This is an imaging method for making organisms appear brighter than their background. It can be used for a range or organisms. Here we describe a method for Drosophila larvae.
Frustrated Total Internal Reflection (FTIR) is a method for tracking animals by making them brighter than their background rather than the traditional method of tracking animals by making them darker than their background. Here we demonstrate the FTIR Imaging Method (FIM) for Drosophila larvae.
Experimental Setup
The FTIR system consists of a Zantiks FTIR plate with an infrared (IR) light source, a Petri dish with agar, a black cover for the dish, and a Zantiks MWP unit. Multiple Petri dishes may be prepared ahead of time so that many larvae can be tested in quick succession on fresh agar. It is important to prepare the agar plates without bubbles or blemishes. The IR light escapes from the agar through the body of the Drosophila larva so that they appear brighter than the background.
The IR light source is powered from the MWP unit, and is connected to CN2 on the lower PCB (this is normally used for the power for the Internal IR which can be disconnected while the FTIR plate is in use).
Experimental Procedure
A Drosophila larva is placed in the centre of a petri dish, and the black cover is placed over the top. The dish can then be placed onto the FTIR plate, and the FTIR plate is put into the MWP unit - please place in the locator, and press to the side and the back.
To improve imaging further one may make an optical connection between the FTIR plate and the agar dish. This can be with water, or glycerol, of other liquid. Please make sure to not trap bubbles between the dish and the plate.
Then push the petri dish into the MWP unit. This is so the plate will be in the same place every time, and so location data is consistent between trials. Please then place the foam front cover over the FTIR plate to isolate the experimental chamber, and finally put the InfraRed light source in place.
Experimental scripts may now be run.
Procedure for tracking Drosophila larvae with FTIR
Results
The resulting video and track can be analysed as required - e.g. for time spent in zones, distance travelled in zones, or latency to the edge of the dish.
