Plan apo 10

Fluorescence images were obtained using the microscope Keyence BZ9000 (Keyence). The exposure times were set and kept the same for all experiments and repetitions. Images were taken using a ×10 objective.
Objective: Nikon ×10 Plan Apo NA 0.45/4.00 mm
Resolution: 8-bit
Format: 1360 × 1024 px
Light source: mercury vapour lamp
Cells were counted by adjusting the threshold of the 8-bit images and then using the Analyze Particles function in ImageJ. The ration of transfected cells in GFP channel and total number of cells in Hoechst channel revealed transfection efficiency of a sample. All date sets were depicted as mean ± standard deviation. At least three replicates per slide and at least three slides were tested for each transfection sample.

High-content image analysis was used to measure the effects of pharmacological agents on ZMEL cell proliferation and differentiation.
Using a GE INCell 6000, 2 fields were captured per well using a Nikon 10 × Plan Apo objective, 0.45NA. Images of the Hoechst signal was captured via the 4,6-diamidino-2-phenylindole channel with 0.1 and 0.3 seconds exposure in order to identify the nucleus and cell body respectively. The FITC channel was used for GFP with a 1 second exposure, and the dsRed channel used for tdTomato, with a 2 second exposure. Using GE Workstation software, nuclei were identified using a top-hat segmentation of the first (shortest exposure time) Hoechst image and cell bodies using a global threshold of the second (longer exposure time) Hoechst image, with identified nuclei as seed objects. GFP-positive cells were classified as having an average intensity within the nuclear mask of >500 in the GFP channel and tdTomato-positive cells with an average intensity >360 in the dsRed channel. Measurements were captured for all cells as well as GFP and tdTomato cells separately. Cell elongation is defined as the ratio of the minor to major axis of the cell. Major axis is the longest straight-line interval that can be drawn in the cell mask, while the minor axis is the longest straight line perpendicular to the major axis.

Confocal images were acquired using the Nikon A1–Rsi Confocal Laser Scanning Microscope (Nikon Instruments, Inc., Tokyo, Japan) configured on a Nikon Eclipse Ti inverted microscope. Images were collected using either a Nikon 10× Plan Apo (NA 0.45) objective, a Nikon 20× Plan Apo VC (NA 0.75) objective, a Nikon 40× Plan Fluor (NA 1.30) oil objective, or a Nikon 60× Apo (NA 1.40) oil objective. Image acquisition was performed using Nikon NIS–Elements AR software (version 5.20.02). Green fluorescence was excited using a 488 nm diode laser, and fluorescence emission was detected through a 525/50 nm bandpass emission filter. Red fluorescence was excited using a 561 nm diode laser, and fluorescence emission was detected through a 595/50 nm bandpass emission filter. For each data set, a confocal series through the thickness of the tissue section was collected. For the 20× objectives, confocal images were collected in 1.5 µm increments through an average thickness of 30 µm. For the 40× objectives, confocal images were collected in 1 µm increments through an average thickness of 20 µm. For each confocal series, a Maximum Intensity Projection image was generated, representing the brightest intensity pixels through the Z–depth.