Fluorescence – Calcium Imaging Microscope – RatioMaster

PTI RatioMaster

Fluorescence microscope for intracellular Calcium, Sodium and Magnesium ion concentrations

PTI RatioMaster is the most comprehensive system for measuring intracellular calcium, sodium, magnesium ion concentrations or pH and membrane potential. PTI Ratiomaster includes all the tools required for data acquisition, analysis and calibration of intracellular dyes or intrinsic fluorophores.

Segment: Scientific
Division: Molecular and Microanalysis
Base product
Manufacturing Company: HORIBA Scientific

PTI RatioMaster is a monochromator-based wide-field microscope system for measuring ratio-fluorescence or fluorescence dye intensity of labeled proteins in nanomolar concentrations. Ratio-fluorescence microscopy is the ultimate tool to study dynamic events in live cells and cellular structures. Unlike conventional intensity based fluorescence microscopy, ratio-fluorescence eliminates the effects of photo-bleaching, cell path length variation, non-uniformity of dye loading, non-uniform field of view, making ratio-fluorescence the most accurate method to determine intracellular ion concentrations. More importantly, PTI RatioMaster is fast enough to provide essential dynamic information about ion concentrations and interactions with other molecules, drugs or added reagents.

The PTI RatioMaster is capable of dynamic ratio fluorescence measurements on a millisecond timescale. A xenon arc lamp provides high intensity, continuous broadband illumination. Alternating excitation wavelengths are selected by a computer-controlled high-speed random access monochromator coupled to a (user specified/supplied) inverted or upright, fluorescence microscope with a liquid light guide. Emitted light is collected from the sample and passed through a photometer having a bilateral translatable iris and a viewing eyepiece to a switchable analog/photon counting photomultiplier detector. Analog detection is used when emitted light is relatively intense; low light levels are detected by photon counting. All system functions are under computer control. Data is collected and analyzed by proprietary Windows “FelixGX” based advanced fluorescence software.

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

Features

  • Data Integrity — We know how valuable your data is, which is why we use an XML file to archive and store experimental data. No proprietary binary formats allow the researcher to manipulate data with peace of mind that their data remains in its original form and can be opened in any spreadsheet program.
  • Open Architecture FelixGX™ software also allows for maximum flexibility for your future needs with our modular software architecture. Components can be added to the system to increase functionality. This feature allows researchers to get started inexpensively and then add functionality later as their research grows.
  • Day to Day Routine -  Session templates for common experiments are provided. In addition, you can create your own templates. Ready to run common experiments “out of the box” with minimal input from the user. Just push acquire and go!
  • Modular Design -  If the out of the box acquisition protocols do not quite fit your applications, the PTI RatioMaster Macro editor allows you to create your own specialized experiments by a drag and drop command library into a loop logic. This means you do not have to be a programmer or understand a scripting language to implement user defined macros. You simply select the function you want and then apply it to the loop. This allows the researcher to increment the start excitation wavelength, emission wavelength, number of points per second, increase temperature and a host of other acquisition modes. The Macro editor is simply one of the easiest to use ever designed by HORIBA!

Features of the Software

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

Lamp Specifications

Lamp Power Supply

PTI RatioMaster lamp power supplies are highly-regulated DC units that provides very stable power for xenon, mercury, and mercury/xenon compact arc lamps as well as tungsten-halogen lamps.

Lamp power capacity75 to 200 watts
Height100 mm (3.9 inches)
Width100 mm (3.9 inches)
Length210 mm (8.3 inches)
Weight1.9 kg (4.2 pounds)
Window diameter (D)65 mm

DeltaRAM X™ Random Access Monochromator

PTI RatioMaster's exciting new DeltaRAM X™ represents the next bold step in the evolution of light sources. The compact, proprietary (patented) single monochromator design permits the selection of any single wavelength in two milliseconds or less. It is ideally suited for multi-wavelength applications as well as excitation scanning. It is easily controlled via a single low voltage signal line. Includes a 2-meter liquid light guide, for use with most microscopes and other sample handling devices. DeltaRAM X™ delivers powerful excitation wavelength from 250–650 nm under synch-lock computer control. Synch-lock control, locks the DeltaRAM X™ monochromator to the camera exposure or frame readout. The DeltaRAM X™ saves you money by not requiring purchase of additional excitation filters for each dye you wish to use. Synch-lock allows accurate timing to be retained between camera and illuminator.

