Minimal requirements for reporting microscopy methods, including machine-readable metadata, shaded (LiMi-model metadata specifications), and representative examples to facilitate microscopy methods reporting.
| Categories . | . | Examples . | Machine-readable LiMi-model alignment . |
|---|---|---|---|
| Specimen setup | |||
| Sample mounting | Cover glass (cover glass number or thickness; coating) | Samples were grown on #1.5H cover glass (Marienfeld), coated with 1 mg/ml collagen type I (C8919; Sigma-Aldrich) | CoverGlass/CoverGlassNo CoverGlass/Thickness CoverGlass/Coating |
| Mounting medium or imaging medium (name and manufacturer) | Prior to imaging, samples were mounted in SlowFade Glass mounting medium (Thermo Fisher Scientific) | MountingMedium/Model MountingMedium/Manufacturer | |
| Sample labelinga | Fluorescent protein (specific variant or probe) | mGFPmut3, GCaMP6f | |
| Dye (name, manufacturer, and concentration) | MitoTracker Green at 1 µg/ml final Secondary antibody conjugated to Alexa Fluor 647 | | |
| Hardware setup | |||
| Microscope stand | Description (manufacturer; model; inverted or upright) | Microscopy imaging experiments were performed on a Nikon Ti2 inverted microscope stand | MicroscopeStand/Manufacturer MicroscopeStand/Model MicroscopeStand subtype (Inverted, Upright) |
| Modalities and modules/add- on | Specify the modalities and modules usedb | Microscope stand was equipped with a Yokogawa spinning disk CSU-W1 and a SORA module | Pixels/Channel/IlluminationType (Wide-field_Fluorescence, Confocal_Fluorescence_array-raster- scan, Confocal_Fluorescence_spinning disk) |
| For phase contrast imaging, we used a phase contrast objective and a respective phase plate in the condenser | |||
| Fluorescent images were captured on a Zeiss Observer.Z1 widefield microscope equipped with an Apotome module | |||
| Objective and additional magnification | Full designation (description, specification) found on the barrel of the objective (magnification, numerical aperture, correction type, and immersion type) | Images were acquired using a 100×/1.45 DIC Plan Apochromat, oil immersion objective | Objective/Magnification Objective/LensNA Objective/Correction Objective/ImmersionType |
| Additional Magnification (magnification changer) | In addition, a 1.5× Optovar was inserted in the Lightpath | MagnificationChanger | |
| Light source | Manufacturer and model (for non-laser light sources) Type (for non-laser light sources) (e.g., LED, mixed metal halide, and mercury) | Samples were illuminated using an LED light engine (Spectra X, Lumencor) | LightSource subtype (Arc, Filament, GenericLightSource, Laser, LightEmittingDiode, MultiLaserEngine) LightSource/Manufacturer |
| Specify the excitation wavelength used (for laser-based) | DAPI excitation was performed using a 405 nm diode laser | LightSource/Model LightSource/Laser LightSource/PeakWavelength | |
| Wavelength selection | Specific filters or filter cubes (excitation filter center wavelength/FWHM, emission filter center wavelength/FWHM, and optionally company, filter name) | Alexa Fluor 488 was imaged using filter cube 38 HE (Zeiss, BP 470/40 Ex, DC495 dichroic, BP525/50Em) | FilterCube Filter/TransmittanceRange/Wavelength Filter/TransmittanceRange/FWHMBandwidth Filter/Manufacturer Filter/Model |
| Adjustable wavelength selector (e.g., spectral detection in a point scanner), cut on/cutoff wavelengths | eGFP emission was detected with a spectral detector between 500 and 544 nm, with a spectral width of 8.9 nm for the single detection elements | Dichroic/TransmittanceRange/Wavelength | |
| Detection system (as applicable) | Camera (manufacturer and model) | An Orca Flash 4.0 (Hamamatsu) monochrome camera | Camera/Manufacturer Camera/Model |
