Microscopy and image acquisition

3.5.1 Sample preparation during live platelet imaging

The SiR-actin or SiR-tubulin labeled or CM Green stained platelet samples were prepared at the microscope (see section 3.5.2) shortly before starting the live imaging. The fibrinogen- coated coverslips were washed thrice with 100 µL warm HT-BSA buffer and were anchored in the coverslip holder that was placed inside a preheated, humid, stage top incubator (see section 3.5.2 for details). Before adding the platelets, traces of liquid on the coverslips were gently blotted out with a piece of tissue. Then 90 µL of the labeled and/or treated platelet solution was pipetted onto the coverslips. This step was followed by the addition of 10 µL of thrombin solution (final concentration of 4 U/mL, thrombin from human plasma, 1 KU, ≥

53 2800 NIH units/mg protein, Sigma-Aldrich, Munich, Germany) to trigger the platelet spreading. Details of imaging are described in the following sections.

3.5.2 Bright-field imaging and epifluorescence imaging

The imaging of both fixed and live platelets was performed using an inverted microscope (IX 81, Olympus, Hamburg, Germany) with built-in shutters that was equipped for both bright-field and epifluorescence imaging in various ranges of magnifications (10X - 100X) and was also equipped with a digital camera for recording the imaging. The SiR-actin live spreading experiments and the fixed-platelet time series (see section 3.3.1) were recorded with a charge-coupled device (CCD) Retiga 6000 camera (QImaging, Surrey, BC, Canada). Some of the SiR-actin and all the SiR-tubulin live experiments with the pharmacological inhibitors were recorded with a complementary metal-oxide semiconductor (CMOS) ORCA-Flash 4.0 V2 C11440-22CU camera (Hamamatsu Photonics Deutschland GmbH, Herrsching am Ammersee, Germany).

For imaging both, the fixed-platelet time series, and the SiR-actin and SiR-tubulin live, time-lapse movies of platelet spreading and cytoskeletal reorganization, a 60X oil immersion objective (UPLSAPO 60XO, numerical aperture NA = 1.35, working distance WD = 0.15 mm, Olympus, Hamburg, Germany) was used.

During live imaging of platelets, a stage top incubator (INUG2E-ONICS, Tokai Hit, Shizuoka-ken, Japan) was mounted on top of the microscope stage. The incubator and the 60X objective were preheated to 37°C and the atmosphere inside the incubator was saturated with water vapor and 5% CO2, to maintain physiological conditions for the platelets.

For the SiR-actin/SiR-tubulin time-lapse experiments, the platelet plasma membrane was visualized by either bright-field (BF; in case of singly labeled SiR-actin/SiR-tubulin platelets) or by epifluorescence in the FITC channel (in case of CM Green stained platelets). The F-actin/MT reorganization was subsequently visualized by epifluorescence imaging in the Cy5 channel. In few of the experiments, instead of imaging the platelet plasma membrane, the underlying labeled fibrinogen coating was imaged in the FITC channel together with the F-actin/MT reorganization in the Cy5 channel.

A sketch of our microscope setup is shown in Figure 3.3. A halogen lamp (12 V, 100 W, Olympus, Hamburg, Germany) provided the illumination source for the bright-field imaging.

The transmitted light is focused on the platelet samples via a condenser and magnified by the objective. For the epifluorescence imaging, the MT20 illumination system with a 150 W

54 xenon arc lamp provided the illumination source (Olympus, Hamburg, Germany). During the imaging, a FITC/Cy5- dualband filterset (AHF Analysentechnik, Tübingen, Germany) was used which consisted of an excitation filter (470/622 Dualband Exciter H) that allowed for excitation of the fluorescently labeled platelet samples around 470 nm (FITC) and a second one around 622 nm (Cy5). The excitation light was further filtered by a dichroic mirror/beam splitter (497/655 H Dualband Beamsplitter) which selectively allowed light only above 497 nm and light only above 655 nm to pass through. An emission filter (537/694 Dualband Emitter H) selectively allowed the emission from the samples around 537 nm and around 694 nm. The spectra of the optical filters, the probes, and the dyes used are shown in Figure 3.4.

The imaging parameters for the SiR-actin/SiR-tubulin-labeled platelet time-lapse movies in Cy5, FITC (fibrinogen coating) and BF channels were 50 ms of illumination time, with epifluorescence lamp intensity of 23.1% and BF lamp power of 4 V. The imaging parameters for the CM Green-stained platelet time-lapse movies in Cy5 (SiR-actin) and FITC (CM Green) channels were 36 ms of illumination time, with epifluorescence lamp intensity of 11.49%. Time-lapse images of both the plasma membrane or fibrinogen coating (in BF or FITC channel) and the F-actin/MT (in the Cy5 channel) were simultaneously recorded, with an interval of 10 seconds between each frame and for a total time period of 60-120 minutes.

The imaging parameters for the fixed-platelet time series in Cy5 (F-actin), FITC (vinculin/myosin) and BF channels were 100 ms of illumination time, with epifluorescence lamp intensity of 100 % and BF lamp power of 4 V.

55 Figure 3.3: Microscope setup with bright-field and epifluorescence imaging

Plasma membrane of platelets was imaged by bright-field microscopy which used a halogen lamp as the light source. The fluorescently labeled cytoskeletal components were imaged by epifluorescence microscopy which used a xenon arc lamp as the light source and highly selective excitation and emission filters. The images were recorded by a digital camera.

56 Figure 3.4: Spectra of the filters and fluorophores used for imaging

A) Excitation and emission spectra of STAR 635 phalloidin and vinculin/myosin secondary antibodies conjugated to Alexa Fluor 488 used for the fixed platelet time series. The spectral raw data values were obtained from Abberior and ThermoFisher [2, 2, 6]. B) Excitation and emission spectra of SiR-actin/SiR-tubulin used to visualize the F-actin/MT during live imaging. The spectral raw data values were obtained from Spirochrome [4]. C) Excitation and emission spectra of CM Green used to visualize platelet plasma membrane during live imaging. The graph was taken from [5] as the spectral raw data values were not available. D) The spectra of the dualband FITC/Cy5 filterset used for the platelet experiments to match the excitation and emission wavelengths of all the fluorophores used for imaging. The spectral raw data values were obtained from AHF Analysentechnik [3].

57

3.5.3 Bright-field and epifluorescence image processing

All time-lapse images and fixed-platelet images recorded with the microscope (see section 3.5.2) were processed to adjust their brightness and contrast, using ImageJ [130]. The fluorescence intensity of the SiR-actin and SiR-tubulin probes increases with increasing F-actin or MT content. During the initial spreading stages, the platelets had less F-F-actin/MT content which increased as they spread over time. Hence the time-lapse fluorescence images were not of equal brightness. Thus, each of the epifluorescence images was individually adjusted for their brightness and contrast whereas the brightness and contrast values for all BF images (belonging to one single dataset) were adjusted to the same values. For the fixed-platelet double staining series (see section 3.3.1), the fluorescence images in different channels (F-actin in Cy5 and vinculin/myosin in FITC) were overlayed in ImageJ to reveal the overlapping regions.