Summary of metrics and troubleshooting
| Monitoring points (measuring frequency) . | Metrics . | Limit values . | Possible reasons for out of limit values: Proposed solutions . |
|---|---|---|---|
| Point spread function (once per mo) | FWHM XY exp/theory | <1.5 | Dirty or damaged objective: Meticulous front lens cleaning. Damaged objective: Close examination of the front lens (scratches, lens detachment); send to repair. Pinhole and collimator lens misalignment (LSCM): Check the alignment. Optical aberrations: Check the manufacturer correction specifications (APO, PlanAPO…); remove DIC or additional lens from the light path; clean optical elements in the light path; regulate correction collar for NA, temperature, or cover slip thickness. Sample: Adjust stage and sample flatness; choose #1.5 (0.17 mm thick) cover slip; image beads near cover slip; use appropriate mounting or immersion medium at the right temperature; make sure there are no bubbles in the immersion oil. Vibrations-drifts: Use an anti-vibration optical table and regulate air pressure properly; do a short time-lapse of a single bead. Galvanometric scanners (LSCM): Calibrate bidirectional mode; check at mono-directional mode. Excitation light alignment and polarization: Check sources coalignment and inspect light guide (polarization, bends). |
| FWHM Z exp/theory | <1.5 | ||
| LAR | Case-dependent illumination source | ||
| Field illumination (twice per yr) | Uniformity (U) | >50% | Damaged objective: Close examination of the front lens (scratches, lens detachment); send to repair. Large camera sensor chip size (WF, SDCM): Crop the central field of view of the camera sensor chip. Incorrect position of the field diaphragm (WF): Centering of the field diaphragm. Sources and system alignment: Adjusting the collector lens or liquid light guide position; checking possible bending of the optical fibers; coalignment of the light sources; pinhole alignment; beam size at the back focal plane of the objective. UV misalignment: Check the manufacturers’ correction specifications of the optical path components. |
| Centering (C) | >20% | ||
| Co-registration (twice per yr) | Ratio rexp/rref | 1 | Chromatic aberrations: Check the constructor correction specifications of the considered lens (APO, PlanAPO…). Damaged objective: Close examination of the front lens (scratches, lens detachment); send to repair. Sources and system alignment: Dichroic mirror and filter positions; additional lenses and light sources alignment. Micro-lenses disk (SDCM): Check the constructor correction specifications of the optical path components. |
| Illumination power stability (once per yr) | Stability STABpower | >97% | Laser dying: Thorough check by the constructor. Electrical instabilities: AC-powered light source; check the power grid stability of the room. Environmental variations (thermal, air flow, humidity...): General verification of environmental and vibration conditions. Fiber polarization: Check the bends of the optical fibers; proper alignment of laser in fiber. Defective optical components (AOTF, AOM, fiber): Thorough check by the constructor. |
SDnormalized intensity | <0.2 | ||
| Stage drift (once per yr) | Va: Mean velocity after stabilization | [0–50] nm/min | Environmental variations (thermal, air flow, humidity...): General verification of environmental conditions; protect the stage from thermal shifts (cover, temperature-controlled chamber, or incubator); let the stage stabilize before starting experiments. Stage control components not properly working: Check joystick at “no move” position, check controller status. Mechanical instability: Tighten the mechanical elements; thorough check by the constructor. |
| Vb: Mean velocity before stabilization | [0–100] nm/min | ||
| τstab | <120 min | ||
| Stage positioning repeatability (once per yr) | σpositioning deviation | Stage specifications | Environmental variations (thermal, air flow, humidity...): General verification of environmental conditions. Mechanical instability: Clean pieces of broken cover slip; tighten mechanical elements; thorough check by the constructor. Objective touches sample insert if position far from center: Change insert or limit movements to central positions. Not enough oil on the objective: Add enough oil beneath the whole sample before starting an experiment. Stage drift: Let the stage stabilize before starting a multiposition experiment. |
| Camera read noise (once per yr) | VAR STABnoise | >90% >97% | Electronics issue; detector temperature; detector aging; cosmic rays: Thorough check by the constructor. |
| Monitoring points (measuring frequency) . | Metrics . | Limit values . | Possible reasons for out of limit values: Proposed solutions . |