| Point detector (type) | A 32-channel GaAsP detector was used to detect the emitted signal | PointDetector subtype (Multialkali/GaAsP)PhotomultiplierTube, PhotoDiode, HybridPhotoDetector) | |
| Acquisition setup | All acquisition settings optimized to acquire the image must be reported, and should include but not be limited to: | ||
| Acquisition settings | Camera-based: exposure time | The DAPI channel was captured with 30 ms exposure time (camera based) | Pixels/Plane/ExposureTime |
| Point detector–based: pinhole size (in AU), pixel dwell or scan speed, simultaneous or sequential acquisition | The images were captured sequentially, with a pixel dwell time of 2 µs and a 1 AU pinhole and no averaging | PinholeSettings/Aperture Pixels/Plane/PixelDwellTime ConfocalScannerSettings/ScanningFrequency ConfocalScannerSettings/MultiChannelMode(Parallel or Sequential) | |
| Final effective image pixel size (in the image) | Final image pixel size was 0.065 um/pixel | Pixels/PhysicalSizeX Pixels/PhysicalSizeY | |
| Z-stack settings (z-step increment, number of steps, total range) | Images were acquired as 15 µm range z-stacks with a 100 nm z-step interval | Pixels/PhysicalSizeZ Pixels/SizeZ | |
| Time series settings (time increment and total acquisition time) | Time-lapse imaging was performed for 2.5 h with a 10 min interval | Pixels/Plane/TimeIncrement | |
| Tiling settings | Tiling was performed with a 10% overlap | | |
| Acquisition software | Name, manufacturer, and version | NIS-Elements AR V5.21 (Nikon) Micro-manager 2.0.0 | AcquisitionSoftware/Name AcquisitionSoftware/Developer AcquisitionSoftware/Version |
| Categories . | . | Examples . | Machine-readable LiMi-model alignment . |
|---|---|---|---|
| Specimen setup | |||
| Sample mounting | Cover glass (cover glass number or thickness; coating) | Samples were grown on #1.5H cover glass (Marienfeld), coated with 1 mg/ml collagen type I (C8919; Sigma-Aldrich) | CoverGlass/CoverGlassNo CoverGlass/Thickness CoverGlass/Coating |
| Mounting medium or imaging medium (name and manufacturer) | Prior to imaging, samples were mounted in SlowFade Glass mounting medium (Thermo Fisher Scientific) | MountingMedium/Model MountingMedium/Manufacturer | |
| Sample labelinga | Fluorescent protein (specific variant or probe) | mGFPmut3, GCaMP6f | |
| Dye (name, manufacturer, and concentration) | MitoTracker Green at 1 µg/ml final Secondary antibody conjugated to Alexa Fluor 647 | | |
| Hardware setup | |||
| Microscope stand | Description (manufacturer; model; inverted or upright) | Microscopy imaging experiments were performed on a Nikon Ti2 inverted microscope stand | MicroscopeStand/Manufacturer MicroscopeStand/Model MicroscopeStand subtype (Inverted, Upright) |
| Modalities and modules/add- on | Specify the modalities and modules usedb | Microscope stand was equipped with a Yokogawa spinning disk CSU-W1 and a SORA module | Pixels/Channel/IlluminationType (Wide-field_Fluorescence, Confocal_Fluorescence_array-raster- scan, Confocal_Fluorescence_spinning disk) |
| For phase contrast imaging, we used a phase contrast objective and a respective phase plate in the condenser | |||
| Fluorescent images were captured on a Zeiss Observer.Z1 widefield microscope equipped with an Apotome module | |||
| Objective and additional magnification | Full designation (description, specification) found on the barrel of the objective (magnification, numerical aperture, correction type, and immersion type) | Images were acquired using a 100×/1.45 DIC Plan Apochromat, oil immersion objective | Objective/Magnification Objective/LensNA Objective/Correction Objective/ImmersionType |
| Additional Magnification (magnification changer) | In addition, a 1.5× Optovar was inserted in the Lightpath | MagnificationChanger | |
