Z-stacking (also known as focus stacking) is a digital image processing method commonly used to overcome this obstacle. The technique combines multiple images taken at different focal distances to provide a composite image with a greater depth of field (i.e. the thickness of the plane of focus) than any of the individual source images. It is particularly useful for capturing in-focus images of objects under high magnification.
Z-stacking is commonly done using confocal microscopy where the field of view is restricted both axially and longitudinally, much like in a pin hole camera, such that in-focus “slices” of the object can be acquired and z-stacked to form a composite 3D image of the object. However, because the excitation light illuminates the entire structure, photobleaching and phototoxic effects extend to all planes. While the lack of longitudinal restriction seen in widefield microscopy helps to eliminate these complications, parts of the object will appear in-focus and parts out-of-focus. In this case, z-stacking is still possible, but requires the use of deconvolution, a technique to get rid of this out-of-focus information by applying a mathematical algorithm. This provides sharper images that can be combined to yield more realistic 3D impressions of the structure of interest. In the application note referenced here, we demonstrate this technique to perform z-stacking of images of HCT116 tumoroids in a 3D cell culture scaffold.
Multiple images were captured with the Cytation 3 at different z-planes through the 3D cellular structures.The freeware program CombineZP was then used to perform z-stacking of the images. The process was easy to perform and provides a lower cost method to create clear final images that can be used for cellular analysis.
By: BioTek Instruments, Brad Larson, Principal Scientist