axion vert a1 inverted microscope  (Carl Zeiss)

Journal: bioRxiv

Article Title: Winding-Up of Fibrin Fibers as a Novel Mechanism of Platelet-Mediated Fiber Compaction

doi: 10.64898/2026.02.15.705975

Figure Lengend Snippet: Platelets become labelled by fluorescent fibrin fibers during clot retraction A) Time course of unconstrained clot retraction by platelets (4x10 in 400 μl of 12% plasma in PBS) for a total of 60 minutes at room temperature. Experiments have been performed 6x using blood from different donors and typical examples of blood from two different donors are shown (experiments III and IV are repetitions of I and II the day after; although a longer lag-phase is observed, the final retraction volume is similar for the four experiments). B) Unconstrained clot retraction (1x10 platelets per ml in 50% plasma/50% PBS) in presence of fibrinogen-Alexa 488. Image acquisition was started immediately after thrombin addition at a focal plane 100 μm above the bottom of the well and image stacks were collected (61 focal planes, step size 0.5 μm) for a time period of 20:27 min (100 frames with a time interval of 12.5 sec). See also associated video 1. The experiment has been performed twice using platelets of the same donor. Shown are the first, intermediate and last time points of a depth color-coded time-lapse video (left panel; scale bar 10 μm) and the maximal intensity projections (MIPs) of the same time points (right panel; scale bar 10 μm). C) Constrained clot retraction (1x10 platelets per ml in 50% plasma/50% PBS, fibrinogen-Alexa 488 final concentration 12.5 μg/ml) between two holders. Clots were induced by addition of thrombin (2.5U/ml final), fixed at the indicated retraction times, embedded in gelatin, flash frozen and cryosections (14 μm) were stained for the integrin subunit αIIb (magenta; scale bar 5 μm). The time course was performed twice using PRP from two different donors (retraction assays were repeated, although not for all time points, more than eight times using blood from different donors, with consistent results). Image acquisition was performed using a wide-field epi fluorescence microscope (BX41; Olympus) equipped with a Plan 100x/1.25 NA oil objective, a camera (DP70; Olympus), and the acquisition software analySIS (Olympus).

Article Snippet: The geometric optical aberrations were corrected both in excitation and detection light paths in the open loop mode with a deformable mirror (AlpAO DM97-15) inserted between the confocal scanning head and the microscope inverted stand (Nikon Ti2E).

Techniques: Clinical Proteomics, Concentration Assay, Staining, Fluorescence, Microscopy, Software

Journal: bioRxiv

Article Title: Winding-Up of Fibrin Fibers as a Novel Mechanism of Platelet-Mediated Fiber Compaction

doi: 10.64898/2026.02.15.705975

Figure Lengend Snippet: Live imaging of spread platelets winding-up fluorescent fibrin fibers A-C: Shown are three experiments using blood from different donors. A) Washed platelets were adjusted to 5x10 platelets per 2 ml PBS and 5 μl plasma was added as well as fibrinogen-Alexa 488. Thrombin was added to induce fibrin fiber formation and platelet activation. After 10 min, the suspension was transferred into a petri dish (WPI Fluoro), centrifuged and installed in the microscope incubator at 37°C to start image acquisition (see also video 9, upper panel). The first and last time points of the time-lapse video are shown; acquired using the fluorescence and transmission channels. Projections of four focal fluorescent image planes of fibrin fibers (green) and projections of the transmission planes as well as the merged channels are shown. An arrow indicates a forming kink in a fibrin fiber (scale bar 10 μm). Below are shown Individual time points of the time-lapse video zooming on the rectangle indicated in the panels above. This region shows rotational fiber movements. B) Independent experiment under conditions as described in A except that the petri dish was kept at room temperature to slow down the platelet mediated fiber reorganizations (scale bar 10 μm). An arrow shows platelet mediated fiber compaction. Below are shown Individual time points of the time-lapse video zooming on the rectangle indicated in the panels above. This region shows a fibrin fiber getting curved by the platelet and finally ruptures followed by a rotational movement of the ruptured fiber. C) Platelets of a third donor (not washed, i.e., 5 μl PRP with 4x10 platelets per 2 ml PBS) and fibrinogen-Alexa 488 were added to 2 ml of PBS and the experiment was continued as in B (scale bar 10 μm). Below are shown Individual time points of the time-lapse video zooming on the rectangle indicated in the panels above. This region shows fibrin fibers getting coiled around the pseudo-nucleus of the spread platelet. The last zoomed image shows a temporal color-coded projection of the transmission channel to illustrate the rotational movement of the pseudo-nucleus (scale bar 5 μm). D) Samples shown in B and C were fixed 3h after the start of time-lapse acquisitions and stained for the integrin subunit aIIb. Shown are fibrin fibers (green) integrin staining (magenta) and the merge (samples B and C, upper and lower panels, respectively; scale bar 10 μm). E) Preformed fluorescent fibrin fibers were prepared in the absence of platelets. PRP was adjusted to 2.5x10 platelets per ml with PBS and 1800 μl was transferred into a petri dish (WPI Fluoro). The dish was centrifuged to allow spreading of the platelets and the supernatant was replaced by the preformed fibrin fibers. The dish was installed in the microscope incubator to start image acquisition (see also video 9, last panel). The experiment was performed twice with blood of the same donor. The last fluorescent image of the time-lapse video is shown (left image, scale bar 10 μm). Fifteen of the fibrin accumulations in the middle of the platelets are rotating in a counterclockwise direction (indicated as "1"), four rotate clockwise ("2") and for 16 no clearly visible turn is observed ("3"). A projection of all fluorescent time points, temporal color-coded using the LUT spectral, is shown. White corresponds to the sum of all the colors at each time point, meaning no movement during the time-lapse video. (right image, scale bar 10 μm).

Article Snippet: The geometric optical aberrations were corrected both in excitation and detection light paths in the open loop mode with a deformable mirror (AlpAO DM97-15) inserted between the confocal scanning head and the microscope inverted stand (Nikon Ti2E).

Techniques: Imaging, Clinical Proteomics, Activation Assay, Suspension, Microscopy, Fluorescence, Transmission Assay, Staining