Linking the genotypes and phenotypes of cancer cells in heterogenous populations via real-time optical tagging and image analysis

Dynamic Visualization of TGF-β/SMAD3 Transcriptional Responses in Single Living Cells

“…Experiments were performed 16-24 h after plating on the glass-bottom dishes. The cells were simultaneously imaged using 460 nm (dynGFP; 10 mW/cm 2 ) and 532 nm (RFP-NLS10; mW/cm 2 ) excitation for 1 day at 1 frame per 3 min with the 1x objective (numerical aperture = 0.5) in the Ultrawide field-of-view optical microscope [36]. The field of view size (~2.5 × 3.5 mm) was chosen for imaging containing a fraction of the cells.…”

“…The movement of CAGA 12 -dynGFP B16F10 cells expressing red fluorescent protein (RFP) containing a nuclear localization signal (NLS) was tracked by RFP signal and analyzed using the mTGMM (modified Tracking using Gaussian Mixture Model) cell-tracking algorithm [36]. In brief, the intensity profile of individual nuclei/cell was modelled as a 2D Gaussian distribution.…”

Microscopy-based single-cell proteomic profiling reveals heterogeneity in DNA damage response dynamics

“…We used U2OS cells expressing 53BP1-mScarlet as a model system and monitored IR-induced DDR dynamics via 53BP1 foci changes, imaged through clustered mScarlet-fluorescence. To screen a large quantity of cells and identify subpopulations of cells with different intracellular 53BP1 foci changes over time, we implemented the UFO microscope ( You et al., 2022 ) in the FUNpro pipeline ( Figure 1 A) to image thousands of cells with 0.8 μm spatial resolution, sufficient to resolve nuclear DDR foci. Given that DDR foci has so far mainly been detected using confocal microscopy, we confirmed that the foci detected by UFO show a high correlation (Spearman’s correlation ρ = 0.94; Figure 2 C) with the foci detected by confocal microscopy (with a size larger than 1 μm in diameter and a signal-to-noise ratio [SNR] higher than 2.0) ( Figure 2 ).…”

“…Given that DDR foci has so far mainly been detected using confocal microscopy, we confirmed that the foci detected by UFO show a high correlation (Spearman’s correlation ρ = 0.94; Figure 2 C) with the foci detected by confocal microscopy (with a size larger than 1 μm in diameter and a signal-to-noise ratio [SNR] higher than 2.0) ( Figure 2 ). We then developed and implemented an automatic DDR foci-tracking algorithm ( STAR methods ; Figures 1 B and S1 ; Table S1 ) in the modified tracking with Gaussian mixture model (mTGMM [ You et al., 2022 ]) cell-tracking algorithm to quantify changes in DDR foci while tracking cellular movement in a real-time fashion. We tracked individual cell migration and division and intracellular dynamics for thousands of cells (∼57,000 cells/min) to register foci dynamics for individual cells over hundreds of image frames (24 h of imaging; Figure 1 B).…”

“…The ultrawide field-of-view optical (UFO) microscope is a custom-built microscope and has been described in detail previously ( You et al., 2022 ). Here, we upgraded the system to have a higher spatial resolution by implementing an objective with a large field-of-view (FOV) and relatively high numerical aperture (Olympus MVP Plan Apochromat 1×, 0.5 NA) in conjunction with a large chip-size cMOS camera with small pixel-size (Grasshopper3, FLIR, 4096 × 3000 pixels, 3.25 μm/pixel).…”