|---|---|---|---|
| Point spread function (once per mo) | FWHM XY exp/theory | <1.5 | Dirty or damaged objective: Meticulous front lens cleaning. Damaged objective: Close examination of the front lens (scratches, lens detachment); send to repair. Pinhole and collimator lens misalignment (LSCM): Check the alignment. Optical aberrations: Check the manufacturer correction specifications (APO, PlanAPO…); remove DIC or additional lens from the light path; clean optical elements in the light path; regulate correction collar for NA, temperature, or cover slip thickness. Sample: Adjust stage and sample flatness; choose #1.5 (0.17 mm thick) cover slip; image beads near cover slip; use appropriate mounting or immersion medium at the right temperature; make sure there are no bubbles in the immersion oil. Vibrations-drifts: Use an anti-vibration optical table and regulate air pressure properly; do a short time-lapse of a single bead. Galvanometric scanners (LSCM): Calibrate bidirectional mode; check at mono-directional mode. Excitation light alignment and polarization: Check sources coalignment and inspect light guide (polarization, bends). |
| FWHM Z exp/theory | <1.5 | ||
| LAR | Case-dependent illumination source | ||
| Field illumination (twice per yr) | Uniformity (U) | >50% | Damaged objective: Close examination of the front lens (scratches, lens detachment); send to repair. Large camera sensor chip size (WF, SDCM): Crop the central field of view of the camera sensor chip. Incorrect position of the field diaphragm (WF): Centering of the field diaphragm. Sources and system alignment: Adjusting the collector lens or liquid light guide position; checking possible bending of the optical fibers; coalignment of the light sources; pinhole alignment; beam size at the back focal plane of the objective. UV misalignment: Check the manufacturers’ correction specifications of the optical path components. |
| Centering (C) | >20% | ||
| Co-registration (twice per yr) | Ratio rexp/rref | 1 | Chromatic aberrations: Check the constructor correction specifications of the considered lens (APO, PlanAPO…). Damaged objective: Close examination of the front lens (scratches, lens detachment); send to repair. Sources and system alignment: Dichroic mirror and filter positions; additional lenses and light sources alignment. Micro-lenses disk (SDCM): Check the constructor correction specifications of the optical path components. |
| Illumination power stability (once per yr) | Stability STABpower | >97% | Laser dying: Thorough check by the constructor. Electrical instabilities: AC-powered light source; check the power grid stability of the room. Environmental variations (thermal, air flow, humidity...): General verification of environmental and vibration conditions. Fiber polarization: Check the bends of the optical fibers; proper alignment of laser in fiber. Defective optical components (AOTF, AOM, fiber): Thorough check by the constructor. |
SDnormalized intensity | <0.2 | ||
| Stage drift (once per yr) | Va: Mean velocity after stabilization | [0–50] nm/min | Environmental variations (thermal, air flow, humidity...): General verification of environmental conditions; protect the stage from thermal shifts (cover, temperature-controlled chamber, or incubator); let the stage stabilize before starting experiments. Stage control components not properly working: Check joystick at “no move” position, check controller status. Mechanical instability: Tighten the mechanical elements; thorough check by the constructor. |
| Vb: Mean velocity before stabilization | [0–100] nm/min | ||
| τstab | <120 min | ||
| Stage positioning repeatability (once per yr) | σpositioning deviation | Stage specifications | Environmental variations (thermal, air flow, humidity...): General verification of environmental conditions. Mechanical instability: Clean pieces of broken cover slip; tighten mechanical elements; thorough check by the constructor. Objective touches sample insert if position far from center: Change insert or limit movements to central positions. Not enough oil on the objective: Add enough oil beneath the whole sample before starting an experiment. Stage drift: Let the stage stabilize before starting a multiposition experiment. |
| Camera read noise (once per yr) | VAR STABnoise | >90% >97% | Electronics issue; detector temperature; detector aging; cosmic rays: Thorough check by the constructor. |
Summary of the basic metrics, the recommended measuring frequency, the experimental limit values, the possible reasons associated with their exceeding values, and troubleshooting advice.