A guide to transport-of-intensity equation (TIE) imaging for biologists

The measurement of dry cell mass (often referred to as "protein") under the microscope can be accomplished using a quantitative phase imaging technique known as Transport of Intensity Equation (TIE) microscopy. This method requires no specialized equipment, relying instead on two slightly defocused brightfield images acquired with a standard optical microscope. The images are processed by the TIE equation to convert the gradient of intensity into phase shifts and ultimately a distribution of protein mass. Beyond its simplicity, a major advantage of TIE over most other quantitative phase methods is its compatibility with fluorescence and with cell volume measurements. When paired with volume data, TIE enables the conversion of protein mass into the biologically significant parameters of protein concentration and intracellular water content. This review emphasizes practical implementation, including calibration, focal plane selection, reproducibility, image size effects, strategies for artifact reduction, as well as the biological relevance of the recovered phase. We also describe an easy-to-use Fiji plugin for solving the TIE equation, eliminating the need for advanced computational tools.