Journal: bioRxiv

Article Title: Cell morphogenesis via shape-guided self-propelled treadmilling actin waves

doi: 10.1101/2024.02.28.582450

Figure Lengend Snippet: a , A U251 cell stained with Alexa Fluor 594 conjugated phalloidin. An enlarged view of the rectangular region is shown to the right. Arrows and arrowheads indicate lamellipodia and filopodia, respectively. b , Time-lapse phase-contrast images of U251 cells. c , Fluorescence time-lapse images of filopodium-type (arrowheads) and lamellipodium-type (arrows) actin waves in U251 cells expressing LifeAct-mCherry obtained by TIRF microscopy. See Supplementary Video 2. d , A fluorescence image of a COS7 cell expressing LifeAct-mCherry obtained by TIRF microscopy: right panels indicate enlarged time-lapse images in rectangular regions 1 and 2. Arrows and arrowheads indicate lamellipodium-type and filopodium-type waves, respectively. See Supplementary Video 3. e - f , Fluorescence time-lapse images of a U251 cell expressing LifeAct-mCherry (obtained by TIRF microscopy) and EGFP-LifeAct (obtained by epifluorescence microscopy): lower panels and f indicate enlarged time-lapse images in rectangular regions 1, 2 and 3. See Supplementary Video 4. Scale bars, 20 µm ( a , b , d ); 5 µm ( a , d enlarged views, c , e ); 1 µm ( e enlarged views, f ).

Article Snippet: The fluorescent speckles of HaloTag-actin and EGFP-LifeAct were observed using a TIRF microscope (Olympus, IX81) equipped with a CMOS camera (Hamamatsu, ORCA Flash4.0LT), an objective lens 150x 1.45 NA (Olympus, UAPON), and MetaMorph software.

Techniques: Staining, Fluorescence, Expressing, Microscopy, Epifluorescence Microscopy

Journal: bioRxiv

Article Title: Cell morphogenesis via shape-guided self-propelled treadmilling actin waves

doi: 10.1101/2024.02.28.582450

Figure Lengend Snippet: a , A fluorescence image of a U251 cell expressing EGFP-LifeAct obtained by TIRF microscopy. Asterisk indicates cellular leading edge. See Supplementary Video 1. b-c , Fluorescence time-lapse images of filopodium-type (arrowheads, b ) and lamellipodium-type (arrows, c ) actin waves in U251 cells expressing LifeAct-mCherry obtained by TIRF microscopy. d-e , Fluorescence time-lapse images of actin waves on the dorsal membrane (blue arrowheads and arrows, d ) and filopodium-type actin waves in cytoplasm (green arrowheads, e ) in U251 cells expressing LifeAct-mCherry obtained by 3D imaging with confocal deconvolution microscopy. See Supplementary Video 5. f , A fluorescent speckle image of HaloTag-actin in a U251 cell obtained by TIRF microscopy; actin filaments were also monitored by EGFP-LifeAct. See Supplementary Video 6. Time-lapse montages of a filopodium-type actin wave in the rectangular region at 10-sec intervals are shown to the right. Green and magenta lines indicate wave forward translocation and actin filament retrograde flow, respectively. Actin polymerisation rate can be calculated as the sum of the forward translocation rate and actin retrograde flow rate (double-headed arrow). g , A diagram showing the translocation mechanisms of actin waves travelling on the ventral plasma membrane (left) . Right panels show a possible mode of actin wave translocation observed in e and . For explanations, see text and Supplementary Discussion. h , Translocation velocities of filopodium-type actin waves ( f , green line) before and after the application of DMSO (control, N = 4 cells: before application, n = 34 waves; after application, n = 32 waves), CK666 (N = 3 cells: before application, n = 49 waves; after application, n = 33 waves) or SMIFH2 (N = 5 cells: before application, n = 52 waves; after application, n = 37 waves). i , Velocities of actin filament retrograde flow ( f , magenta line), actin filament polymerisation ( f , double-headed arrow), and filopodium-type wave translocation ( f , green line) in WT and shootin1b KO U251 cells (WT, N = 11 cells, n = 71 waves; KO#1, N = 9 cells, n = 69 waves; KO#2, N = 10 cells, n = 45 waves). Scale bars, 20 µm ( a ); 5 µm ( f left): 1 µm ( b , c , d , e , f right). Data represent means ± SEM; ***p < 0.01; *p < 0.05; ns, not significant. Statistical analyses were performed using the two-tailed Mann‒Whitney U -test ( h , i ).

