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(A–C) FILM acquisition of binder probes that recognize <t>CD2,</t> ICAM-1 (CD54) in the pSMAC, and CD45 in the dSMAC zones (10 nm pixels with 3-pixel Gaussian blurring). For CD2 and CD45, APC-conjugated antibodies were used; for ICAM-1, a nanobody Fab conjugated with Vio ® 667 was used. Cyan boxed region shows the IF image (103.17 nm pixels) corresponding to the white boxed region. (D–F) Zoom-in of the respective boxed regions for the panels to the left. Localizations are shown as peak-normalized 2D Gaussians with color-coded precision according to the colorbar. (G–I) Zoom-in of the respective boxed regions for the panels to the immediate left. Localizations are shown as crosses (indicating ±1-σ uncertainty in x and y ) with color-coded precision according to the colorbar.
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(A–C) FILM acquisition of binder probes that recognize <t>CD2,</t> ICAM-1 (CD54) in the pSMAC, and CD45 in the dSMAC zones (10 nm pixels with 3-pixel Gaussian blurring). For CD2 and CD45, APC-conjugated antibodies were used; for ICAM-1, a nanobody Fab conjugated with Vio ® 667 was used. Cyan boxed region shows the IF image (103.17 nm pixels) corresponding to the white boxed region. (D–F) Zoom-in of the respective boxed regions for the panels to the left. Localizations are shown as peak-normalized 2D Gaussians with color-coded precision according to the colorbar. (G–I) Zoom-in of the respective boxed regions for the panels to the immediate left. Localizations are shown as crosses (indicating ±1-σ uncertainty in x and y ) with color-coded precision according to the colorbar.
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(A–C) FILM acquisition of binder probes that recognize <t>CD2,</t> ICAM-1 (CD54) in the pSMAC, and CD45 in the dSMAC zones (10 nm pixels with 3-pixel Gaussian blurring). For CD2 and CD45, APC-conjugated antibodies were used; for ICAM-1, a nanobody Fab conjugated with Vio ® 667 was used. Cyan boxed region shows the IF image (103.17 nm pixels) corresponding to the white boxed region. (D–F) Zoom-in of the respective boxed regions for the panels to the left. Localizations are shown as peak-normalized 2D Gaussians with color-coded precision according to the colorbar. (G–I) Zoom-in of the respective boxed regions for the panels to the immediate left. Localizations are shown as crosses (indicating ±1-σ uncertainty in x and y ) with color-coded precision according to the colorbar.
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(A–C) FILM acquisition of binder probes that recognize <t>CD2,</t> ICAM-1 (CD54) in the pSMAC, and CD45 in the dSMAC zones (10 nm pixels with 3-pixel Gaussian blurring). For CD2 and CD45, APC-conjugated antibodies were used; for ICAM-1, a nanobody Fab conjugated with Vio ® 667 was used. Cyan boxed region shows the IF image (103.17 nm pixels) corresponding to the white boxed region. (D–F) Zoom-in of the respective boxed regions for the panels to the left. Localizations are shown as peak-normalized 2D Gaussians with color-coded precision according to the colorbar. (G–I) Zoom-in of the respective boxed regions for the panels to the immediate left. Localizations are shown as crosses (indicating ±1-σ uncertainty in x and y ) with color-coded precision according to the colorbar.
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(A–C) FILM acquisition of binder probes that recognize CD2, ICAM-1 (CD54) in the pSMAC, and CD45 in the dSMAC zones (10 nm pixels with 3-pixel Gaussian blurring). For CD2 and CD45, APC-conjugated antibodies were used; for ICAM-1, a nanobody Fab conjugated with Vio ® 667 was used. Cyan boxed region shows the IF image (103.17 nm pixels) corresponding to the white boxed region. (D–F) Zoom-in of the respective boxed regions for the panels to the left. Localizations are shown as peak-normalized 2D Gaussians with color-coded precision according to the colorbar. (G–I) Zoom-in of the respective boxed regions for the panels to the immediate left. Localizations are shown as crosses (indicating ±1-σ uncertainty in x and y ) with color-coded precision according to the colorbar.

Journal: bioRxiv

Article Title: Ångström Resolution with Flow Immunofluorescence Localization Microscopy (FILM)

doi: 10.64898/2025.12.16.694546

Figure Lengend Snippet: (A–C) FILM acquisition of binder probes that recognize CD2, ICAM-1 (CD54) in the pSMAC, and CD45 in the dSMAC zones (10 nm pixels with 3-pixel Gaussian blurring). For CD2 and CD45, APC-conjugated antibodies were used; for ICAM-1, a nanobody Fab conjugated with Vio ® 667 was used. Cyan boxed region shows the IF image (103.17 nm pixels) corresponding to the white boxed region. (D–F) Zoom-in of the respective boxed regions for the panels to the left. Localizations are shown as peak-normalized 2D Gaussians with color-coded precision according to the colorbar. (G–I) Zoom-in of the respective boxed regions for the panels to the immediate left. Localizations are shown as crosses (indicating ±1-σ uncertainty in x and y ) with color-coded precision according to the colorbar.

Article Snippet: α-tubulin (REA1136 REAfinityTM, h, APC, #130-119-541, Miltenyi Biotec), CD2 (REA972 REAfinityTM, h, APC, #130-116-150, Miltenyi Biotec), CD20 (REA780 REAfinityTM, h, APC, #130-111-339, Miltenyi Biotec), CD28 (REA612 REAfinityTM, h, APC, #130-118-343, Miltenyi Biotec), CD45 (REA1023 REAfinityTM, h/nhp, APC, #130-117-191, Miltenyi Biotec), CD54/ICAM-1 (REA266 REAfinityTM, h, APC, #130-120-711, Miltenyi Biotec), CD81 (REA513 REAfinityTM, h, APC, #130-119-787, Miltenyi Biotec), CD107a/LAMP1 (REA792 REAfinityTM, h, APC, #130-111-847, Miltenyi Biotec), IgD (REA740 REAfinityTM, h, APC, #130-110-644, Miltenyi Biotec), IgM (REAL689 REAdye_leaseTM, h, PE, #130-125-786, Miltenyi Biotec), Pan-Cytokeratin (REA1141 REAfinityTM, h, APC, #130-120-096, Miltenyi Biotec), β-catenin (REA480 REAfinityTM, h, APC, #130-124-444, Miltenyi Biotec), TCRα/β (REA652 REAfinityTM, h, APC, #130-113-535, Miltenyi Biotec), TOM22 (REA1185 REAfinityTM, APC, h/ms, #130-122-079, Miltenyi Biotec), DAPI Staining Solution (#130-111-570, Miltenyi Biotec), and ATTO 643 phalloidin (ATT-AD643-81, ATTO-TEC).

Techniques: