MetaFluor® Imaging System: Software for Fluorescence Ratio Imaging

Fluorescence ratio imaging is the monitoring of live cells in which a fluorescent indicator of intracellular ions is introduced. Indicator dyes have been designed to shift their fluorescence excitation or emission spectrum when binding with specific ions. Images are obtained at two different wavelengths, typically matching the absorption bands at the high and low binding conditions.

By ratioing the intensities in the images, it is possible to construct a map showing the local ion concentrations throughout the field of view. Since the monitoring process is nondestructive, image acquisition can be repeated frequently to trace and monitor the time course of cellular responses.

The MetaFluor® Imaging System is designed for dual-wavelength intracellular ion measurements. The system provides simultaneous display of the raw data, ratio image, graphs of intensities, ratios and ion concentrations, and a non-ratiometric image such as a brightfield or phase-contrast image. Two different ratiometric indicators can be imaged and measured simultaneously.

Custom Configuration

Toolbars, menus, wizards and dialog boxes help move you through the image processing steps quickly. Features such as multiple image windows, flexible device control, synchronization and timing, and journals allow for automated image acquisition and analysis unlike any other system.

With MetaFluor, you customize the set-up once, then let the experiment run by itself. You are able to collect a large amount of data online and process it with either MetaFluor or an analysis-only copy of the software.

Device Control

MetaFluor works with microscopes equipped with epi-fluorescence illumination. The system includes device drivers for numerous commercially-available filter wheels, shutters, monochromators and high speed filter changers for illumination control. Camera drivers are optional. The MetaFluor system's camera drivers support acquisition from a wide variety of digital cameras.

MetaFluor enables sub-region, binning and analog-to-digital (A/D) selection if the camera allows it. Gain and exposure time can be set per wavelength for acquisition. Streaming can be used as an acquisition option. With appropriate wavelength‐switching devices, streaming allows you to acquire a predefined number of images at the maximum frame rate of the camera; the technology for this is patented.

Journaling and Task Automation

Journals are sophisticated and customizable macros that record and execute many tasks without requiring you to know any programming language.

MetaFluor's Journal Editor allows you to create functions to simplify system operations, automate acquisition and device control, set variables and sequence events.

Its Auto-Execute Journal command runs journals at specific points in the acquisition cycle.

Its Sequence Journals command allows the execution of a set of commands at a given time during the acquisition process or at a given stage in the experiment.

User-definable taskbars make it easy to achieve “one-button” control of your system. All of these automation capabilities let you customize MetaFluor to run experiments with your own protocols.

Powerful Real‐Time Processing Acquisition

When acquiring from video sources, MetaFluor can average up to 256 images per time point, significantly reducing random image noise. Background subtraction is also used to improve accuracy by correcting for stray light, camera noise and auto-fluorescence.

Real‐Time Processing

MetaFluor will perform frame integration or averaging and background subtraction on your image as your experiment progresses. Ratio shifts or ion fluxes are observed immediately, providing instant feedback on your experiment.

Calibration

A direct display of intracellular ion concentrations is obtained by using the various calibration options offered; the Grynkiewicz equation (Grynkiewicz et al., 1985) and titration equation for both in situ and in vitro experiments. These calibrations can then be stored for future use.

Grynkiewicz, G, Poenie, M, Tsien, RY. A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260(6): 3440-3450.

Binning And Super Pixels

If the signal is too small or noisy, binning will improve the signal‐to‐noise (S/N) ratio. Binning groups of neighboring pixels creates a “super pixel” with an intensity equal to the sum of the intensity values of the original pixels. Spatial resolution is diminished, but the S/N is enhanced. If your goal is to improve the precision of ion concentration measurements and detect small changes, binning offers a powerful solution.

Interactive Graphs

A display of multiple graphs gives flexibility in the presentation of your experiment's data. MetaFluor enables you to click on graph traces to display a readout of the time and data value for the region nearest to the click.

The Event Mark function is useful to record when drugs or solutions were added, experimental conditions changed, triggers were received or sent or other events occurred. You have the option to associate a timer and an alarm bell to each event. Additionally, for perfused samples, ambient conditions can be logged and tracked.

An annotation is saved within each TIFF file that contains pixel data. The annotation records wavelength-dependent settings. Additional information can be stored in a protocol file.

Export For Data Analysis

If needed, MetaFluor can log and export all measurements to either a text file or to a spreadsheet program such as Microsoft® Excel® spreadsheet software.

Compatible With MetaMorph

Because MetaFluor saves images as TIFFs, you can import them into MetaMorph for further processing and analysis.

Presentation And Publication

Images in MetaFluor can be displayed in monochrome, pseudocolor, or using a variety of user-defined set of values. Ratio images can also be displayed using a special display mode called Intensity Modulated Display, or IMD.

With the IMD mode, color is used to represent the relative ratio value, while the intensity or brightness of the color is used to represent the brightness of the source images. This technique helps automate the process of extracting spatial information from the background, by automatically eliminating background fluorescence from the scene.