Article Snippet: The fluorescent speckles of HaloTag-actin and EGFP-LifeAct were observed using a TIRF microscope (Olympus, IX81) equipped with a CMOS camera (Hamamatsu, ORCA Flash4.0LT), an objective lens 150x 1.45 NA (Olympus, UAPON), and MetaMorph software.

Techniques: Fluorescence, Expressing, Microscopy, Membrane, Imaging, Translocation Assay, Clinical Proteomics, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Cell morphogenesis via shape-guided self-propelled treadmilling actin waves

doi: 10.1101/2024.02.28.582450

Figure Lengend Snippet: a , A Fluorescent speckle image of HaloTag-actin in a U251 cell obtained by TIRF microscopy; actin filaments were also monitored by EGFP-LifeAct. b , A Fluorescent speckle image of HaloTag-actin in a U251 cell obtained by 3D imaging with confocal deconvolution microscopy; actin filaments were also monitored by EGFP-LifeAct. c , Immunoblot analyses of U251 cells with anti-shootin1b, anti-cortactin and anti-L1 antibodies. d , A fluorescence image of a U251 cell stained with anti-shootin1b antibody, Alexa Fluor 594 conjugated phalloidin (for actin filament), and DAPI (for nucleus). e , A fluorescence image of a U251 cell stained with anti-shootin1b antibody and Alexa Fluor 594 conjugated phalloidin (for actin filament) obtained by TIRF microscopy. An enlarged view of the area within the rectangle is shown in the right panel. Arrowheads indicate shootin1b colocalisation with actin filament. f , Translocation velocities of filopodium-type actin waves before and after the application of 0.05 µM cytochalasin B (N = 4 cells, before application: n = 34 waves, after application: n = 28 waves). g , Immunoblot analysis of shootin1b in WT U251 cells, postnatal day 1 rate brain, and shootin1b KO U251 cells. The cell and tissue lysates were immunoblotted with anti-shootin1 and anti-actin antibodies. The arrowhead and asterisk indicate the bands corresponding to shootin1b and shootin1a, respectively. Shootin1a is a splicing variant of shootin1b expressed in neurons. Immunoblots with anti-actin antibody served as loading controls. h , A diagram showing the effects of shootin1b KO on the velocities of actin filament retrograde flow (magenta arrows) and actin wave translocation (green arrows). Scale bars, 20 µm ( b left, d , e left); 5 µm ( b middle, e right); 1 µm ( a , b right). Data represent means ± SEM; ***p < 0.01. Statistical analyses were performed using the two-tailed Mann‒Whitney U-test ( f ).

Article Snippet: The fluorescent speckles of HaloTag-actin and EGFP-LifeAct were observed using a TIRF microscope (Olympus, IX81) equipped with a CMOS camera (Hamamatsu, ORCA Flash4.0LT), an objective lens 150x 1.45 NA (Olympus, UAPON), and MetaMorph software.

