Measure Flow

This workflow allows you to estimate the flow of a fluorescent solution through the aperture of a cantilever depending on the applied pressure. This is achieved by continuously measuring the fluorescent intensity of an area around the cantilever’s aperture over time.

Acquiring data

For the measurement the probe must be in focus and imaging settings must be fixed such that changes of fluorescent intensity are visible but do not saturate the images (otherwise the intensity would be capped).

Region of interest

The region of interest ROI is the area (of the video feed) in which the fluorescent intensity is measured. It is determined by the position of the crosshair on the video feed and the parameter Rectangle size (px).

The ROI should contain only an area of the video feed where intensity change is visible (the area can be determined by briefly applying some pressure using the pressure tool)

Background

The fluorescence intensity immediately before applying pressure will be taken as background intensity and serves as reference for all measurements.

The Initial idle time parameter can be used to delay applying pressure until background intensity has settled. If the measurements contain a lot of negative intensity values, this parameter should be increased.

Pressure sequence

In the pressure sequence editor you can define the pressure pulses that are applied during the measurement.

The editor supports adding either single pulses or generating a sequence of pulses with increasing pressure. In either case, an period of idle pressure is automatically generated after each pulse. This idle period is necessary to let the fluorescence intensity decrease until the background intensity is reached again.

Editor tab	Function
	Generate a pressure pulse with a specified duration, followed by a configurable idle (0 mbar) time.
	Generate a sequence starting with a specified pressure followed by a number of pulses with increasing pressure, including idle time after each pulse.
	Import or export a pressure sequence.

Press to generate the pressure pulse according to the configuration.

Once pulses are generated, they can be re-ordered and adjusted while the editor remains open.

During pulse generation, each pulse can also be assigned a name (in case of generated pulse step sequence, all pulses are given the same name) which will be appended to the measurement data. When evaluating the data this name can then be used to group the pulses.

The whole pressure sequence can be repeated any number of times using the Sequence repetitions parameter.

Live plot

When running the pressure sequence, a live plot displays the measured intensity and the applied pressure:

Example of a pressure sequence with six steps, pressure increasing by 50mbar per step.

Saving data

The collected data can be saved to a text file by pressing Save data and then processed by the flow analysis tool.