Sp5 2 laser scanning microscope

Confocal imaging was performed using a Perkin Elmer VoX-1000 Spinning Disk microscope equipped with a 60 ×/1.4 NA/oil CFI Apochromat TIRF objective, four laser lines (405, 488, 561 and 635 nm) and a Hamamatsu EMCCD camera (C9100-50). The following filter sets were used: excitation: quad-bandpass 405/488/568/640 nm with the matching emission filters for DAPI (445/30 nm), Alexa Fluor 488 (500–548 nm), TRITC (526–623 nm) and Cy5 (664–750 nm). For higher special resolution, images were acquired using a Leica SP5 II laser scanning microscope using a 100 × 1.44 NA HCX PL APO Objective with a pixel size of 86.6 nm and a z-spacing of 125 nm for subsequent deconvolution. For imaging the 405, 488, 561 and 633 nm laser line and spectral detection windows of 425–465 nm (DAPI), 495–558 nm (Alexa 488), 600–660 nm (Alexa 594) and 640–705 nm (Cy5) were used. Images were then deconvolved with wavelength specific point spread functions using ImageJ and the Iterative Deconvolution 3D plugin [58 ]. In addition, a Zeiss Axiovert 200 with a 100 ×/1.4 NA/oil Plan-Apochromat objective was used to image metaphase spreads. Images were recorded using a Zeiss Axiocam mRM, and the following filters were used: DAPI; ex: 350/50 nm; bs: 400 nm; em: 460/50 nm and Alexa Fluor 488: ex: 482/18 nm; bs: 495 nm; 520/28 nm.

Confocal microscopy imaging was performed using a Leica SP5-II laser scanning microscope attached to a Leica DMI6000 inverted epifluorescence microscope and equipped with a ×63 oil immersion lens, 1.4 NA. Images of the microparticles were recorded on fields of view containing groups of 5–20 particles. Two to three fields of view were recorded for each experiment. Experiments were repeated at least three times. Approximately 95% of the stained droplets were consistent with the images shown in the main text.