Table 4.

Troubleshooting table.

Step	Problem	Possible reason	Solution
26 and 29	Hair shadows	Hair fragments lying on	Thoroughly clean the skull surface before imaging, as even the smallest hair will cast a shadow of at least 20–30 µm wide. It can be helpful to clip the scalp hair before the surgery; however, this generates extra hair fragments, which can eventually be deposited on the scalp. Hair clipping can be replaced by thorough cleaning after the incision. Alternatively, depilatory creams can be used; however, these require prolonged anesthesia, which can be problematic with ketamine-xylazine. Regardless, it is crucial to thoroughly clean the skull surface by flushing it with PBS and wiping it with sterile gauze. White hair is particularly difficult to see by eye and therefore special care must be taken when working with white mice
33	No cells detected	One of the following problems may have occurred: The cell pellet was aspirated during washing DiD staining was not efficient	See Step 17 for suggestions on how to avoid this Minimize the amount of serum remaining in the cell suspension. Serum components bind efficiently to DiD and sequester it from the cells, which remain unlabeled. Different cell types require different staining protocols to achieve similar final results. When working with a new cell population, titrate the optimal DiD concentration and check the efficiency of staining by FACS analysis, using, for example, 633-nm excitation and an APC-cy7 filter in order to reduce the problems arising from detection of very bright, off-scale signal from the Cy5 filter/detector
35	Insufficient length of anesthesia	Logistical setup and excessively lengthy image acquisition	Perform the surgery near the microscope or microscopy facility. Develop a series of checkpoints to ensure appropriate positioning of the mouse and an optimized imaging routine. Maintain the same imaging routine to ensure efficiency and reproducibility between experiments. If necessary, image only half of the calvarium (e.g., the right half), assuming that the other half would contain a similar number of HSPCs in similar positions (this reduces the collected data and the sample size (n) for future statistical analysis). Alternatively, try a slightly higher dose of ketamine-xylazine cocktail or substitute the cocktail with isoflurane anesthesia
	Out-of-focus signal	Signal spillage from very bright osteoblasts or difficulty in balancing signals in a z-stack Resolution in the optical plane (xy) is higher than the axial resolution (z) and therefore only vertical bone marrow cavity walls appear clearly demarcated (as opposed to a gradual slice-to-slice shift)	Compare the signal of each structure of interest throughout the image series and make a consistent judgment about what is in focus and what is not as well as where bone and osteoblasts are located within the area of interest (e.g., only consider signal in focus or only consider signal of a certain relative intensity). In many cases, blurred or dim signal in one slice will be strong and clearly in focus in others. Clear and consistent standards for each analysis minimize person-toperson variability. It is expected that measurements repeated by the same or different investigators may differ by few µm
	Unexpected mouse death	An excess amount of gel/saline solution overflows the imaged area, covers the nostrils and causes suffocation Excessive pressure on the mouse head from the cover slip or the objective causes suffocation by compression of the throat An overdose of anesthetics is accidentally administered	Reduce amount of gel/saline solution used Adjust the mouse within the holder in order to avoid localized pressure on the throat Reduce the dose of anesthetic agent
36B(ii)	Cell tracking difficulties	Shifts in the mouse position between imaging sessions and potential alterations within the bone marrow microenvironment	Carefully reposition the mouse and calculate the angle of positional shift in all three dimensions. Acquiring z-stacks for multiple signals can facilitate recognizing the same area through the spatial relationship of multiple components. Little is known about the effects of irradiation and of the imaging itself on the bone marrow microenvironment. Occasionally, we have observed increased numbers of osteoblasts during the second imaging session (C.L.C. and C.P.L., unpublished observations). If you are interested in measuring HSPC-osteoblast distance a few days after transplantation, we recommend not imaging the mouse until the time point of interest