Table 1.
Troubleshooting Guide for QFSM analysis software
| Problem | Possible Cause | Solution |
|---|---|---|
| Noise Model Calibration | ||
| Low Gauss ratio (typically <3) | Cropped region contains parts of cell. | Modify region of interest along the stack depth. |
| Spatial and/or temporal range of the background region is too small. | Acquire a movie containing background only. Run the noise model calibration on this background movie. (a) | |
| Thresholding/Mask Refinement | ||
| Cell mask is not determined properly | Automatic thresholding fails | Use alternate thresholding methods/Set threshold value manually |
| Inhomogeneous background | Crop region of the movie for analysis. | |
| Multiple cells in the movie | Adjust the object number in mask refinement Crop each cell as a separate movie |
|
| Speckle detection | ||
| Misdetected speckles | Low Gauss ratio | See Noise Model Calibration above |
| Low signal to noise ratio | Increase alpha value | |
| Dense speckles are not resolved | Increase number of iterations | |
| Flow Tracking | ||
| Inhomogenous flow | Template size too short/big | Use variable template range to determine adapted size. |
| Integration window too short | Optimize integration window. | |
| Background is perturbing correlation | Use stationary background subtraction | |
| High number of outliers | Decrease outlier threshold | |
| Speckle Tracking | ||
| Short tracks | Track lifetime is too short | Use iterative speckle detection |
| Random flow directionality | Initialization problem |
|
| Kinetic Analysis/Flow Analysis | ||
| Maps | Sparse kinetic events cannot generate sufficient information | Increase time windows for averaging |
(a)
If none of these help then the camera noise is electronically correlated (can happen especially with first generation EM-CCD or sCMOS cameras); or there is a high level of stray light in the microscope.