Measuring Ratio-Fluorescence with the PTI EasyRatioPro

The PTI EasyRatioPro from HORIBA Scientific is an advanced monochromator-based wide-field microscope system, designed for measuring fluorescence dye intensity or ratio-fluorescence of labelled proteins in nanomolar concentrations.

Ratio-fluorescence microscopy is a critical instrument used for analyzing dynamic occurrences in live cells and cellular structures. Compared to the traditional intensity based fluorescence microscopy, ratio-fluorescence helps to remove the impact of cell path length variation, non-uniformity of dye loading, photo-bleaching and non-uniform field of view, making ratio-fluorescence the most precise process to establish intracellular ion concentrations.

The PTI EasyRatioPro is quick in providing vital dynamic data about ion concentrations and interactions with other drugs, molecules, or added reagents. It uses CCD, EMCCD or sCMOS cameras for sensitive and fast acquisition of fluorescence images from as many as 16 different excitation wavelengths, and four emission wavelengths (optionally). It is possible to support over 100 cameras, with more added regularly.

Excitation can be achieved using HORIBA’s patented DeltaRam™ X compact rapid switching monochromator integrated with a liquid light guide to a vertical or inverted fluorescence microscope.

The PTI EasyRatioPro is equipped with HORIBA’s patented Warp Drive, which is can transform imaging by enhancing productivity using its unique tactile control surface.

Key Features

The main features of the PTI EasyRatioPro are:

  • HORIBA’s acquisition engine (AE) can stream imaging data directly to hard disks in real time for acquisition and playback
  • Non-destructive image processing. Processing Plug-ins can be applied during recording or playback
  • Built-in experiment session file for tracking all imaging data on the hard disk RAID or server
  • Daily routines automation is easy using provided session templates for standard experiments, or customization is possible
  • DeltaRam™ X allows virtually infinite excitation wavelengths from 250 to 650 nm
  • Warp drive provides real-time control of about 60 programmable function keys and touch sensitive sliders to control exposure time, gain, binning, and wavelength selection of up to 16 different channels.

Software Features

  • Square, freehand, ellipse, and linear profile drawn area of interest photometry in real-time or in post-acquisition
  • Trace math analysis operations including average, anti-log, differentiate, combine, XY combine, integrate, and linear fit, peak finder
  • Total 1-4 exponential decay fitting with global analysis, exponential analysis method (ESM)
  • Real-time non-destructive plug-ins for ratio-metric calculation, thresholding, and ROI based or image based background subtraction, flat field correction, addition, subtraction, multiplication, division, three channel compositing, palette, calibration, and ROI calculation
  • Real-time generation of user defined event markers and event journaling
  • Calibration editor for formation of calibration curves using point polynomial fitting, Grynkiewics pH and Grynkiewics Ca++ formulas
  • Control of up to 16 excitation, emission, or derived channels
  • Export of image data to accepted formats such as avi, tif, jpg, bmp, png.
  • Warp Drive human user interface control for dedicated control over exposure, electron multiplier, gain, mute, select, solo, record, window functions, jog/shuttle, play, fast forward, rewind, stop, time code, looping, etc.

Applications

EasyRatioPro has been specifically designed for use in ion imaging and is further refined for kinetic imaging.

The key applications of EasyRatioPro include:

  • Measurement of mitochondrial, endoplasmic reticulum, and intracellular free Ca2+
  • Studying Ca2+ homeostasis, Ca2+ wave propagation, Ca2+ mobilization, Ca2+ oscillation, and Ca2+ wave quantitation
  • Measurement of other intracellular ions such as Mg2+, Mn2+, Zn2+, Sr2+, Na+, and Cl-
  • Determination of intracellular, lysosomal and vacuolar pH levels and in-situ calibration of intracellular pH and intracellular [Ca++] and [Na+]
  • Assessment of plasma membrane potential, iron homeostasis membrane potential, and mitochondrial membrane potential
  • Detection by immunofluorescence of PfCRT expression in HEK293 cells
  • Measurement of cell volume and detection of fluorescent Zn2+ in MIN6 cells
  • Identifying cells coexpressing GFP for analysis; mitoGFP and ratiometric GFP (redox-GFP) studies
  • FRET studies of donor excitation spectroscopy, donor emission intensity, emission intensity, ratio of donor/acceptor emission intensity, molecular proximity, acceptor emission intensity, and enzyme-substrate binding.
  • Calcium imaging of single HEK 293 cell, studying changes in availability of CaM
  • Demonstration of selective plasma membrane permeabilization in HEK293 cells by digitonin

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