Standard Cytoo chips are organized as 12x12 blocks, ecah block is made of a regular 12x12 or 9x9 grid of fluorescent fibronectin patterns. The patterns are either identical all over the chip or can have various shapes and sizes (starter chip), the blocks are always 1300 µm squares (Fig. 1).
The cells are seeded on the chip and fixed after some resting time. A proportion of the cells are isolated (one cell per pattern) while some patterns are empty or occupied by more than a cell. Depending on the study the user is typically interested in acquiring the images from isolated cells or dividing cells. Here the intelligent imaging comes into play.
Starter chip are typically used in a preliminary phase to test the cell / pattern compatibility. The different Cytoo shapes can also be used to break the symmetry of the cell environment. We developed a macro that identifies isolated cells during the primary scan and acquire them at high resolution during the secondary scan.
We perform intelligent imaging of the Cytoo chips on the Leica SP5 microscope to detect isolated nuclei (single cell sitting on a pattern), the method is described here. With the typical minimal settings required for the primary scan analysis (20x objective, Zoom set to 1) 2x2 fields of view per Cytoo blocks are necessary to tile each of the Cytoo blocks. The analysis function detects the Cytoo patterns in the pattern channel and computes the occupancy (number of cells) of each pattern by analyzing the images of the DAPI channel.
In our application high resolution images of the isolated cells (one a pattern) were acquired during the secondary scan (typically an overnight scan without user interaction). The analysis function can be easily extended to, for instance, only select the cells that are well spread on the patterns (by analyzing a third channel with a properly chosen marker of the cytoplasm). An additional macro (coming soon) is able to build Score Map from the previous analysis.
of empty and isolated cell per Cytoo block
The fluorescent channel of the Cytoo patterns The DAPI channel with the detected isolated cells
To test the macro download the experiment folder (part1, part2). Uncompress the folder. Download the main CAM macro for block analysis and the block analysis macro. Copy both of of them to Fiji macro folder and run the main macro.
In the first step select the folder ExperimentUVXY (folder where the microscope has saved the images).
In the main dialog box un-tick "Send CAM script" (we are running the macro offline, no microscope connected). Select "Cytoo_Isolated_Nucleus_Confocal" as pre analysis function and make sure the starting well position is (0,0) and the ending well position is (1,1), that is 4 Cytoo blocks in total.
Select the analysis area in the first Cytoo block. Draw a circle around a pattern to define the analysis region (it should be larger than the pattern itself). The four blocks are processed and 4 montages showing the positions of the detected cells are saved to file in the experiment folder. The macro will stop after each block for illustrative purpose to highlight the detected isolated cells. This behavior can be modified in the macro "FixedCAMBlockAnalysisFunctions" at line 179 by setting the variable "DebugMode" to 0.