Techniques: Microscopy, Imaging, Western Blot, Fluorescence, Staining, Translocation Assay, Variant Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Cell morphogenesis via shape-guided self-propelled treadmilling actin waves

doi: 10.1101/2024.02.28.582450

Figure Lengend Snippet: a-b , Fluorescence time-lapse images of filopodium-type actin waves (arrowheads) which entered into pre-existing lamellipodium (arrows, a ) and filopodium (asterisks, b ) in U251 cells expressing EGFP-LifeAct obtained by TIRF microscopy. Entry of the waves into lamellipodia resulted in local actin filament accumulation and lamellipodial extension (arrows, a ). See Supplementary Video 7. c-d , Fluorescence time-lapse images of lamellipodium-type actin wave (yellow arrows, c ) and filopodium-type actin waves (arrowheads, d ) which arrived at the cell periphery in U251 cells expressing LifeAct-mCherry obtained by TIRF microscopy ( c ) and expressing EGFP-LifeAct obtained by epifluorescence microscopy ( d ). They pushed the plasma membrane, resulting in the formation of lamellipodium ( c ) and filopodia ( d ), and continued to move laterally along the membrane. The former merged with the pre-existing lamellipodium (blue arrows, d ). See Supplementary Videos 8 and 9. e , A diagram describing the relationship between the velocities of lateral movement ( V L ) and ventral movement ( V V ) of filopodium-type actin waves. If actin waves move laterally along the membrane driven by directional actin treadmilling, V L is equal to V V cos θ , where θ is the angle of the polymerising actin filaments with respect to the membrane. f , Fluorescence time-lapse images of a filopodium-type actin wave travelling along the lateral membrane (arrowheads) of a U251 cell expressing LifeAct-mCherry obtained by epifluorescence microscopy. See Supplementary Video 10. The right graph shows V L and θ obtained by the time-lapse imaging; V L closely matched V V cos θ (N = 10 cells, n = 21 waves). Scale bars, 5 µm ( c , d , f ); 1 µm ( a , b ).

Article Snippet: The fluorescent speckles of HaloTag-actin and EGFP-LifeAct were observed using a TIRF microscope (Olympus, IX81) equipped with a CMOS camera (Hamamatsu, ORCA Flash4.0LT), an objective lens 150x 1.45 NA (Olympus, UAPON), and MetaMorph software.

Techniques: Fluorescence, Expressing, Microscopy, Epifluorescence Microscopy, Clinical Proteomics, Membrane, Imaging

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Roles of LRRC26 as an auxiliary γ1-subunit of large-conductance Ca 2+ -activated K + channels in bronchial smooth muscle cells

doi: 10.1152/ajplung.00331.2019

Figure Lengend Snippet: Large-conductance Ca2+-activated K+ channel α-subunit (BKα) and BKγ1 form molecular complex on the cell surface in mouse bronchial smooth muscle cells (mBSMCs). A: coimmunoprecipitation assay was performed using rat BSMs (rBSMs). Lysates were precipitated with anti-BKα antibody and blotted using either anti-BKα or anti-BKγ1 antibody. Similar results were obtained from 3 independent experiments. Two rats were used for each experiment. Lane 1, lysates from rBSM; lane 2, samples precipitated with control resin that cannot bind to antibodies; lane 3, samples precipitated with resin that binds to anti-BKα antibody. B: total internal reflection fluorescence (TIRF) imaging of mBSMCs, in which BKα and BKγ1 were labeled with each antibody. Fluorescent signals from particles corresponding to BKα, BKγ1, and colocalization are shown in green, red, and yellow, respectively. Merged image was overlapped with a cell image. C: ratio of BKα particles localized alone or colocalized with BKγ1 to total BKα particles in mBSMCs (BKα alone, 134 particles and colocalized, 214 particles from 15 cells).

Article Snippet: Fluorescently labeled cells were observed by using confocal (AIR, Nikon, Tokyo, Japan) or total internal reflection fluorescence (TIRF) microscopes (Nikon).

