Fluorescence microscopy is perfect for intracellular ion measurements and the most universal of these measurements is intracellular calcium imaging measurements. Two of the most typical dyes for calcium imaging are Fura-2 and Indo-1.
The PTI EasyRatioPro is a monochromator-based wide-field microscope system that has been created for measuring ratio-fluorescence or fluorescence dye intensity of labeled proteins in nanomolar concentrations. Ratio-fluorescence microscopy is the decisive tool to research dynamic events in cellular structures and live cells.
In contrast to conventional intensity-based fluorescence microscopy, ratio-fluorescence removes the effects of cell path length variation, non-uniformity of dye loading, photo-bleaching, non-uniform field of view, making ratio-fluorescence the most accurate technique to establish intracellular ion concentrations.
More notably, the PTI EasyRatioPro is sufficiently fast to provide vital dynamic information about ion concentrations and interactions with other drugs, molecules, or added reagents.
The PTI EasyRatioPro integrates sCMOS, CCD, or EMCCD cameras for sensitive and fast acquisition of fluorescence images of up to 16 types of excitation wavelengths and optionally, four emission wavelengths. (More than 100 cameras are supported, with more added regularly.)
Excitation is via HORIBA’s patented DeltaRam™ X compact rapid switching monochromator coupled by a liquid light guide to an inverted or upright fluorescence microscope. This permits nearly infinite excitation wavelengths from 250 to 650 nm, and acquisition rates of up to 250 for dual excitation measurements or 1000 images per second for dual emission.
Another exclusive novelty of the PTI EasyRatioPro is HORIBA’s patented ‘Warp Drive’ that transforms imaging by boosting productivity via its advanced tactile control surface.
The Warp Drive features real-time control of up to 60 programmable function keys and touch-sensitive sliders to regulate binning, exposure time, gain, and wavelength selection of up to 16 different channels. Optimizing images in real time has never been this simple.
- HORIBA’s Acquisition Engine (AE) streams imaging data straight to hard disks in real time for acquisition and playback. This enables the user to perform experiments unrestricted by the internal RAM of the computer. In contrast to alternatives that can only stream to RAM, a high-speed stream of up to terabytes of data directly to hard disk storage is possible.
- HORIBA understands how valuable one’s data is; hence it employs non-destructive image processing. Processing Plug-ins can be used during record or playback mode. The raw data integrity is preserved, allowing the user to experiment with threshold, background subtraction and other processes to improve the image data while the original data stays on the hard drive.
- The PTI EasyRatioPro modular software architecture is programmed to meet present as well as future needs. Plug-ins can be integrated to boost systems functionality. This also means that one can get started economically and then add functionality later, as and when the research requirements expand.
- Users need not worry about where their data is saved. The PTI EasyRatioPro integrates an experiment session file that keeps track of all imaging data on the hard disk RAID or server. The session maintains track of all image data, exposure times, wavelength, region of interest data, channels, and all essential controls.
- Day-to-day routines automation is easy using the session templates provided for standard experiments. Users can also create their own.
- Ready to function ‘out of the box’ with minimal input from users. Users only have to push record and begin.
- Other features of the software:
- Square, freehand, ellipse, and linear profile drawn area of interest photometry in real-time or in post-acquisition.
- Trace math analysis functions such as average, combine, anti-log, differentiate, XY combine, integrate, linear fit, and peak finder.
- Full 1-4 exponential decay fitting with Global analysis, Exponential Analysis Method (ESM).
- Calibration editor for the development of calibration curves using point polynomial fitting, Grynkiewics Ca++ and Grynkiewics pH formulas.
- Real-time non-destructive plug-ins for ratio-metric calculation, thresholding, ROI-based or Image-based background subtraction, flat field correction, three-channel compositing, addition, subtraction, multiplication, division, calibration, palette and ROI calculation.
- Real-time generation of user-defined event markers and event journaling.
- Control of up to 16 excitation, emission, or derived channels.
- Export of image data to common formats such as bmp, avi, jpg, png, tif.
- Warp Drive human user interface control for dedicated control over exposure, electron multiplier, gain, mute, time code, select, solo, record, jog/shuttle, play, rewind, fast forward, window functions, stop, looping and much more.