High Speed Dynamic Fluorescence Measurements – the PTI RatioMaster

HORIBA Scientific’s PTI RatioMaster is a monochromator-based wide-field microscope system designed for measuring ratio-fluorescence or fluorescence dye intensity of labeled proteins in nanomolar concentrations. It offers researchers solid, reliable, sensitive detection for the collection and testing of ratiometric photometry data for pH, calcium and intracellular ion imaging.

Ratio-fluorescence microscopy helps in removing the effects of photo-bleaching, non-uniformity of dye loading, non-uniform field of view, and cell path length variation, thereby making ratio-fluorescence a highly precise technique to establish intracellular ion concentrations.

A key advantage with the PTI RatioMaster is that it is fast enough to provide vital dynamic data about ion concentrations and interactions with other molecules, drugs or added reagents. It can provide dynamic ratio fluorescence measurements on a millisecond timescale. High intensity, continuous broadband illumination is provided by a xenon arc lamp. Proprietary Windows FelixGX based advanced fluorescence software collects and analyzes the data.

A Sarcomere Length module is optionally available to measure alterations in sarcomere length and whole cell length as a function of time.

Key Features

The following are the main features of the PTI RatioMaster:

  • Data integrity is maintained using XML files to archive and store experimental data
  • Open architecture FelixGX™ software offers maximum flexibility for user’s future needs along with HORIBA’s modular software architecture
  • Session templates for common experiments are provided. Customized templates are also possible
  • It has a modular design, with PTI RatioMaster Macro editor to create customized experiments using a simple user-friendly drag and drop command library into a loop logic.

Software Features

  • Square, single area of interest photometry in real-time
  • Trace math analysis operations
  • 3D graphing and plotting
  • Real-time generation of user defined event markers and event journaling
  • Real-time non-destructive ratiometric calculation, and background subtraction
  • Control of numerous excitation, emission channels
  • Numerous derived channels and customizable control panels
  • Lookup tables and equation calibration of raw data

Applications

The key applications of PTI RatioMaster include:

  • Measurement of mitochondrial, lysosomal, endoplasmic reticulum, and intracellular free Ca2+
  • Studying Ca2+ homeostasis, waves, mobilization, oscillation, and spikes
  • Measurement of other intracellular ions such as Mg2+, Ba2+, Zn2+, Sr2+, Mn2+, Na+, and Cl-
  • Assessment of plasma membrane potential and mitochondrial membrane potential
  • FRET studies of donor excitation spectroscopy, donor emission intensity, ratio of donor/acceptor emission intensity, molecular proximity, acceptor emission intensity, and enzyme-substrate binding
  • Measuring respiratory burst and reactive oxygen species (ROS), oxygen consumption in cultured cells, nitric oxide (NO), and intracellular GSH
  • Identifying cells coexpressing GFP for analysis
  • Excitation spectrum of Fura-AM
  • Analyzing intracellular, phagosomal, lysosomal and Vacuolar pH
  • Fura-2 titration
  • Simultaneous measurement of Ca2+ and myocyte cell length

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