Techniques: Co-Immunoprecipitation Assay, Fluorescence, Imaging, Labeling

Journal: The Journal of Neuroscience

Article Title: Secretagogue Stimulation of Neurosecretory Cells Elicits Filopodial Extensions Uncovering New Functional Release Sites

doi: 10.1523/JNEUROSCI.2634-13.2013

Figure Lengend Snippet: Activity-dependent filopodial extension drives the increase in footprint surface area in neurosecretory cells. A, B, Bovine chromaffin (A) and PC12 (B) cells expressing GFP-GPI were examined by TIRF microscopy, imaged at 2 Hz and stimulated with Ba2+ (2 mm) or vehicle treated as indicated. Top, Time-lapse of the GFP-GPI fluorescence in the footprint. Bottom, GPI-GFP footprint area (green) with an overlay of the pretreatment area (white with red outline). Scale bars, 5 μm. Arrowheads indicate outgrowing filopodia. C, Changes in surface area over time were plotted as indicated. D, Average change in footprint area (normalized to initial area before treatment) for stimulated chromaffin and PC12 cells and vehicle-treated controls 400 s after stimulation (n = 6/10/8/5). E, Bovine chromaffin cells were treated with vehicle or 2 mm BaCl2 for 8 min and the region of the cell containing the cell footprint was analyzed by electron microscopy. Note the numerous filopodia emerging from the footprint in stimulated cells. The inset shows a region of membrane extension containing SGs (arrowheads). Proximity to the plasma membrane is confirmed by the profiles of clathrin-coated pits containing ruthenium red stain, indicative of continuity with the plasma membrane (arrows). Bottom, Outlines of the cells in the top. Left: Ratio of outline length to visible surface before and stimulation. Right: Counts of objects outside the cell surface in unstimulated and stimulated conditions (n = 6).

Article Snippet: Transfected cells on glass-bottomed culture dishes (MatTek) were visualized with a total internal reflection fluorescence (TIRF) microscope (Marianas, SDC Everest; Intelligent Imaging Innovations) fitted with a 100× oil-immersion objective (numerical aperture = 1.46, Carl Zeiss Pty Ltd) using an EMCCD camera (QuantEM 512sc) and Slidebook software (version 4.2.0.27/28).

Techniques: Activity Assay, Expressing, Microscopy, Fluorescence, Electron Microscopy, Staining

Journal: The Journal of Neuroscience

Article Title: Secretagogue Stimulation of Neurosecretory Cells Elicits Filopodial Extensions Uncovering New Functional Release Sites

doi: 10.1523/JNEUROSCI.2634-13.2013

Figure Lengend Snippet: Vesicular fusion does not account for the entire footprint enlargement. SG granule fusion was measured in bovine chromaffin cells expressing NPY-mCherry. Cells were examined by TIRF microscopy and imaged at 2 Hz before and for 7 min after stimulation (black arrow) with Ba2+ (2 mm). A, Individual fusion events were defined as the disappearance of NPY-positive SGs, followed by a cloud-like dispersal of NPY-mCherry to the extracellular space, corresponding to a spike in overall NPY-mCherry intensity. B, SG fusion was measured in bovine chromaffin cells expressing NPY-mCherry and VAMP2-pHluorin. Cells were examined by TIRF microscopy and imaged at 2 Hz before and for 7 min after stimulation with Ba2+ (2 mm). C, Validation of the NPY-mCherry assay described in A by comparison with VAMP2-pHluorin overall fluorescence intensity change. To count fusion events with VAMP2-pHluorin, we determined the average fluorescence intensity change due to alkalinization of the pHluorin moiety during a fusion event by calculating the difference in fluorescence intensity between two adjacent frames before and during a fusion event and averaging over a number of events. We then assigned this value to each individual fusion event detected using the NPY-mCherry assay (orange). Note that these results do not significantly differ from the direct VAMP2-pHluorin fluorescence measurement (green). D, Chromaffin and PC12 cells were cotransfected with NPY-mCherry and VAMP2-pHluorin and analyzed by TIRF microscopy. Arrowheads in the inset indicate vesicles where both markers colocalize. The graph depicts the percentage of VAMP2-pHluorin-positive vesicles that also contain NPY-mCherry and vice versa (n = 7). E, Chromaffin and PC12 cells cotransfected with GFP-GPI and NPY-mCherry and stimulated with Ba2+ (2 mm). The extent of footprint enlargement based on the actual GFP-GPI measurements (green) was compared with the estimated membrane increase based on the measured number of SG fusion events (NPY-mCherry assay) multiplied by the predicted membrane surface area of a 300 nm SG (orange; n = 4–6). Note that the actual footprint increase is significantly larger than that of the estimate based on fusion alone.