Systems not synch-locked can be plagued with synchronization problems or latency due to operating events or user clicking events. Try this in another imaging software package, click and drag a window, either the illuminator will stop moving, or images will stop being acquired until the mouse button is released. This does not happen with PTI RatioMaster's sync lock!

Excitation wavelength range250–650 nm
Optimum scanning range290–610 nm
Wavelength selection speed< 2 milliseconds point-to-point
Resolution< 1 nm
Accuracy+ 3/-1 nm
Wavelength bandwidthAdjustable from 0–24 nm
Stray light rejection> 104
Light delivery0.5 meter quartz fiber optic bundle
Liquid light guide2 meter length 2 mm core diameter
Microscope adapterUser specified

Liquid Light Guide

Spectral Transmission Curves

Specific Properties

SeriesDia.PropertiesRecommended Light SourcesApplicationsTemp Range
3002, 3 ,5 mmSuperior transmission of up to 5 W of UV radiation. Suitable for rugged environmentsHg and Xe short arc, Tungsten HalogenUV Epoxy curing and fluorescence inspection, at lengths up to 20 meters.-5 to +35°C
3802, 3, 5 mmExcellent transmission from NUV to far red, at lengths to 30 m. Suitable for rugged environmentsXe short arc, Tungsten Halogen, Metal HalideOutstanding white light illumination-5 to +35°C
20002, 3, 5 mmTransmission of high power NIR in the multi-watt range. Protection required from high power radiation below 420 nm. Integrated long pass dichroic filters availableXe short arc, Tungsten Halogen, Nd-YAG or diode lasersVisible or NIR illumination at lengths to 10 meters+5 to +35°C
8005 mmEnhanced flexibility, no losses even with small bend radius. Protection required from high-powered radiation below 420 nm. Integrated long pass dichroic filters availableTungsten Halogen, Xe short arcVery High NA white light illumination, excitation of HPD in medicine.+5 to +35°C

 

Mechanical Dimensional Information

Core DiameterD1D2D3L1L2
2 mm4 mm8 mm5.5 mm6.7 mm20 mm
3 mm5 mm9 mm7 mm20 mm24 mm
5 mm7 mm10 mm9.5 mm20 mm24 mm

Photometer

When you need to perform microscope photometry, what do you use? Most labs end up acquiring some microscope accessory intended for another purpose—such as photography.

Unfortunately, these devices are rarely up for the task. Microscope photometry requires absolute light-tightness. It also helps to have a device designed to accommodate a photomultiplier housing—or even two. Additionally, it should hold filters, have an adjustable iris, and provide a means to view the image being measured.

Our microscope photometers do all of this—and more. They are specifically designed for sensitive photometric measurements. And they couple easily to virtually any popular microscope.

The Model D-104B is a single-channel photometer. The D-104C is a dual-channel photometer that allows simultaneous measurements at two different wavelengths via a dichroic cube.

No system is complete without an analog/digital photon counting detector. In order to complete a PTI RatioMaster system, an analog/digital photon counting detector, selected from only our best quality and performing photo-multipliers, the 814 series. You may also select one of the excellent Red sensitive PMT's to complement the system.

Analog/Digital Photon Counting

Model 814 is a compact PMT housing for analog or digital (photon-counting) detection. The detection mode is switchable, and the built-in LCD can also selectively display high voltage or analog signal level. Features power on/off switch, high voltage adjust, LCD high voltage/analog signal display, detection mode and display mode switches, gain, time constant and offset adjustment controls, BNC signal output connection, BNC external high voltage adjust input connection, light-tight RF shielded case, internal direct anode output mode control, and socket for most 1-1/8 inch side-on photomultiplier tubes. Includes a separate wall-mount line voltage power supply. This detection system excels at applications like the following:

  • Live-cell imaging
  • High-speed emission ratio imaging
  • Quantitative FRET, FRAP, FISH
  • Luminescence

Analog Mode:

This mode can be used for medium to high levels of light intensity. The maximum PMT current is 10 microamperes. The output is a DC signal between +13 Volts and -13 Volts.

Photon-Counting (Digital) Mode:

This mode can be used for low to medium light levels up to 4 million counts per second. The output is a TTL level, active low signal. The signal line must be terminated at 50 ohms. The output is a frequency corresponding to the number of photons impinging on the photocathode.

