Fig 2: csf1ra and csf1rb have distinct expression patterns in zebrafish. (A) Coexpression of csf1ra (red) and mpeg1 (green) visualized in 72 hpf Tg(fms:GAL4;UAS:mCherry);Tg(mpeg1:EGFP) triple transgenic embryos: (Aa) whole embryo, (Ab) head, (Ac) caudal hematopoietic tissue (CHT) region. (B) HCR WISH of 72 hpf embryos for csf1rb (red) and mpeg1 (green). (Ba) whole embryo, (Bb) head, (Bc) CHT region. Fluorescence images were taken on Dragonfly 503 microscope (Andor) using Zyla-4.2 sCMOS camera with magnification ×10 and processed with the Fusion software, FIJI, and Adobe Photoshop. (C) qRT-PCR analysis of pooled zebrafish embryos showing the expression dynamics of csf1ra and csf1rb in zebrafish development. Pool of 15 to 20 embryos per sample in 2 to 6 biological replicates. The expression was normalized to mob4 gene and to the time point with the highest expression (14 dpf for csf1ra and 7 dpf for csfrb). (D) qRT-PCR analysis of adult zebrafish tissues. Pool of 3 to 5 fish organs per sample in 3 to 5 biological replicates. The expression was normalized to ef1a gene. (E) scRNA-seq data showing the expression of csf1ra and csf1rb in whole kidney marrow (WKM) cell populations (pool of 2 to 3 WKMs per sample). BC, B-cells; BP, blood progenitors; E, erythroid cells; EC, endothelial cells; EP, erythroid progenitors; G, granulocytes; KSC, kidney support cells; M/M, monocytes & macrophages; MP, myeloid progenitors; NKC, NK cells; T, thrombocytes; TC, T-cells.

Fig 3: Distribution of uniformity and centering field illumination flatness metrics and their influence on biological imaging. (A) Uniformity (U) and centering (C) distribution are classified into microscopy modalities, with representative field illumination pattern examples. (B) Statistical analysis of A, according to the modality and the excitation channel. The mean value is shown as a cross, the SD as a box and the whiskers correspond to the min and max. (C) U–C distribution classified into excitation source types with the statistical analysis study. (D–F) Influence of field illumination flatness on image quality of biological structures. The objective is a Plan-Apo 60×/1.4 (Nikon) on a spinning disk microscope (Dragonfly-Andor). (D) Field illumination pattern and U–C values for a plastic slide extracted by MetroloJ_QC, the corresponding image for a DAPI stained nuclei sample, and a 2 × 2 tile scan. (E) Same configuration as D for the GFP channel and actin Alexa Fluor 488 labeling. (F) The 50% central area of D with the U–C calculated metrics.

Fig 4: Indirect immunofluorescence staining of a-tubulin (green) and F-actin (red), and simultaneous DAPI chromosome staining (blue) of EA.hy926 cells. (A) Mitosis in control (i: interphase; ii: metaphase; iii: anaphase; iv: metaphase and cytokinesis) and 26Z treated cells with IC50 (130 nM) and IC80 (300 nM) concentrations for 24 h. Microphotographs were obtained with an Andor Dragonfly 505 Confocal Microscope. (B) Cytoskeleton structure of adherent cells in control conditions and after treatment with 26Z at IC50 (130 nM) and IC80 (300 nM) for 24 h. Images were acquired with an Axiovert 200 M inverted microscope. Scale bars, 10 µM. Arrowheads—multipolar spindles, arrows—membrane blebbing and apoptotic bodies.

Fig 5: THP-1 macrophage cells invaded by remodeled E. coli ?MurQ KU in 2D and 3D culture. E. coli ?MurQ KU (EQKU) cells are remodeled by incorporating the azido bacterial cell wall precursor in the cell wall with recycling enzymes AmgK and MurU. (A) EQKU cells labeled with DBCO-488 using SPAAC were invaded into macrophage cells and observed with Andor Dragonfly microscope. Bacteria (green) can be visualized within macrophages (purple) as shown by ortho view above. Scale bar = 5 µm (B,C) The CuAAC condition bacteria were invaded into THP-1 macrophage cells and fixed prior to the CuAAC click labeling (bacteria, green) and subsequent staining with Hoechst (nucleus, blue). The SPAAC condition bacteria were remodeled with the DBCO-488 (bacteria, green) and then invaded into the THP-1 cells. The cells were then fixed and stained with Hoechst (nucleus, blue). The 2D samples (B) were also stained with Phalloidin-TRITC (F-actin, purple). Images were taken on a Zeiss LSM800 Confocal Microscope with single plain snaps for 2D images and ortho-projections (XYZ) of Z-stacks of the cells in the synthetic hydrogels. Images are representative of a minimum of three fields viewed per replicate with at least two technical replicates, and experiments were conducted in at least three biological replicates. (B) Scale bars = 10 µm (C) Scale bars = 5 µm.