Article Snippet: Transfected cells on glass-bottomed culture dishes (MatTek) were visualized with a total internal reflection fluorescence (TIRF) microscope (Marianas, SDC Everest; Intelligent Imaging Innovations) fitted with a 100× oil-immersion objective (numerical aperture = 1.46, Carl Zeiss Pty Ltd) using an EMCCD camera (QuantEM 512sc) and Slidebook software (version 4.2.0.27/28).

Techniques: Expressing, Microscopy, Fluorescence

Journal: The Journal of Neuroscience

Article Title: Secretagogue Stimulation of Neurosecretory Cells Elicits Filopodial Extensions Uncovering New Functional Release Sites

doi: 10.1523/JNEUROSCI.2634-13.2013

Figure Lengend Snippet: Footprint expansion does not depend on regulated exocytosis. NPY-mCherry was expressed in PC12 cells, DKD-PC12 cells, and DKD-PC12 cotransfected with Munc18–1-GFP. A, Cells were imaged at 2 Hz by TIRF microscopy before and for 7 min after 2 mm Ba2+ stimulation and fusion numbers were counted and normalized to footprint area. Note the rescue of exocytosis elicited by Munc18–1-GFP reexpression in DKD-PC12 cells. B, GFP-GPI was expressed in DKD-PC12 cells and DKD-PC12 cotransfected with (untagged) Munc18–1. Cells were examined by TIRF microscopy and imaged at 2 Hz before and during Ba2+ (2 mm) stimulation. Top, Time-lapse of the GFP-GPI fluorescence in the footprint. Bottom, GPI-GFP footprint area (green) with an overlay of the prestimulation area (white with red outline). Scale bars, 5 μm. C, Footprint area change for GFP-GPI-transfected DKD PC12 cells expressing empty vector or Munc18–1 and wild-type (wt)-PC12 cells after Ba2+ (2 mm) stimulation (400 s). Note that all three populations exhibit similar increases in footprint area. The numbers in the bars indicate the number of cultures analyzed.

Article Snippet: Transfected cells on glass-bottomed culture dishes (MatTek) were visualized with a total internal reflection fluorescence (TIRF) microscope (Marianas, SDC Everest; Intelligent Imaging Innovations) fitted with a 100× oil-immersion objective (numerical aperture = 1.46, Carl Zeiss Pty Ltd) using an EMCCD camera (QuantEM 512sc) and Slidebook software (version 4.2.0.27/28).

Techniques: Microscopy, Fluorescence, Transfection, Expressing, Plasmid Preparation

Journal: The Journal of Neuroscience

Article Title: Secretagogue Stimulation of Neurosecretory Cells Elicits Filopodial Extensions Uncovering New Functional Release Sites

doi: 10.1523/JNEUROSCI.2634-13.2013

Figure Lengend Snippet: Interfering with actin polymerization and myosin II function reduces footprint enlargement. Bovine chromaffin (A, B) and PC12 (C, D) cells coexpressing GFP-GPI and NPY-mCherry were treated with cytochalasin D (CytoD) or blebbistatin (Blebbi) for 20 min before examination by time-lapse TIRF microscopy. Cells were then stimulated with Ba2+ and imaged at 2 Hz. A, B, Time-lapse profile of the GFP-GPI fluorescence in the footprint of chromaffin (A) or PC12 (B) cells treated as indicated. Bottom, GPI-GFP footprint area (green) with an overlay of the prestimulation area (white with red outline). Scale bars, 5 μm. B, D, Footprint area change under indicated conditions 400 s after Ba2+ stimulation or vehicle treatment for chromaffin (B) and PC12 cells (D) based on either the actual GFP-GPI measurements (black bars) or the corresponding estimated (est.) footprint increase based on fusion events (white bars). Note that for both blebbistatin and cytochalasin D treatments in chromaffin cells, the measured and estimated footprint area changes do not differ significantly. The numbers inside the bars indicate the numbers of cultures analyzed.

