Using Volocity, cytosolic Ca²⁺ alterations were monitored using the ratiometric dye, fura-2 AM. In response to the mitochondrial uncoupler FCCP and NMDA, leakage of mitochondrial Ca²⁺ from mitochondria is observed in hippocampal cultures, accompanied by an increased cytosolic Ca²⁺ concentration. Image courtesy of Dr. Bettina Platt, University of Aberdeen.

Solutions for Ratiometric Imaging

Acquisition and Analysis

Ratiometric images can be acquired using Volocity Acquisition our advanced 3D image capture software. Volocity Quantitation has a wide variety of tools which can be used to import and analyze ratiometric images from any source. Our Ratio Plugin software is required to optimize both Volocity Acquisition and Quantitation, providing you with a fully on-line 3D ratiometric imaging solution.

Measurements

Your results are stored in a ‘Measurement Item’ which automatically charts the intensity values from the ROIs selected as well as providing the raw data for further analysis.  The image data can be reanalyzed as required by using the ratio measurement features in Volocity Quantitation. Charts and data can be exported and used for publication.

3D Ratio Imaging

Recent advances in Volocity have added a new dimension to your ratio imaging by providing the software tools required to perform ratio imaging in three dimensions. The Ratio Plugin allows the creation of 3D acquisition protocols that can capture ratio image pairs at high speed and in Z.  In the live Video preview, a live graphical plot of each ROI is provided, with each ROI shown in a different color for ease of navigation. You have the option to view one acquisition channel at a time, or you can choose to see acquisition channels, plus the ratioed image with a rainbow CLUT (Color Look Up Table) applied and the intensity modulated channel, all at the same time.  These two feedback features allows you to follow the course of your experiment in real time and monitor events to make sure that your protocol is working correctly.