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