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RatioMaster™
Fluorescence Microscopy

 
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Applications

RatioMaster™ is up for the task for many applications
RatioMaster

Software Applications

 

[Ca2+] signals in cardiac myoblasts and myotubes

More Applications

  • IP3-induced intracellular
  • [Ca2+]c and mitochondrial [Ca2+]m
  • IP3-induced intracellular [Ca2+]c and mitochondrial[Ca2+]m
  • Visualization of Mitochondria by mitoGFP
  • [Ca2+]m signals in permeabilized myoblasts and myotubes
  • IP3-induced intracellular [Ca2+]c and mitochondrial [Ca2+]m

 

Excitation spectrum of Fura-AM

RatioMaster

Complete hydrolysis of the membrane permeable form of Fura-2 (Fura-2/AM) to Fura-2 is critical for accurate measurement Fura-2/AM has a different excitation spectrum compared with Fura-2. The scanning property of our monochromator makes it easy to check the status of Fura-2/AM hydrolysis.

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Fura-2 Titration

The fluorescence excitation spectrum of Fura-2 shifts to the lower wavelength upon Ca++ binding. This is a major characteristic of an ion-specific ratio fluorescent dye.

RatioMaster

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In-Situ Calibration of Intracellular [Ca2+]i

RatioMaster

By measuring the ratio of fluorescent intensity at 340/380 nm, [Ca2+]i can be measured over several orders of magnitude and with a high degree of precision.

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Simultaneous measurement of Ca2+ and myocyte cell length with Felix 32

RatioMaster

The above figure shows data traces from the simultaneous collection of fluorescence and cell length from a cardiac myocyte. Myocyte was loaded with Indo-1 and excited at 365 nm wavelength using the RatioMasterT RM-5 dual emission detection system. The blue trace shows the calcium ratio increase and decrease with the cell contraction . The contraction data (Green trace) can be correlated with sample accuracy with the fluorescence data since both signals were collect simultaneously.

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FRET for molecular proximity assay

FRET occurs because the seprase and the plasminogen activator receptor co-localize on the membrane of malignant melanoma cells. FRET was detected via a fluorescently labeled antibody targeted to each receptor. The results of this FRET: The acceptor's excitation spectrum gains spectral features of the donor's excitation spectrum. Not a real FRET, but similar. Instrument: PTI RM3.

RatioMaster

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FRET for Binding Assay

RatioMaster

FRET occurs when the EYFP fusing RII protein kinase binds to the ECFP fusing Ht31 protein (kinase anchoring protein). The results of this FRET: The donor (ECFP) emission fluorescence decreases. The acceptor (EYFP) emission fluorescence increases. Instrument: PTI's FRET RM pro.

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FRET to measure Ca2+ in ER

FRET occurs when ER Ca2+ binds to the "chameleon 3er", causing it to fold thereby bringing the two dyes in close proximity. The results of the FRET: The donor (CFP) emission fluorescence decreases. The acceptor (YFP) emission fluorescence increases. The emission ratio of Acceptor/Donor increases. InsP3 triggers the release of ER Ca2+, thus decrease the level of FRET. Instrument: PTI's FRET RM pro.

RatioMaster

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Excitation Ratio

PTI

Excitation Ratio is used to set up and run experiments for intracellular ion determinations using excitation-shifted probes such as Fura-2 for calcium and BCECF for pH. In this experiment, the excitation source must alternate between two different excitation wavelengths that are characteristic of the probe. The emission intensity at both excitation wavelengths is measured at a longer emission wavelength and the ratio of these intensities is calculated. The ratio is proportional to the concentration of the ion under investigation.

Excitation 1, 2
Enter the excitation wavelengths in the text boxes. Your instrument will automatically alternate between excitation wavelength 1 and excitation wavelength 2. The rate of alternation is dependent upon the illuminator type. The patented PTI DeltaRAM V can provide up to 250 ratios/sec while the DeltaScan X can produce ultra-fast switching allowing for 650 ratios/sec. A model 101 monochromator must move from one excitation wavelength to the other at the slewing speed set in the hardware configuration.

Emission
Enter the emission wavelength in the text box. If your instrument has two emission monochromators, FeliX32™ will ask for two emission wavelengths. The wavelengths you enter will be the wavelengths to which the monochromators will automatically move prior to data acquisition. If your system uses filters for wavelength selection, simply enter the peak wavelengths of the filters in these boxes.

Enable Single Point Screening
This is used to collect single data points into a spreadsheet display.

