The super depth of field microscope breaks through the depth of field limitation of traditional microscopes through optical system optimization or digital image synthesis technology, achieving clear observation of large depth samples. Its debugging requires a combination of optical adjustment, parameter optimization, and software algorithms. The debugging process is elaborated in detail from four aspects: hardware calibration, software settings, lighting control, and imaging verification.

1、 Hardware calibration and mechanical parameter adjustment

1. Matching the objective lens with the working distance

-Objective lens selection: Low magnification objectives (such as 2 ×~5 ×) are preferred due to their large field of view and natural deep depth of field, making them suitable for super depth of field observation. Although high-power objective lenses have high resolution, their depth of field is extremely shallow, and they need to be used in conjunction with digital synthesis technology.

-Working distance adjustment: By lifting the stage or focusing knob, the distance from the front end of the objective lens to the surface of the sample should be slightly greater than the nominal working distance (such as leaving a 12mm margin when the working distance of the objective lens is 10mm), to avoid the lens touching the sample.

2. Align the spotlight with the light path

-Aperture adjustment of spotlight: Reduce the aperture of the spotlight to 60%~80% of the objective field of view, reduce stray light interference, and improve contrast. If it is an adjustable aperture stop, it needs to match the numerical aperture (NA) of the objective lens (such as NA0.2, the aperture corresponding to the objective lens is ≤ 0.2).

-Optical axis calibration: Use specialized centering specimens (such as cross grids) to adjust the position of the light source, ensuring that the light is evenly and symmetrically incident on the objective lens, and avoiding edge vignetting effects.

3. Camera parameter initialization

-Sensor focusing: Adjust the object distance through the real-time preview interface to make the sample form a clear image on the sensor target surface. If equipped with autofocus function, multiple points need to be selected on the sample surface for calibration to avoid defocusing caused by local tilting.

-Resolution verification: Shoot a resolution test target (such as a USAF target), check if the image can distinguish the highest line width, and confirm that the objective camera combination reaches the theoretical resolution.

2、 Software parameter optimization and depth of field synthesis

1. Depth of Field Expansion Mode Setting

-Multi focus stack: Enable the software's "depth of field synthesis" function, set the number of shots (usually 5-15), focus step (adjusted according to the height difference of the sample, such as 10 μ m/step), and synthesis algorithm (such as maximum clarity fusion or multi frame alignment overlay).

-Dynamic range optimization: For high reflectivity or dark field samples, enable HDR (High Dynamic Range) mode to preserve details in bright and dark areas through multi exposure synthesis.

2. Adjustment of image enhancement parameters

-Denoising processing: Under low light conditions, increase the gain value appropriately, but synchronously enable denoising algorithms (such as dark current correction or spatiotemporal filtering) to avoid speckle noise.

-Sharpening and Contrast: Limited sharpening (intensity<20%) is applied to composite images to enhance edge details; Contrast adjustment is based on histogram equalization to avoid color distortion caused by excessive stretching.

3. Automated process configuration

-Z-axis scanning preset: Enter the sample height range, and the software automatically calculates the focal point distribution, suitable for multi-layer structures or concave convex surfaces.

-Region of Interest (ROI) Selection: Box the key observation area to prioritize clarity and reduce computation time for irrelevant regions.

3、 Lighting system regulation and uniformity optimization

1. Balance between light source intensity and color temperature

-Intensity grading adjustment: Set the brightness of the light source according to the sample transmittance grading (such as using low-power LED for transparent samples and high brightness halogen lamps for metal reflective samples), to avoid overexposure or underexposure.

-Color temperature correction: Use a standard whiteboard to capture reference images and adjust the color temperature of the light source to 5500K~6500K to ensure consistent color reproduction.

2. Kola lighting and uniform light treatment

-Kola effect application: By adjusting the conjugation between the spotlight and the light source, the filament image is uniformly projected on the sample plane, eliminating circular light spots.

-Diffuser assistance: Add frosted glass or diffusion film to the light path to further soften the light and solve the glare problem in high reflective areas.

3. Polarization and dark field mode

-Polarized light adjustment: Rotate the polarizer to the extinction state, highlighting the sample's stress birefringence or crystal anisotropy.

-Dark field illumination: Turn off direct light and only retain scattered light, used to observe submicron particles or small defects.

4、 Imaging verification and troubleshooting

1. Depth of Field Quantization Detection

-Ladder ruler test: Place calibration samples engraved with steps of different heights, take composite images, measure the maximum height difference that can be clearly distinguished, and verify whether the nominal depth of field (such as ± 5mm) is achieved.

-Longitudinal resolution evaluation: By observing the separation degree of different layers through tilted samples, ensure that the synthesis algorithm does not introduce artifacts.

2. Common problem solutions

-Edge blur: Check if the objective lens is perpendicular to the sample or increase the focal step.

-Color deviation: recalibrate white balance and clean lens stains.

-Composite failure: Confirm the alignment accuracy of multiple frames of images to avoid displacement exceeding the threshold caused by vibration.

3. Maintenance and long-term stability

-Regular cleaning: Use a dust-free cloth to wipe the objective lens and spotlight to avoid dust scattering and affecting contrast.

-Calibration review: Monthly check the alignment of the optical axis and the calibration of the focal length to prevent mechanical drift.