Protocol

Introduction

The protocol settings are extremely important for a correct analysis of the data. It needs to contain all the relevant timing of events, such as the time of the stimulation, its properties and when to stop the analysis, i.e., when the ammonium application starts.

As mentioned in the data structure page, the Excel information sheet (Infosheet) has three different entries: the protocol, the associated units and the general information, as shown in the figure below.

infosheet
1: Infosheet example. In the blue box indicate some of the protocol fields, in particular the protocol indicate a field stimulation with ammonium application; the red box indicates the units for the protocol fields, and in the green box are indicated the general information.

The data in the Infosheet applies to all the cells within that Excel file. It is very important that all cells contain data that was acquired using the parameters as set in the protocol section. If cells need to be loaded that use a slightly different protocol, those cells need to be collected in a separate Excel file with a correct Infosheet. Excel files with different protocols can be loaded sequentially into the program and all analyses will work as expected.

Template creator

Purpose

The purpose of the template creator is to give the user an overview of how to name the datasheets, the layout expected by the program and moreover facilitate the writing of the protocol information. The template creator can be found in the main interface under the "Help" menu. As can be seen from the figure below, the interface is divided in two blocks: the sheetname creator and the infosheet creator.

emptytemplate
Figure 2: The empty template creator interface. See the text for more descriptions.

Sheetname creator

The Sheetname creator helps the user to create a template of the name to use for the data sheeets in Excel. It asks for the date (in the format year month day, two digits each), a condition that has a relevant meaning to divide the data in at least two groups. Note that the program will automatically divide the data according to every different condition it finds in the Excel file. Note that the condition label is case-sensitive: 'WT' and 'Wt' will be considered different groups! The coverslip ID could be filled in with two kinds of ID, either a number or the position in the plate; in case of only the number the template creator will add the prefix "cs"; this is used by the program to group cells per cover slip when exporting for multilevel analysis. Lastly, the cell number identifies which cell was imaged within a cover slip.

The dual color option will create two identical Excel sheets with the only difference being an "_g" and "_r" after the cell number.

sheetname
Figure 3: An example of the sheetname for both the single color (bottom line), and the dual color (top line).

Infosheet creator

This part of the interface is needed to create the information sheet, it has both the protocol information and the general inormation about the setup and settings. Different fields that require a time point or interval have a drop menu where the user can choose if the entered value is in units of frames, seconds or Hz. Then, as specified in the info structure, during the loading fase the program will automatically convert values and units in their most suitable format.

In the interface there are several check boxes: the field stimulation will activate the boxes for the electrical stimulation; the chemical stimulation will do the same for specifying any sort of chemical stimulation, this will be saved in the Excel file as "chemical start" and "chemical duration", allowing the user to specify in the "chemical name" the compound that was used; the synaptic marker will change the layout of the data sheet to include the synaptic data; the multi block protocol will copy the information of the stimulations twice, the user then has to adjust them in the Excel file. Lastly, the ROIs frame will create the Excel sheet where the frame numbers for the manual detection can be entered.

infosheet_fig
Figure 4: Example of a fully filed infosheet creator.

ROIs frame

With the label "ROIs frame" the program expect the first frame when the event was detected in ImageJ, or in any other imaging analysis program. More information could be found in the manual detection page.