximum values. The grey areas highlight the IQR of the control conditions. NS, not significant. *** P < 0.001. .

Article Snippet: Frequency-domain FLIM–FRET was performed using a LIFA fast frequency-domain FLIM system (Lambert Instruments) attached to an inverted microscope (Zeiss AXIO Observer.D1) with sinusoidally modulated (40 MHz) epi-illumination (1 W for 405 nm or 3 W for 470 nm) from a temperature-stabilized multi-light-emitting diode (LED) system (Lambert Instruments) and a ×63/1.15 objective (Zeiss, Objective LD C-Apochromat ×63/1.15 W Corr M27).

Techniques: Control, Western Blot, Transfection, Expressing

Journal: Nature Cell Biology

Article Title: Dynamic regulation of integrin β1 phosphorylation supports invasion of breast cancer cells

doi: 10.1038/s41556-025-01663-4

Figure Lengend Snippet: a – d , Representative images (left) and quantification of apparent FRET efficiency (right) for intermolecular FLIM–FRET of the following tagged protein pairs, Dok1–Clover/Cofilin–mRFP ( a ), CTTN–mEmerald/Dok1–mScarlet ( b ), Dok1–Clover/mScarlet–TKS5 ( c ) and CTTN–mEmerald/Cofilin–mRFP ( d ). FRET between mScarlet and the donor-tagged protein was used as a negative control for all pairs (for a , n = 85 (Dok1/mScarlet) and 95 (Dok1/Cofilin) cells pooled from five biological replicates; for b , n = 65 (CTTN/mScarlet) and 68 (CTTN/Dok1) cells pooled from three biological replicates; for c , n = 62 cells for each condition pooled from three biological replicates; for d , n = 65 (CTTN/mScarlet) and 70 (CTTN/Cofilin) cells pooled from three biological replicates; unpaired two-tailed Student’s t -test with a Welch’s correction). Scale bars, 20 μm. e , Representative images of mice with MM231 ITGB1(WT or YYFF) cells stably expressing the luciferase/EGFP construct. Oral gavage of Vehicle or SHP099 (100 mg kg −1 ) proceeded for 5 days from the day of injection. f , A box and whisker plot highlighting the endpoint metastatic burden as an average (Avg) radiance value from the luciferase signal of the MM231 cells in e ( n = 9 mice tracked per group). g , Representative lung sections stained for EGFP-positive MM231 cells. Scale bars, 2 mm; insets: 200 μm. h , Quantification of pulmonary nodule number (that is, clusters of greater than ten cells) in lungs from EGFP-positive MM231 cells ( n = 10 mice per group). i , Quantitative real-time PCR of the RNA samples collected from the MM231 ITGB1(WT or YYFF) cells stably expressing the luciferase/EGFP construct. The mice were designated as either ‘metastatic’ or ‘low signal’ after setting a threshold for ‘metastatic’ as having an expression fold change >1 compared with the mean of the WT/vehicle control with human GAPDH normalized to mouse/human GAPDH ( n = 10 mice/group). The boxplots represent the median and IQR. The whiskers extend to min and max values. The grey areas highlight the IQR of the control conditions. *** P < 0.001. .

Article Snippet: Frequency-domain FLIM–FRET was performed using a LIFA fast frequency-domain FLIM system (Lambert Instruments) attached to an inverted microscope (Zeiss AXIO Observer.D1) with sinusoidally modulated (40 MHz) epi-illumination (1 W for 405 nm or 3 W for 470 nm) from a temperature-stabilized multi-light-emitting diode (LED) system (Lambert Instruments) and a ×63/1.15 objective (Zeiss, Objective LD C-Apochromat ×63/1.15 W Corr M27).

Techniques: Negative Control, Two Tailed Test, Stable Transfection, Expressing, Luciferase, Construct, Injection, Whisker Assay, Staining, Real-time Polymerase Chain Reaction, Control

Journal: bioRxiv

Article Title: Exploration of mScarlet for development of a red lifetime sensor for calcium imaging

doi: 10.1101/2024.12.22.628354

Figure Lengend Snippet:

Article Snippet: For lifetime measurements, we used a Lambert Instruments FLIM Attachment (LIFA) setup, composed of an Eclipse Ti microscope (Nikon) with a Lambert Instruments Multi-LED for excitation, a LI2CAM camera, a LIFA signal generator (all Lambert Instruments) to synchronize the light source and the camera, and was controlled by the LI-FLIM software (version 1.2.13).

Techniques: Fluorescence, Standard Deviation

Journal: bioRxiv

Article Title: Exploration of mScarlet for development of a red lifetime sensor for calcium imaging

doi: 10.1101/2024.12.22.628354

Figure Lengend Snippet: The fluorescence lifetime was measured in bacterial lysate in the presence (0.1 mM CaCl 2 ) and absence (9.5 mM EDTA) of calcium at room temperature. The difference in phase and modulation lifetime between the two conditions (+Ca 2+ minus −Ca 2+ ) is plotted against the average intensity of the lysates in the two conditions. Each point represents the measurement of a single lysate originating from a single colony. Variants indicated by a cube were rationally designed previously, variants indicated by a dot are newly found in the screen and variants indicated by a gray circle were not sequenced.

Article Snippet: For lifetime measurements, we used a Lambert Instruments FLIM Attachment (LIFA) setup, composed of an Eclipse Ti microscope (Nikon) with a Lambert Instruments Multi-LED for excitation, a LI2CAM camera, a LIFA signal generator (all Lambert Instruments) to synchronize the light source and the camera, and was controlled by the LI-FLIM software (version 1.2.13).

