The aim of the “Primary Screen” is to find and locate candidates or structures of interest. This is achieved with low resolution, low optical sectioning performance and with the aim of altering the sample as little as possible. On the other hand, the primary scan has to be performed at maximum scan speed so as not to miss a short event in a large number of objects. Typically, samples for screening grow in multi-well plates. These are polymer stage inserts containing a number of cavities (here named “wells”). In each well a single individuum or a small number of e.g. cells is usually growing. The grid structure of the well plate allows easy object relocation and change monitoring. To fit to varying sample sizes, well plates with 6, 24 or 96 wells, etc. are available. For example, zebra fish embryos at different stages of development are typical objects analyzed in multi-well plates (where one fish embryo is seeded into one well). The aim of Primary Screen is to get sufficient information about every well at appropriate time resolution. This information is then used to recognize and decide which samples in which wells are of interest and should be investigated further in a so called Secondary Screen (see below).
Primary screening is performed by observing large fields of view. Sometimes a camera is suitable for this task. Confocal provides better contrast, modern systems offer low magnification optics for imaging a complete well at once. Confocal imaging at large pinhole settings is sufficient (this allows out-of-focus events to be found, too). Two-channel recording is also helpful, such as a fluorescence image and a transmitted light image simultaneously. All parameters needed for a primary scan are stored in a “scan job”, which is retrievable for continuing the screening process after analyzing a rare event – or for starting a new experiment the next day.
Before starting a matrix screen experiment, the system needs to know the start coordinates and the distances of the wells in x- and y-direction. As wells are aligned in a regular Cartesian pattern, the system can then automatically move from well to well, either in a regular manner from upper left to lower right, or in a predefined sequence, where all wells are addressed after manual selection by the operator. Different scan jobs can be created and freely combined at multiple positions. For reproducibility these screens, xy coordinates as well as all other relevant imaging scan parameters are storable in a template. The advantage of storing parameters in templates is obvious: if you exchange the multi-well plate, only the start coordinate has to be readjusted.
This automation saves a significant amount of time; more experiments can be run in a shorter time in a standardized way.