Digital Photometer Specifications

Maximum count rate4 MHz
Pulse pair resolution250 ns
OutputTTL level active low pulse
Rise time20 ns
Fall time100 ns
Pulse width220 ns

Model 814 PMT Housing

Model 814 is a compact PMT housing for analog or digital (photon-counting) detection. The detection mode is switchable, and the built-in LCD can also selectively display high voltage or analog signal level.

Features power on/off switch, high voltage adjust, LCD high voltage/analog signal display, detection mode and display mode switches, gain, time constant and offset adjustment controls, BNC signal output connection, BNC external high voltage adjust input connection, light-tight RF shielded case, internal direct anode output mode control, and socket for most 1–1/8 inch side-on photomultiplier tubes. Includes a separate wall-mount line voltage power supply.

Analog Mode:

This mode can be used for medium to high levels of light intensity. The maximum PMT current is 10 microamperes. The output is a DC signal between +13 Volts and -13 Volts. This signal is usually measured with a voltmeter, a recorder, or the analog-to-digital converter in a general-purpose I/O board in a computer.

Photon-Counting (Digital) Mode: 

This mode can be used for low to medium light levels up to 4 million counts per second. The output is a TTL level, active low signal. The signal line must be terminated at 50 ohms. The output is a frequency corresponding to the number of photons impinging upon the photocathode, and is usually measured with a counter in a general-purpose I/O board in a computer.

Direct PMT Output Mode: 

This mode applies the signal from the photomultiplier anode directly to the output BNC. This is generally used only when the highest-speed response is desired. Internal digital or analog signal processing is completely bypassed. The peripheral equipment connected to the housing must be properly configured to accept the signal directly from the PMT itself.

High Voltage Power Supply Specifications

Input+/- 15 VDC, 250 mA
High voltage-200 to -1,100 VDC manually adjustable (LCD display indicates actual cathode voltage)
External high voltage adjust0 to +5 VDC (0 = -200 V, 5 = -1,100 V), continuously adjustable
Input regulation+/- 0.05% max. (for 15 V +/-1 V input)
Load regulation+/- 0.05% max.
Ripple100 mV p-p max.
Temperature coefficient+/- 0.01% max. (+5 to +40° C)
Drift+/- 0.03%/hr max. (after 15 minute warm-up)

PMT

Your choice of 2 PMTs

R 1527
Spectral Response185 to 680 nm
Photocathode MaterialLow noise bialkali
Effective Area of PMT8 x 24 mm
Supply Voltage1250 V dc

Cathode Sensitivity

Quantum Efficiency at 300 nm (Peak)

Luminous

Radiant 400 nm (Peak)

19 % typ.

60 µA/lm typ.

60 mA/W typ.

Anode Sensitivity

Luminous

Radiant 400 nm

400 A/lm typ.

4 x 105 A/W typ.

Gain6.7 x 106 typ.
Anode Dark Current 
(After 30minute Storage in the darkness)
0.1 nA

R 928
Spectral Response185 to 900 nm
Photocathode MaterialMultialkali
Effective Area of PMT8 x 24 mm
Supply Voltage1250 V dc

Cathode Sensitivity

Quantum Efficiency at 260 nm (Peak)

Luminous

Radiant 400 nm

25.4 % typ.

250 µA/lm typ.

74 mA/W typ.

Anode Sensitivity

Luminous

Radiant 400 nm

2500 A/lm typ.

7.4 x 105 A/W typ.

Gain1 x 107 typ.
Anode Dark Current 
(After 30minute Storage in the darkness)
3 nA

Analog PMT Specifications

Gain settings1, 0.1, 0.01, 0.001 µA = 1 V
Time constant settings0.05, 0.5, 5, 50 and 500 msec
Offset correction+/-50 nA
Inherent noise50 pA p-p typical, with 0.05 msec time constant
Dimensions (h x w x d)7.39 x 4.04 x 4.02 inches

Digital PMT Specifications

Maximum count rate4 MHz
Pulse pair resolution250 ns
OutputTTL level active low pulse
Rise time20 ns
Fall time100 ns
Pulse width220 ns
Dimensions (h x w x d)7.39 x 4.04 x 4.02 inches
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Intracellular Ion Kinetic Solutions Brochure
CategoryDocuments
Size 6.03 MB
FiletypePDF

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