• If your tissue bleaches quickly, making live image work difficult
• Double labeling: if you need to look for co-localization of cells in two channels (such as DAPI to identify cells and GFP to identify astrocytes)
• When auditing other users’ probe runs

All microscope systems are subject to a phenomenon called "thermal drift." Once power is on, the hardware begins to heat up, which can cause the focal plane to shift by several micrometers. This results in problems for users who set up their focus map before the system has fully warmed up: over the course of the acquisition, the focus map may drift by several micrometers, making the resulting images unusable.

To avoid this problem, power on your system at least an hour before setting up your focus maps.

Thermal drift during acquisitions can also be caused by HVAC ducts positioned over/near your microscope.

Typically, we recommend measuring tissue thickness while counting. However, when working from image stacks, the accuracy of your tissue thickness measurements is limited by the Z distance between image planes - that is, if you have 3 micrometers between planes, your tissue thickness measurements may be off by +/- 3μm.

Because of this, it may help to:

1. Manually measure your tissue thickness at 5-10 sites per section (the DAPI channel or another channel with a lot of cells works well for this),
2. Manually enter your tissue thickness before you start counting.

This can be done using step 8 of the SRS Image Stack workflow, step 5 of the Optical Fractionator workflow, or by using the Edit Tissue Thickness button in the Optical Fractionator results window.

When using the Optical Fractionator, in order to give each cell an equal chance to be counted once and only once, it's critical to identify a unique point of the cell that will be marked.

If working directly from the live image, this is usually the top of the cell or the top of the nucleus. When working from SRS image stacks, it becomes time consuming to acquire enough planes to accurately find the FIRST plane where the top of the cell is in focus. Instead, most researchers set their Z scaling so that there are 2-3 planes per cell, and then mark the first image plane where their cell appears in focus.

It's worth noting that tissue shrinkage during processing impacts cells, but the distance between planes of your image stack still refers to the absolute distance in Z. For example, a 20μm cell may shrink during labeling to be only 5 or 6μm-thick. To ensure that this cell will appear in 2-3 planes of the image stack, you need to set the distance between planes to 2μm. If the distance between planes is set to 8μm, there's a chance that the cell could fall between planes and never be counted - this would introduce bias in the form of an underestimation of the total number of cells present.