custom built tirf microscope  (Nikon)

Journal: bioRxiv

Article Title: Metal-induced energy transfer uncovers activation-induced axial reorganization of signaling complexes inside cells

doi: 10.64898/2026.04.23.719849

Figure Lengend Snippet: (A) Schematic of the surface functionalization strategy: mEGFP-ALFAnb captured into PLL-ALFA bNDAs is stained with EN ATTO643 for MIET-based axial distance measurements. The MIET substrate comprised a 30 nm silica spacer. (B) Representative TIRF microscopy image of EN ATTO643 -stained bNDAs on a MIET substrate. Inset shows a magnified view of the marked region. Scale bars: 10 µm; inset: 1 µm. (C) Representative fluorescence lifetime images of EN ATTO643 on glass (left) and MIET substrates (right). Insets show magnified views of the marked region. Scale bars: 5 µm; insets: 1 µm. (D) Representative normalized fluorescence decay curves of ATTO643 on glass (top) and MIET (bottom) substrates. (E) Per-nanodot fluorescence lifetime distributions on MIET (red; n = 8098 nanodots) and glass substrates (grey; n = 6975 nanodots). Solid lines represent Gaussian fits. (F) Axial distance distribution of ATTO643 from the silica surface ( n = 8098 nanodots). Solid line represents Gaussian fit.

Article Snippet: Imaging was performed on the TIRF microscope described above at 25°C using 561 nm excitation in imaging buffer containing 50 pM Cy3B-conjugated imager strand F3 (Massive Photonics).

Techniques: Staining, Microscopy, Fluorescence

Journal: bioRxiv

Article Title: Metal-induced energy transfer uncovers activation-induced axial reorganization of signaling complexes inside cells

doi: 10.64898/2026.04.23.719849

Figure Lengend Snippet: (A) Schematic of the model transmembrane construct ALFAnb-mEGFP-TMD-HaloTag captured into PLL-ALFA NDAs via its extracellular ALFAnb. The extracellular mEGFP stained with EN ATTO643 and the cytosolic HaloTag labeled with HTL-JFX549 report axial distances d 1 and d 2 from the substrate surface, respectively. (B) Representative dual-color TIRF microscopy images showing mEGFP, HTL-JFX549, and merged fluorescence channels with corresponding intensity line profiles along the indicated white dashed lines. Insets show magnified views of the marked region. Scale bars: 10 µm; insets: 1 µm. (C) Representative raw fluorescence lifetime images of EN ATTO643 on glass and MIET substrates (20 nm silica spacer). Insets show magnified views of the marked region. Scale bars: 10 µm; insets: 1 µm. (D) Representative results from single-nanodot fluorescence lifetime analysis of the cell shown in C on glass and MIET substrates. Insets show magnified views of the marked region. Scale bars: 10 µm; insets: 1 µm. (E) Per-nanodot fluorescence lifetime distributions on MIET substrates for EN ATTO643 (red; n = 3428 nanodots, 9 cells) and HTL-JFX549 (magenta; n = 3022 nanodots, 7 cells). Solid lines represent Gaussian fits. (F) MIET calibration curves for EN ATTO643 (red) and HTL-JFX549 (magenta). Dashed lines indicate the axial distances d 1 and d 2 corresponding to the measured lifetimes. (G) Axial distance distributions of EN ATTO643 ( d 1 , red) and HTL-JFX549 ( d 2 , magenta) from the silica surface. Solid lines represent Gaussian fits. Δd marks the axial separation of both fluorescent reporters across the plasma membrane.

Article Snippet: Imaging was performed on the TIRF microscope described above at 25°C using 561 nm excitation in imaging buffer containing 50 pM Cy3B-conjugated imager strand F3 (Massive Photonics).

Techniques: Construct, Staining, Labeling, Microscopy, Fluorescence, Clinical Proteomics, Membrane

Journal: bioRxiv

Article Title: Metal-induced energy transfer uncovers activation-induced axial reorganization of signaling complexes inside cells

doi: 10.64898/2026.04.23.719849

Figure Lengend Snippet: (A) Schematic of the experimental design for probing activation-dependent axial changes of the GP130 IDR in bNDAs. ALFAnb-GP130ΔECD-mEGFP and JAK1-HT labeled with HTL-JFX549 are co-recruited into PLL-ALFA bNDAs, with kinase activity controlled by Ruxo (i) and tyrosine phosphorylation (pTyr, orange) detected with an anti-pTyr antibody (pTyrAb-Dy647) (ii). (B) Representative TIRF microscopy images showing ALFAnb-GP130ΔECD-mEGFP, JAK1-HT labeled with HTL-JFX549, and pTyrAb-Dy647 staining in the presence (+Ruxo, top) and absence (-Ruxo, bottom) of Ruxolitinib. Insets show magnified views of the marked regions. Scale bars: 10 µm; insets: 1 µm. (C) Single-nanodot correlation analysis of GP130ΔECD-mEGFP and JAK1-HT (HTL-JFX549) fluorescence intensities, color-coded by pTyrAb-Dy647 intensity, for +Ruxo (top; n = 7729 nanodots, 15 cells) and -Ruxo (bottom; n = 8174 nanodots, 21 cells) conditions. (D) Representative results from single-nanodot fluorescence lifetime analysis of EN ATTO643 on MIET substrates (20 nm silica spacer) in the presence (+Ruxo, top) and absence (-Ruxo, bottom) of the inhibitor. Insets show magnified views of the marked regions. Scale bars: 10 µm; insets: 1 µm. (E) Per-nanodot fluorescence lifetime distributions under +Ruxo (grey; n = 4460 nanodots, 9 cells) and -Ruxo (red; n = 2689 nanodots, 5 cells) conditions. Solid lines represent Gaussian fits. (F) Axial distance distributions for +Ruxo (grey) and -Ruxo (red) conditions, calculated from the fluorescence lifetimes shown in (E). Solid lines represent Gaussian fits. (G) Model of the three axial states of the GP130 IDR: the resting state with bound JAK1(FS) (10.5 nm), the Ruxo-inhibited state with full-length JAK1 (11.2 nm), and the activated state (6.8 nm). Distances refer to the IDR C-terminus above the inner plasma membrane leaflet.

Article Snippet: Imaging was performed on the TIRF microscope described above at 25°C using 561 nm excitation in imaging buffer containing 50 pM Cy3B-conjugated imager strand F3 (Massive Photonics).

Techniques: Activation Assay, Labeling, Activity Assay, Phospho-proteomics, Microscopy, Staining, Fluorescence, Clinical Proteomics, Membrane