Journal: ACS Photonics

Article Title: Simultaneous Multicolor Multifocal Scanning Microscopy

doi: 10.1021/acsphotonics.3c00205

Figure Lengend Snippet: Multicolor multifocal scanning microscopy (MSM). (a) Optical setup of MSM. Multiple laser lines propagate and enter the microlens array (MLA) at different angles to form a multicolor foci excitation array at the sample plane (left inset). Right inset illustrates the experimental excitation pattern that contains an interposed two-color foci array ( d = 1.6 μm and θ = 4°). DM, dichroic mirror; TS, telescope; RL, relay lens; OBJ, objective lens; TL, tube lens; and CAM, camera. (b) Data processing of MSM, containing image tracking, pinholing, pixel reassignment (scaling), summing to form the intermediate image (INT), and deconvolution. The overlay of tracked image stacks forms the wide-field (WF) image. Scale bar: 3 μm.

Article Snippet: In brief, the wide-field microscope was equipped with multicolor laser lines (488 and 647 nm, Coherent OBIS LX) and a 100×, 1.45NA objective lens (Nikon CFI Plan Apochromat Lambda 100× Oil).

Techniques: Microscopy

Journal: ACS Photonics

Article Title: Simultaneous Multicolor Multifocal Scanning Microscopy

doi: 10.1021/acsphotonics.3c00205

Figure Lengend Snippet: Multicolor imaging of phantom samples using MSM. (a,b) Wide-field (a) and MSM (b) images of 100 nm Tetraspek fluorescent beads with emission peaks at 515 nm (green) and 680 nm (dark red). (c–l) Zoomed-in wide-field (c), intermediate (e,i), and MSM (g,k) images of the bead as marked in panel (a,b), and on the right, their corresponding FWHMs by Gaussian fitting showing the enhanced resolution in both spectral channels. (m–p) Wide-field (m) and super-resolution (n,o) images exhibited two nearby beads separated at 206 nm below the diffraction limit that were resolvable by MSM (p). (q,r) Wide-field (q) and MSM (r) images of 6 μm surface-stained fluorescent microspheres. (s) Zoomed-in montage wide-field and super-resolution image of the boxed region as marked in panel (q), exhibiting enhanced resolution and contrast by MSM. (t,u) Cross-sectional intensity profiles along the yellow lines as marked in panels (q,r), showing resolved structures in both 515 nm (t) and 680 nm (u) channels. Scale bars: 5 μm (a), 300 nm (c), 3 μm (m), and 5 μm (q,s).

Article Snippet: In brief, the wide-field microscope was equipped with multicolor laser lines (488 and 647 nm, Coherent OBIS LX) and a 100×, 1.45NA objective lens (Nikon CFI Plan Apochromat Lambda 100× Oil).

Techniques: Imaging, Staining

Journal: ACS Photonics

Article Title: Simultaneous Multicolor Multifocal Scanning Microscopy

doi: 10.1021/acsphotonics.3c00205

Figure Lengend Snippet: Super-resolution multicolor imaging of microtubules in HeLa cells using MSM. (a,b) Wide-field (a) and super-resolution (b) images of microtubules immune-stained for both 488 and 647 nm excitations. The arrows point to the thick nucleus region that exhibited enhanced image contrast and resolution of microtubules in the MSM image. (c,d) Zoomed-in wide-field (c) and super-resolution (d) images of the corresponding yellow boxed region as indicated in panel (a). (e) Cross-sectional intensity profiles of a microtubule filament in wide-field (black) and two-color super-resolution images. (f–k) Zoomed-in wide-field (f,i) and super-resolution (g,j) images of the corresponding boxed regions as marked in panel (a). Panels (h,k) show the cross-sectional intensity profiles along the corresponding dashed lines in panels (f,g) and (i,j), respectively. Scale bars: 10 μm (a), 5 μm (c), 2 μm (f), and 1 μm (i).

Article Snippet: In brief, the wide-field microscope was equipped with multicolor laser lines (488 and 647 nm, Coherent OBIS LX) and a 100×, 1.45NA objective lens (Nikon CFI Plan Apochromat Lambda 100× Oil).

Techniques: Imaging, Staining

Journal: ACS Photonics

Article Title: Simultaneous Multicolor Multifocal Scanning Microscopy

doi: 10.1021/acsphotonics.3c00205

Figure Lengend Snippet: Super-resolution multicolor imaging of peroxisomes and mitochondria in HeLa cells using MSM. (a,b) Wide-field (a) and super-resolution (b) images of peroxisomes (green) and mitochondria (red) labeled with GFP and MitoTracker, respectively. (c–e) Zoomed-in wide-field (c) and super-resolution (d,e) images of the corresponding boxed region as indicated in panel (a). (f,g) Zoomed-in wide-field (f) and super-resolution (g) images of the corresponding yellow boxed region as indicated in panel (a), exhibiting enhanced optical sectioning and resolution. (h) Cross-sectional intensity profiles of mitochondria along the line as marked in panel (a), revealing fine structural details using MSM (red). (i,j) Transverse ( x and y , respectively) cross-sectional intensity profiles of the peroxisome (indicated by the arrow in panel (c)) in both wide-field and super-resolution images. (k) Cross-sectional intensity profiles across the cluster of peroxisomes as indicated by the line in panels (c,d), showing the resolution of sub-diffraction-limited structures. Scale bars: 10 μm (a), 2 μm (c), and 1 μm (f).

Article Snippet: In brief, the wide-field microscope was equipped with multicolor laser lines (488 and 647 nm, Coherent OBIS LX) and a 100×, 1.45NA objective lens (Nikon CFI Plan Apochromat Lambda 100× Oil).

Techniques: Imaging, Labeling

Journal: ACS Photonics

Article Title: Simultaneous Multicolor Multifocal Scanning Microscopy

doi: 10.1021/acsphotonics.3c00205

Figure Lengend Snippet: Multicolor MSM for live-cell imaging and with extended field of view. (a,b) Wide-field (a) and MSM (b) images of mitochondria (red) and lysosomes (green) of living HeLa cells. Recording images at a frame rate of 200 Hz, super-resolution sequences were formed every 1.3 s. (c) Zoomed-in time-lapse sequences of lysosomes and mitochondria in the boxed region in panel (c). The dashed lines marked the initial mitochondrial structure at t = 0, indicating the delicate motion of the organelle over time ( Supplementary Videos S1 and S2 ). (d) Wide-field (top) and super-resolution (bottom) images of the nucleus (green) and microtubules (red) of HeLa cells across an extended field of view (>400 μm × 130 μm). Scale bars: 10 μm (a,b), 3 μm (c), and 50 μm (d).

Article Snippet: In brief, the wide-field microscope was equipped with multicolor laser lines (488 and 647 nm, Coherent OBIS LX) and a 100×, 1.45NA objective lens (Nikon CFI Plan Apochromat Lambda 100× Oil).

Techniques: Live Cell Imaging