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Emission Ratio

Emission Ratio is used to set up and run experiments for intracellular ion determinations using emission-shifted probes such as Indo-1 for calcium and SNAFL for pH. In this experiment, a constant excitation wavelength is used and two emission wavelengths must be selected. This is normally done with two monochromators in a cuvette system, but one monochromator can be utilized. In a microscope-based system, the two emission wavelengths are selected using a dichroic assembly in the photometer. The emission intensity at both emission wavelengths is measured and the ratio of these intensities is calculated. The ratio is proportional to the concentration of the ion being determined.

Excitation
Enter the excitation wavelength in the text box. The wavelengths you enter will be the wavelengths to which the monochromators will automatically move prior to data acquisition. If your system uses filters for wavelength selection, simply enter the peak wavelengths of the filters in these boxes.

Emission 1, 2
Enter the emission wavelengths in the text boxes. Your instrument will automatically alternate between wavelength 1 and wavelength 2. The rate of alternation is dependent upon the configuration. Dual emission systems provide up to 1000 ratios per second. Single monochromator emission systems slew between the wavelengths to provide up to 1 ratio per second. A model 101 monochromator must move from one excitation wavelength to the other at the slewing speed set in the hardware configuration.

Enable Single Point Screening
This is used to collect single data points into a spreadsheet display.

PTI

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Excitation Scan

PTI

In an Excitation Scan, the excitation monochromator is scanned between two wavelengths while the emission monochromator is fixed. The emission intensity is measured as a function of excitation wavelength. Due to the nature of fluorescence, the emission wavelength is set at a wavelength that is longer than the excitation wavelength range (red-shifted).

Start and Stop
Enter the initial excitation wavelength and the final excitation wavelength for the scan in these text boxes. Emission Enter the emission wavelength in the text box. If your instrument has two emission monochromators, FeliX32™ will ask for two emission wavelengths.

Length
This shows the length of the scan that will be run. If the starting wavelength and the length are entered, FeliX32T will calculate the ending wavelength corresponding to these parameters.

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Emission Scan

In an Emission Scan, the emission wavelength is scanned between two wavelengths while the excitation monochromator is fixed. The emission intensity is measured as a function of excitation wavelength. Due to the nature of fluorescence, the excitation wavelength is set at a shorter wavelength than the emission wavelength range.

Excitation
Enter the excitation wavelength in the text box. Start and End wavelength Enter the emission wavelength scanning range in the Emission 1 text boxes. If the system is equipped with two emission monochromators, FeliX32T will request two wavelength ranges.

Length
This shows the length of the scan that will be run. If the starting wavelength and the length are entered, FeliX32T will calculate the ending wavelength corresponding to these parameters. For dual emission systems, the length of the scan will be the identical for both emission channels. FeliX32T will adjust the emission ranges to ensure they both have the same length.

PTI

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Multiple Dyes

The Multiple Dyes function is used to set up and run experiments for intracellular ion determinations using several indicators in combination, such as Fura-2 for calcium and BCECF for pH. In this experiment, the excitation light source must alternate between four different excitation wavelengths that are characteristic of the two probes (e.g. 340, 380, 440, 490 nm). In addition the isosbestic wavelength for Fura-2 is frequently monitored at 361 nm to obtain a calcium-independent signal.

The emission intensity resulting from excitation at the above five wavelengths is measured at longer emission wavelengths (510 and 525 nm, respectively) and the ratio of these intensities is calculated. The ratio is proportional to the concentration of the ion under investigation. Any combination of up to 10 excitation and 10 emission wavelengths may be defined to accommodate the simultaneous measurement of both excitation and emission-shifted dyes.

Use
Use the checkboxes to select the number of wavelength pairs for the experiment.

Ex
Enter the excitation wavelengths in these text boxes.

Emi. 1 and Emi. 2
Enter the emission wavelengths in these text boxes. If your system only has a single emission channel, the system will only display a single column for entering emission wavelengths.

Enable Single Point Screening
This is used to collect single data points into a spreadsheet display.

PTI

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Timebased

PTI

In a Timebased experiment, the excitation and emission wavelengths remain fixed throughout the experiment. The emission intensity is measured as a function of time. Timebased experiments typically involve kinetic measurements.

Excitation
Enter the excitation wavelength in the text box.

Emission
Enter the emission wavelength in the text box. If your instrument has two emission channels, FeliX32T will ask for two emission wavelengths. The wavelengths you enter will be the wavelengths to which the monochromators will automatically move prior to data acquisition. If your system uses filters for wavelength selection, simply enter the peak wavelengths of the filters in these boxes.

Enable Single Point Screening
This is used to collect single data points into a spreadsheet display.

 

 

 
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