| Light source | Manufacturer and model (for non-laser light sources) Type (for non-laser light sources) (e.g., LED, mixed metal halide, and mercury) | Samples were illuminated using an LED light engine (Spectra X, Lumencor) | LightSource subtype (Arc, Filament, GenericLightSource, Laser, LightEmittingDiode, MultiLaserEngine) LightSource/Manufacturer |
| Specify the excitation wavelength used (for laser-based) | DAPI excitation was performed using a 405 nm diode laser | LightSource/Model LightSource/Laser LightSource/PeakWavelength | |
| Wavelength selection | Specific filters or filter cubes (excitation filter center wavelength/FWHM, emission filter center wavelength/FWHM, and optionally company, filter name) | Alexa Fluor 488 was imaged using filter cube 38 HE (Zeiss, BP 470/40 Ex, DC495 dichroic, BP525/50Em) | FilterCube Filter/TransmittanceRange/Wavelength Filter/TransmittanceRange/FWHMBandwidth Filter/Manufacturer Filter/Model |
| Adjustable wavelength selector (e.g., spectral detection in a point scanner), cut on/cutoff wavelengths | eGFP emission was detected with a spectral detector between 500 and 544 nm, with a spectral width of 8.9 nm for the single detection elements | Dichroic/TransmittanceRange/Wavelength | |
| Detection system (as applicable) | Camera (manufacturer and model) | An Orca Flash 4.0 (Hamamatsu) monochrome camera | Camera/Manufacturer Camera/Model |
| Point detector (type) | A 32-channel GaAsP detector was used to detect the emitted signal | PointDetector subtype (Multialkali/GaAsP)PhotomultiplierTube, PhotoDiode, HybridPhotoDetector) | |
| Acquisition setup | All acquisition settings optimized to acquire the image must be reported, and should include but not be limited to: | ||
| Acquisition settings | Camera-based: exposure time | The DAPI channel was captured with 30 ms exposure time (camera based) | Pixels/Plane/ExposureTime |
| Point detector–based: pinhole size (in AU), pixel dwell or scan speed, simultaneous or sequential acquisition | The images were captured sequentially, with a pixel dwell time of 2 µs and a 1 AU pinhole and no averaging | PinholeSettings/Aperture Pixels/Plane/PixelDwellTime ConfocalScannerSettings/ScanningFrequency ConfocalScannerSettings/MultiChannelMode(Parallel or Sequential) | |
| Final effective image pixel size (in the image) | Final image pixel size was 0.065 um/pixel | Pixels/PhysicalSizeX Pixels/PhysicalSizeY | |
| Z-stack settings (z-step increment, number of steps, total range) | Images were acquired as 15 µm range z-stacks with a 100 nm z-step interval | Pixels/PhysicalSizeZ Pixels/SizeZ | |
| Time series settings (time increment and total acquisition time) | Time-lapse imaging was performed for 2.5 h with a 10 min interval | Pixels/Plane/TimeIncrement | |
| Tiling settings | Tiling was performed with a 10% overlap | | |
| Acquisition software | Name, manufacturer, and version | NIS-Elements AR V5.21 (Nikon) Micro-manager 2.0.0 | AcquisitionSoftware/Name AcquisitionSoftware/Developer AcquisitionSoftware/Version |
Sample preparation is essential for rigorous and reproducible experiments but is covered in other sections of the Materials and methods. This includes detailed protocols for growth, transfection, and imaging conditions (for live-cell experiments), as well as labeling methods such as fixation, permeabilization, and antibody concentrations. We include the fluorescent protein variant or dye used, as this information, often omitted, is crucial for evaluating the hardware setup and acquisition settings.
Modality refers to a microscopy technique such as wide-field, confocal, light-sheet, two photon, STED, etc. Modules are hardware components that enable a modality, such as a confocal scan unit or TIRF arm.