Article Snippet: Transfected cells on glass-bottomed culture dishes (MatTek) were visualized with a total internal reflection fluorescence (TIRF) microscope (Marianas, SDC Everest; Intelligent Imaging Innovations) fitted with a 100× oil-immersion objective (numerical aperture = 1.46, Carl Zeiss Pty Ltd) using an EMCCD camera (QuantEM 512sc) and Slidebook software (version 4.2.0.27/28).

Techniques: Microscopy, Fluorescence

Journal: The Journal of Neuroscience

Article Title: Secretagogue Stimulation of Neurosecretory Cells Elicits Filopodial Extensions Uncovering New Functional Release Sites

doi: 10.1523/JNEUROSCI.2634-13.2013

Figure Lengend Snippet: Newly added footprint area contains high levels of F-actin. A, PC12 cells cotransfected with Lifeact-RFP (LA-RFP; red) and GFP-GPI (green) were examined by TIRF microscopy and imaged at 2 Hz before and for 7 min after stimulation with Ba2+ (2 mm). Bottom, Ratio of Lifeact-RFP:GFP-GPI fluorescence in pseudocolor to indicate changes in F-actin concentration. B, 3D surface intensity plot of Lifeact-RFP:GFP-GPI ratio before and 400 s after stimulation, highlighting the increased F-actin concentration (red) at the edge of the footprint and the decrease in F-actin in the center of the footprint. C, Quantification of Lifeact-RFP and GFP-GPI ratios using regions of interest from the center or the expanding edge of PC12 cell footprints (n = 3). After stimulation, the Lifeact-RFP:GFP-GPI ratio in the initial footprint area decreases while simultaneously increasing in the added footprint area.

Article Snippet: Transfected cells on glass-bottomed culture dishes (MatTek) were visualized with a total internal reflection fluorescence (TIRF) microscope (Marianas, SDC Everest; Intelligent Imaging Innovations) fitted with a 100× oil-immersion objective (numerical aperture = 1.46, Carl Zeiss Pty Ltd) using an EMCCD camera (QuantEM 512sc) and Slidebook software (version 4.2.0.27/28).

Techniques: Microscopy, Fluorescence, Concentration Assay

Journal: The Journal of Neuroscience

Article Title: Secretagogue Stimulation of Neurosecretory Cells Elicits Filopodial Extensions Uncovering New Functional Release Sites

doi: 10.1523/JNEUROSCI.2634-13.2013

Figure Lengend Snippet: SGs move into the newly added footprint area. A, Time-lapse of PC12 cells cotransfected with NPY-mCherry(NPY-Ch) and GFP-GPI, imaged at 2 Hz before and for 7 min after stimulation with Ba2+ (2 mm). Top, NPY-mCherry-positive SGs appear outside the initial area (green dashed line) and are evenly distributed across the entire footprint (green outline). Bottom and side, x-y-t reconstruction of NPY-mCherry-positive SGs, with new SGs appearing in both the initial and the newly added area after stimulation. Note that the SG density (number of granules per unit area) remains constant over time (small inset) in all areas. B, Time-lapse TIRF microscopy of a bovine chromaffin cell cotransfected with Lifeact-GFP (LA-GFP) and NPY-mCherry showing the distribution of F-actin and SGs before and 400 s after stimulation. Right, Trajectories of SGs in the added footprint membrane area show that they are entering the newly forming area that also contains Lifeact-GFP positive structures (bottom). Scale bar, 5 μm. C,D, Maps of NPY-mCherry positive SGs trajectories in a Ba2+-stimulated chromaffin cell color coded for time after stimulation (C) and square displacement (D), as indicated. The white outline indicates the footprint area before stimulation. E, Mean square displacement (MSD) analysis of SGs in the original footprint and added areas. Note that there is no significant difference in mobility between either SG populations. F, SGs undergo regulated exocytosis in the newly added area. Time-lapse profile of a bovine chromaffin cell transfected with Lifeact-GFP (LA-GFP) and NPY-mCherry (red outline of the initial footprint area) stimulated with Ba2+ at t = 0 and imaged at 2 Hz. Arrowheads depict a SG entering an added area and undergoing fusion (pseudocolored in the inset). In the merged insets, the GFP channel is replaced with an outline of the limit of the footprint. Scale bar, 5 μm. G, Quantification of fusion events per area in both the original footprint and the added area. During footprint expansion, starting 120 s after stimulation, the number of fusions/area in the new area does not differ significantly from those in the initial footprint. Scale bar, 5 μm.

