Immersol 518f oil

The phantom is fabricated using two-photon laser lithography, in which a focused laser beam is scanned within liquid resin. The resin within the laser’s focal volume is locally polymerized. Adjusting the scanning trajectory and the exposure time of the laser beam enables simultaneous control over the 3D printed geometry (accuracy at the order of 100 nm) and RI (accuracy at the order of $5\times {10}^{-4}$ , maximal $\mathrm{\Delta }$ RI = 0.03 within the structure) in three dimensions. We used Photonic Professional GT (Nanoscribe GmbH) equipped with a 1.3 NA 100 $\times$ microscope objective and piezo scanning stage. The phantom is fabricated in the IP-Dip resin (Nanoscribe GmbH) on top of a #1.5H coverslip (dip-in configuration⁵⁴ ). After fabrication the structure was developed in PGMEA (Propylene glycol monomethyl ether acetate; 12 min), followed by isopropyl alcohol (10 min) and then blow-dried. The full methodology for fabrication and validation of the features can be found in our previous work^{33 (link)}.
To conduct our TPM imaging experiments, the microphantom was immersed in Zeiss Immersol 518F oil (RI ${}_{632}$ nm = 1.5123), which provides similar RI contrast as in the case of cells immersed in culture medium. By using immersion oils with varying RI, it is possible to adjust the scattering properties of the microphantom post-fabrication.

L4 stage or young adult worms were mounted on 2% agarose pads with 10 mM levamisole (CAS 16595-80-5; Sigma-Aldrich). A Zeiss LSM i880 microscope with Airyscan equipped with 40× Fluar objective (N.A. 1.3) or 60× Plan-apochromat objective (N.A. 1.4) using Immersol 518F oil (Carl Zeiss) was used to capture both the fluorescent and differential interference contrast (DIC) images at super-resolution. Images were viewed and processed with ZEN software. GFP was excited at 488 nm, and RFP or TagRFP was excited at 561 nm. GFP emission was captured with the BP495-550 filter, and RFP or TagRFP emission was captured with the LP570 filter. To determine the subcellular localization of GFP::TSP-14A, and the co-localization of TSP-14A with TSP-12, quantitative colocalization analysis was performed using the JACoP plugin of the open-source Fiji software [40 (link)]. For each image, the Costes threshold regression was used as the reference to establish a threshold, and three randomly selected square regions were chosen for each image pair. For each genotype, more than 10 worms were imaged and analyzed. Co-localization analysis was conducted by calculating both Pearson’s correlation coefficient (PCC) and Mander’s overlap coefficient (MOC) [41 (link)].

May-Grunwald Giemsa staining was used to analyse megakaryocyte differentiation, and erythrocyte differentiation. Cytospins were prepared from 5.0 x 10⁴ differentiating megakaryocytes and were fixed in methanol for 3 minutes. After fixation, cytospins were stained in a 50% eosin methylene blue solution according to May-Grunwald (Sigma Aldrich, Seelze, Germany) for 15 minutes, rinsed in water for 5 seconds, and nuclei were counterstained with 10% Giemsa solution (Merck kGaA, Darmstadt, Germany) for 20 minutes. Megakaryocyte maturation can be characterised by polyploidisation. Erythrocyte differentiation can be characterised by enucleation to produce reticulocytes. Micrographs were acquired with an Axiostar plus microscope (Carl Zeiss, Sliedrecht, the Netherlands) fitted with an 100x/1.3 NA EC Plan Neofluor oil objective using Immersol 518F oil (Carl Zeiss), a Canon Powershot G5 camera (Canon Nederland, Hoofddorp, the Netherlands), and Canon Zoombrowser EX image acquisition software. Photoshop CS5 was used for image processing (Adobe Systems Benelux, Amsterdam, The Netherlands).

Cells were fixed for 3 min in −20°C methanol or 8 min in 20°C 4% paraformaldehyde. Primary antibodies used in this study include rabbit α-NuMA and rat α-BrdU (Abcam, Cambridge, MA), mouse α-p150^glued and rat α-β4 integrin (BD Biosciences, San Jose, CA), mouse α-pankeratin (AE13), mouse α-Gata 3 and rat α-α tubulin (all from Santa Cruz Biotechnology, Dallas, TX), m α-β-tubulin (Sigma), rabbit α-keratin 6 (Covance), chicken α-keratin 5/14 (lab generated), rabbit α-phospho histone H3 and rabbit anti-pSMAD1/5 (Cell Signaling Technology, Danvers, MA), and rabbit α-activated caspase 3 (R&D Systems, Minneapolis, MN). Images were collected using a Zeiss motorized Axio Imager Z1 fluorescence microscope with Apotome attachment, an AxioCam MRm camera, a 10x, 20x, 40x, and 63×/1.4 numerical aperture (NA) Plan Apochromat objective, Zeiss Immersol 518F oil, and AxioVision Digital Image Processing Software. Photoshop (Adobe) and ImageJ software were used for postacquisition processing.

Differential interference contrast (DIC) and fluorescence images were captured with an Axioimager A1 (Carl Zeiss) equipped with epi-fluorescence [Filter Set 13 for GFP (excitation BP 470/20, beam splitter FT 495, emission BP 503–530) and Filter Set 20 for Cherry (excitation BP 546/12, beam splitter FT 560, emission BP 575–640)] and an AxioCam monochrome digital camera (Carl Zeiss). Images were processed and viewed using Axio-vision Rel. 4.7 software (Carl Zeiss). A 63 × objective (Plan-Neofluar NA1.30) was used with Immersol 518F oil (Carl Zeiss). Confocal images were captured by a Zeiss 880 inverted laser scanning confocal microscope with 488 nm (emission filter BP 503–530) and 543 nm (emission filter BP 560–615) lasers, and images were processed and viewed using Zen software (Carl Zeiss). All images were taken at 20°C.