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( a ) Time-lapse STED images obtained by conventional STED (top, Δt = 2.3 μs) and UNet-RCAN using fast STED data (bottom, Δt = 0.054μs). β-tubulin (STAR635P, magenta) and histone (Alexa 594, green) were imaged in U2OS cells. ( b ) Photobleaching analysis of two-color STED images used in (a). Shaded areas are standard deviations of fluorescence intensities ( n = 5). ( c ) Live-cell STED recording of mitochondrial cristae <t>in</t> <t>HeLa</t> cells ( Δt = 1 μs) labeled with PK <t>Mito</t> Orange. ( d ) Denoising and deconvolved STED recording of mitochondrial cristae in COS-7 cells ( Δt = 1 μs) labeled with PK Mito Orange ( e ) Fluorescence time traces of mitochondrial cristae in HeLa cells over 250 consecutive frames (2 s/frame). The fluorescence time trace of STED recording with Δt = 90 μs is displayed for comparison. ( f ) Line profiles along a dashed line in (d). ( g ) 2D- and 3D-UNet-RCAN prediction results for a noisy z-stack of 3D-STED imaging of TOM20 (Atto647N). The z pixel size is 65 nm. ( h ) Denoising results for xz time-lapse STED recording of membrane fusion dynamics ( Δt = 2 μs) over 300 frames in comparison to ground-truth data ( Δt = 20 μs). Scale bars, 5 μm (a,g), 2 μm (c,d,h) and 1 μm for the magnified regions in (g).
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( a ) Time-lapse STED images obtained by conventional STED (top, Δt = 2.3 μs) and UNet-RCAN using fast STED data (bottom, Δt = 0.054μs). β-tubulin (STAR635P, magenta) and histone (Alexa 594, green) were imaged in U2OS cells. ( b ) Photobleaching analysis of two-color STED images used in (a). Shaded areas are standard deviations of fluorescence intensities ( n = 5). ( c ) Live-cell STED recording of mitochondrial cristae <t>in</t> <t>HeLa</t> cells ( Δt = 1 μs) labeled with PK <t>Mito</t> Orange. ( d ) Denoising and deconvolved STED recording of mitochondrial cristae in COS-7 cells ( Δt = 1 μs) labeled with PK Mito Orange ( e ) Fluorescence time traces of mitochondrial cristae in HeLa cells over 250 consecutive frames (2 s/frame). The fluorescence time trace of STED recording with Δt = 90 μs is displayed for comparison. ( f ) Line profiles along a dashed line in (d). ( g ) 2D- and 3D-UNet-RCAN prediction results for a noisy z-stack of 3D-STED imaging of TOM20 (Atto647N). The z pixel size is 65 nm. ( h ) Denoising results for xz time-lapse STED recording of membrane fusion dynamics ( Δt = 2 μs) over 300 frames in comparison to ground-truth data ( Δt = 20 μs). Scale bars, 5 μm (a,g), 2 μm (c,d,h) and 1 μm for the magnified regions in (g).
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Image Search Results


( a ) Time-lapse STED images obtained by conventional STED (top, Δt = 2.3 μs) and UNet-RCAN using fast STED data (bottom, Δt = 0.054μs). β-tubulin (STAR635P, magenta) and histone (Alexa 594, green) were imaged in U2OS cells. ( b ) Photobleaching analysis of two-color STED images used in (a). Shaded areas are standard deviations of fluorescence intensities ( n = 5). ( c ) Live-cell STED recording of mitochondrial cristae in HeLa cells ( Δt = 1 μs) labeled with PK Mito Orange. ( d ) Denoising and deconvolved STED recording of mitochondrial cristae in COS-7 cells ( Δt = 1 μs) labeled with PK Mito Orange ( e ) Fluorescence time traces of mitochondrial cristae in HeLa cells over 250 consecutive frames (2 s/frame). The fluorescence time trace of STED recording with Δt = 90 μs is displayed for comparison. ( f ) Line profiles along a dashed line in (d). ( g ) 2D- and 3D-UNet-RCAN prediction results for a noisy z-stack of 3D-STED imaging of TOM20 (Atto647N). The z pixel size is 65 nm. ( h ) Denoising results for xz time-lapse STED recording of membrane fusion dynamics ( Δt = 2 μs) over 300 frames in comparison to ground-truth data ( Δt = 20 μs). Scale bars, 5 μm (a,g), 2 μm (c,d,h) and 1 μm for the magnified regions in (g).

Journal: bioRxiv

Article Title: Deep learning enables fast, gentle STED microscopy

doi: 10.1101/2023.01.26.525571

Figure Lengend Snippet: ( a ) Time-lapse STED images obtained by conventional STED (top, Δt = 2.3 μs) and UNet-RCAN using fast STED data (bottom, Δt = 0.054μs). β-tubulin (STAR635P, magenta) and histone (Alexa 594, green) were imaged in U2OS cells. ( b ) Photobleaching analysis of two-color STED images used in (a). Shaded areas are standard deviations of fluorescence intensities ( n = 5). ( c ) Live-cell STED recording of mitochondrial cristae in HeLa cells ( Δt = 1 μs) labeled with PK Mito Orange. ( d ) Denoising and deconvolved STED recording of mitochondrial cristae in COS-7 cells ( Δt = 1 μs) labeled with PK Mito Orange ( e ) Fluorescence time traces of mitochondrial cristae in HeLa cells over 250 consecutive frames (2 s/frame). The fluorescence time trace of STED recording with Δt = 90 μs is displayed for comparison. ( f ) Line profiles along a dashed line in (d). ( g ) 2D- and 3D-UNet-RCAN prediction results for a noisy z-stack of 3D-STED imaging of TOM20 (Atto647N). The z pixel size is 65 nm. ( h ) Denoising results for xz time-lapse STED recording of membrane fusion dynamics ( Δt = 2 μs) over 300 frames in comparison to ground-truth data ( Δt = 20 μs). Scale bars, 5 μm (a,g), 2 μm (c,d,h) and 1 μm for the magnified regions in (g).

Article Snippet: The HeLa cells were stained with DMEM containing 250 nM PK Mito Orange (Confocal.nl) for 40 min, followed by three washing steps in DMEM.

Techniques: Fluorescence, Labeling, Comparison, Imaging, Membrane