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Exploration of mScarlet for development of a red lifetime sensor for calcium imaging

doi: 10.1101/2024.12.22.628354

Figure Lengend Snippet: The fluorescence lifetime was measured in bacterial lysates in the presence (0.1 mM CaCl 2 ) and absence (9.5 mM EDTA) of calcium at room temperature. The difference in phase and modulation lifetime between the two conditions (+Ca 2+ minus −Ca 2+ ) is plotted against the average intensity of the lysates in the two conditions. Each point represents the measurement of a single lysate originating from a single colony. Colors and shapes are indicative of the mutations, except for variants indicated by a cube: these were rationally designed previously or found in the previous screen. Mutations are indicated by letters in the legend, f.e. 5511mut C_I has mutations A30C and L266I. Variants indicated by a gray circle are non-sequenced mutants.

Article Snippet: For lifetime measurements, we used a Lambert Instruments FLIM Attachment (LIFA) setup, composed of an Eclipse Ti microscope (Nikon) with a Lambert Instruments Multi-LED for excitation, a LI2CAM camera, a LIFA signal generator (all Lambert Instruments) to synchronize the light source and the camera, and was controlled by the LI-FLIM software (version 1.2.13).

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Exploration of mScarlet for development of a red lifetime sensor for calcium imaging

doi: 10.1101/2024.12.22.628354

Figure Lengend Snippet: The fluorescence lifetime was measured in bacterial lysates in the presence (0.1 mM CaCl 2 ) and absence (9.5 mM EDTA) of calcium at room temperature. The difference in phase and modulation lifetime between the two conditions (+Ca 2+ minus −Ca 2+ ) is plotted against the average intensity of the lysates in the two conditions. The screen was performed on two days and data of these days are plotted separately. Each point represents the measurement of a single lysate originating from a single colony. Colors and shapes are indicative of mutations, except for variants indicated by a cube: these were rationally designed previously. Mutations are indicated by letters in the legend, f.e. 5496mut G_Y has mutations S30G and L265Y. Mutant sensors indicated by a gray circle were not sequenced.

Article Snippet: For lifetime measurements, we used a Lambert Instruments FLIM Attachment (LIFA) setup, composed of an Eclipse Ti microscope (Nikon) with a Lambert Instruments Multi-LED for excitation, a LI2CAM camera, a LIFA signal generator (all Lambert Instruments) to synchronize the light source and the camera, and was controlled by the LI-FLIM software (version 1.2.13).

Techniques: Fluorescence, Mutagenesis

Journal: bioRxiv

Article Title: Exploration of mScarlet for development of a red lifetime sensor for calcium imaging

doi: 10.1101/2024.12.22.628354

Figure Lengend Snippet: Bacteria expressed simultaneously a red candidate sensor and mTq2 coded on a pFR plasmid. Red and cyan fluorescence, and the fluorescence lifetime of the red candidate sensor were measured in bacterial lysate in the presence (0.1 mM CaCl 2 ) and absence (9.5 mM EDTA) of calcium at room temperature. The difference in phase and modulation lifetime (Δτ ϕ and Δτ M ) between the two conditions (+Ca 2+ minus −Ca 2+ ) are plotted on the x-axis. Red fluorescence was divided over the cyan fluorescence (RFP/CFP) and the average is plotted on the y-axis. Each point represents the measurement of a single lysate originating from a single colony. Colors and shapes are indicative of the mutations as indicated in the legend, f.e. 5511mut LV_IA and has mutations E29L and G267A with respect to 5511mut V_I. Mutant sensors indicated by a gray circle were not sequenced. Of two earlier variants, 5511 and 5496, no RFP/CFP ratio was measured as these were not expressed with pFR.

Article Snippet: For lifetime measurements, we used a Lambert Instruments FLIM Attachment (LIFA) setup, composed of an Eclipse Ti microscope (Nikon) with a Lambert Instruments Multi-LED for excitation, a LI2CAM camera, a LIFA signal generator (all Lambert Instruments) to synchronize the light source and the camera, and was controlled by the LI-FLIM software (version 1.2.13).

Techniques: Bacteria, Plasmid Preparation, Fluorescence, Mutagenesis

Journal: bioRxiv

Article Title: Exploration of mScarlet for development of a red lifetime sensor for calcium imaging

doi: 10.1101/2024.12.22.628354

Figure Lengend Snippet: Bacteria expressed simultaneously a red candidate sensor and mTq2 coded on a pFR plasmid. Red and cyan fluorescence, and the fluorescence lifetime of the red candidate sensors were measured in bacterial lysate in the presence (0.1 mM CaCl 2 ) and absence (9.5 mM EDTA) of calcium at room temperature. The difference in phase and modulation lifetime (Δτ ϕ and Δτ M ) between the two conditions (+Ca 2+ minus −Ca 2+ ) are plotted on the x-axis. Red fluorescence was divided over the cyan fluorescence (RFP/CFP) and the average RFP/CFP ratio of both states is plotted on the y-axis. Each point represents the measurement of a single lysate originating from a single colony. Colors and shapes are indicative of the mutations as indicated in the legend, f.e. 5496mut IG_YP and has mutations L29I and G266P with respect to 5496mut G_Y. Mutant sensors indicated by a gray circle were not sequenced. Of two earlier variants, 5511 and 5496, no RFP/CFP ratio was measured as these were not expressed with pFR

Article Snippet: For lifetime measurements, we used a Lambert Instruments FLIM Attachment (LIFA) setup, composed of an Eclipse Ti microscope (Nikon) with a Lambert Instruments Multi-LED for excitation, a LI2CAM camera, a LIFA signal generator (all Lambert Instruments) to synchronize the light source and the camera, and was controlled by the LI-FLIM software (version 1.2.13).

Techniques: Bacteria, Plasmid Preparation, Fluorescence, Mutagenesis