Thunder imager 3d live cell and 3d cell culture

Pre- and post-expansion LED-based WF imaging of tissues was performed using the THUNDER Imager 3D Live Cell and 3D Cell Culture (Leica Microsystems). Low-magnification pre- and post-expansion whole-tissue overviews were performed with a ×20 objective (NA, 0.40). High-magnification pre- and post-expansion images were obtained using multiple objectives, including a ×40 objective (NA, 1.10), ×63 objective (NA, 1.10) and ×100 objective (NA, 1.47) after optimizing the LED intensity and exposure times. To enable post-expansion SRRF processing, time stacks (each consisting between 20 and 200 images depending on the experimental requirements) were obtained for each ROI.

To visualize the GFP-LC3-RFP reporter signal expression of Hy19636_GLRM PDAC cells, tissues were fixed in 2% PFA solution at 4 °C overnight, incubated in PBS containing 30% sucrose and embedded in Tissue-Tek OCT compound (Sakura Finetek) on dry ice. For further analysis, 7-μm sections were used. Widefield imaging was performed using the THUNDER Imager 3D Live Cell and 3D Cell Culture (Leica Microsystems) equipped with a 40× 1.10 NA water immersion objective. LED power and exposure time and other system-specific settings were first optimized using positive control tissue and not changed between image acquisitions of the different groups to provide optimal comparability. For each tissue section, an average of five regions of interest were randomly selected and imaged for subsequent quantitative analysis. ImageJ imaging software was used for file navigation, adjustment of colour balance and image analysis. GFP and RFP quantification was determined using ImageJ v.2.1.0/1.53c. The mean fluorescence intensity for each respective signal was determined per slide in at least five areas per tumour.

Pre- and post-expansion LED-based WF imaging of nanorulers was performed using the THUNDER Imager 3D Live Cell and 3D Cell Culture (Leica Microsystems) fitted with a ×100 objective (numerical aperture (NA), 1.47). LED intensity and exposure times for each condition were systematically optimized. To allow for subsequent pre- and post-expansion SRRF processing, time-stacked images (with each time stack consisting of 50 images) were obtained. Pre- and post-expansion confocal imaging of the nanorulers was performed using the Zeiss LSM 800 confocal microscope with AiryScan using the optimized ×63 objective (NA, 1.4) at 12-fold digital zoom with subsequent AiryScan processing using the ZEN2.6 (blue edition) software.