Article Snippet: Transfected cells on glass-bottomed culture dishes (MatTek) were visualized with a total internal reflection fluorescence (TIRF) microscope (Marianas, SDC Everest; Intelligent Imaging Innovations) fitted with a 100× oil-immersion objective (numerical aperture = 1.46, Carl Zeiss Pty Ltd) using an EMCCD camera (QuantEM 512sc) and Slidebook software (version 4.2.0.27/28).

Techniques: Microscopy, Transfection

Journal: bioRxiv

Article Title: The K2: Open-source simultaneous triple-color TIRF microscope for live-cell and single-molecule imaging

doi: 10.1101/2022.12.19.521031

Figure Lengend Snippet: Render of the K2 open-source simultaneous triple-color TIRF microscope for live-cell and single-molecule imaging. For illustration purposes, the two enclosure boxes to the left and right of the central cube are rendered transparent and their lids are removed. The handheld sample stage joystick controller is placed on the optical bench only for illustration purposes and usually located on the computer desk.

Article Snippet: The sample stage of the K2 single-molecule TIRF microscope is equipped with a three-axis piezo stick-slip positioner (SLS-5252, Smaract) and a two-piece aluminum sample holder that accommodates square (18 mm to 26 mm side length), circular (25 mm to 37 mm diameter) and rectangular (62 mm to 80 mm length, up to 35 mm width) samples.

Techniques: Microscopy, Imaging

Journal: bioRxiv

Article Title: The K2: Open-source simultaneous triple-color TIRF microscope for live-cell and single-molecule imaging

doi: 10.1101/2022.12.19.521031

Figure Lengend Snippet: Optical pathway of the K2 microscope: I) The central cube houses the sample stage, objective, dichroic mirror for splitting excitation and emission light, and the tube lens. II) The excitation pathway launches a four-color laser beam with circular polarization and flat-top beam shape into the central cube. The angle of incidence is controlled using a motorized stage to switch between epifluorescence, HILO- and TIRF-imaging. A flip-in lens can be used to focus the beam at the sample plane for bleaching experiments. III) The detection pathway features a triple-color image splitter, projecting the field-of-view onto different regions of the sCMOS camera chip for simultaneous triple-color imaging. Two mirrors on magnetic mounts can be used to bypass the triple-color image splitter. IV) The focus stabilization pathway uses an infrared laser to detect and compensate for axial drift of the sample. V) A pre-assembled laser combiner box delivers the excitation laser beam via a single-mode fiber to the setup.

Article Snippet: The sample stage of the K2 single-molecule TIRF microscope is equipped with a three-axis piezo stick-slip positioner (SLS-5252, Smaract) and a two-piece aluminum sample holder that accommodates square (18 mm to 26 mm side length), circular (25 mm to 37 mm diameter) and rectangular (62 mm to 80 mm length, up to 35 mm width) samples.

