Monochromator-Based Wide-Field Microscope System for Measuring Ratio-Fluorescence - PTI RatioMaster

The PTI RatioMaster is a monochromator-based wide-field microscope system for measuring fluorescence or ratio-fluorescence dye intensity of labeled proteins in nanomolar concentrations. Ratio-fluorescence microscopy is the decisive tool to research dynamic events in live cells and cellular structures.

In contrast to conventional intensity-based fluorescence microscopy, ratio-fluorescence removes the effects of photo-bleaching, non-uniformity of dye loading, cell path length variation, and non-uniform field of view, making ratio-fluorescence the most accurate technique to establish intracellular ion concentrations.

More significantly, the PTI RatioMaster is sufficiently fast to provide vital dynamic information regarding ion concentrations and interactions with other drugs, molecules, or added reagents. The PTI RatioMaster is designed to handle dynamic ratio fluorescence measurements on a millisecond timescale. A xenon arc lamp offers high-intensity, continuous broadband illumination.

Alternating excitation wavelengths are chosen by a computer-controlled high-speed random access monochromator combined with a (user specified/supplied) upright or inverted, fluorescence microscope with a liquid light guide.

The emitted light is collected from the sample and conveyed via a photometer possessing a bilateral translatable iris and a viewing eyepiece to a switchable analog/photon counting photomultiplier detector.

Analog detection is used when the emitted light is moderately intense; low light levels are detected using photon counting. The computer controls all system functions. Data is collected and tested by proprietary Windows ‘FelixGX’-based advanced fluorescence software.

An optional Sarcomere Length module is available to measure the variations in sarcomere length and full cell length as a function of time. This permits correlation of contractility information with calcium concentration.

Features

  • Data Integrity - HORIBA is aware of the importance of one’s data, which is why the company uses an XML file to archive and store experimental data. No proprietary binary formats allow the scientist to manipulate data with calmness, knowing that their data remains in its primary form and can be opened in any spreadsheet program.
  • Open Architecture FelixGX™ Software - Allows maximum flexibility for the user’s future requirements with HORIBA’s modular software architecture. Components can be incorporated into the system to boost functionality. This feature allows scientists to get started economically and then integrate functionality later as their research expands.
  • Day-to-Day Routine - Session templates for popular experiments are provided. Users can also create their own templates. Ready to use common experiments with limited input from the user. Users simply need to push acquire and begin.
  • Modular Design - If the out-of-the-box acquisition protocols do not quite match one’s applications, the PTI RatioMaster Macro editor allows users to develop their own dedicated experiments by a drag and drop command library into a loop logic.
    • User need not be programmer or comprehend a scripting language to execute user-defined macros.
    • Easily select the function needed and then apply it to the loop.
    • Increment the start excitation wavelength, number of points per second, emission wavelength, increase temperature, and a multitude of other acquisition modes. The Macro editor is one of the easiest to use HORIBA designs.

Features of the Software

  • Trace math analysis functions such as antilog, integrate, combine, XY combine, average, differentiate, linear fit, and peak finder, 1-4 Exponential fitting, FRET Calculator, and Global Analysis
  • Square, single area of interest photometry in real-time.
  • Real-time non-destructive ratiometric calculation and background subtraction.
  • Real-time generation of user-defined event markers and event journaling.
  • Control of unlimited excitation, emission channels.
  • Export of data to common formats such as .ang, .ana, .spc, or text files.
  • Lookup tables and equation calibration of raw data.
  • Unlimited derived channels.
  • 3D graphing and plotting.
  • Customizable control panels.

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