Techniques: Microscopy, Imaging

Journal: bioRxiv

Article Title: The K2: Open-source simultaneous triple-color TIRF microscope for live-cell and single-molecule imaging

doi: 10.1101/2022.12.19.521031

Figure Lengend Snippet: Central cube of the K2 microscope, showing the TIRF objective mounted on a thermal spacer, the sample stage with sample holder, and the dichroic mirror for splitting emitted fluorescence from excitation laser light inside the cube. The plexiglass cover encloses the objective and sample for added thermal stability and protection, and also provides a brightfield light source. Inset: The placement of the detection port on the central cube determines a beam height of 65 mm. The beam height of the excitation path is matched by placing the optical elements on an elevated breadboard, allowing the user to interchange alignment tools and pillar posts between the excitation and detection pathways.

Article Snippet: The sample stage of the K2 single-molecule TIRF microscope is equipped with a three-axis piezo stick-slip positioner (SLS-5252, Smaract) and a two-piece aluminum sample holder that accommodates square (18 mm to 26 mm side length), circular (25 mm to 37 mm diameter) and rectangular (62 mm to 80 mm length, up to 35 mm width) samples.

Techniques: Microscopy, Fluorescence

Journal: bioRxiv

Article Title: The K2: Open-source simultaneous triple-color TIRF microscope for live-cell and single-molecule imaging

doi: 10.1101/2022.12.19.521031

Figure Lengend Snippet: Detection pathway options of the K2 TIRF. Left: triple-color detection pathway with adjustable slit aperture and image splitter for simultaneous imaging of up to three color channels. Right: single-color full field-of-view path.

Article Snippet: The sample stage of the K2 single-molecule TIRF microscope is equipped with a three-axis piezo stick-slip positioner (SLS-5252, Smaract) and a two-piece aluminum sample holder that accommodates square (18 mm to 26 mm side length), circular (25 mm to 37 mm diameter) and rectangular (62 mm to 80 mm length, up to 35 mm width) samples.

Techniques: Imaging

Journal: bioRxiv

Article Title: The K2: Open-source simultaneous triple-color TIRF microscope for live-cell and single-molecule imaging

doi: 10.1101/2022.12.19.521031

Figure Lengend Snippet: a ) Schematic of the qgFocus lock: an infrared laser beam is focused off-center at the back focal plane of the objective and is totally internally reflected at a coverslip. Upon an axial movement Δ Z of the coverslip, the reflection of the beam is shifted laterally by a distance Δ d , which is translated into a distance Δ d ′ through downstream optics. A sensor registers the intensity profile of the back reflected beam and determines its center by a Gaussian fit. A control loop acting on the sample stage moves the sample such that the displacement of the currently measured intensity profile and of the referenced intensity profile is approaching zero. b ) Render of the focus stabilization pathway of the K2 TIRF, mounted alongside the excitation optics on an elevated breadboard. Inset: Separation of incoming infrared laser beam and back-reflected beam by a beamsplitter cube.

Article Snippet: The sample stage of the K2 single-molecule TIRF microscope is equipped with a three-axis piezo stick-slip positioner (SLS-5252, Smaract) and a two-piece aluminum sample holder that accommodates square (18 mm to 26 mm side length), circular (25 mm to 37 mm diameter) and rectangular (62 mm to 80 mm length, up to 35 mm width) samples.

Techniques:

Journal: bioRxiv

Article Title: The K2: Open-source simultaneous triple-color TIRF microscope for live-cell and single-molecule imaging

doi: 10.1101/2022.12.19.521031

Figure Lengend Snippet: Picture of the K2 TIRF microscope. From right to left, there are the excitation and focus lock pathways on an elevated breadboard, the central cube with the objective, sample stage and a plexiglass cover, and the triple- and single-color detection pathways with the camera on the left. The excitation and detection pathways are enclosed in light-tight aluminium boxes to reduce background light levels, minimize laser safety issues, and to prevent dust from building up.

Article Snippet: The sample stage of the K2 single-molecule TIRF microscope is equipped with a three-axis piezo stick-slip positioner (SLS-5252, Smaract) and a two-piece aluminum sample holder that accommodates square (18 mm to 26 mm side length), circular (25 mm to 37 mm diameter) and rectangular (62 mm to 80 mm length, up to 35 mm width) samples.

Techniques: Microscopy