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Ca++ or FRET Ratio Microscopy

Ca++ or FRET Ratio Microscopy

Detail Description

VisiFluor Ca++ or FRET  Ratio Imaging Microscopy

Integrated Systems for Ratiometric Experiments - High Precision Measurement of Ion-Concentrations or FRET: Fluorescence ratio imaging involves the introduction of a fluorescent indicates into living cells to allow the monitoring of these cells through a microscope using a scientific grade high  speed and high sensitivty camera. Indicator dyes have been designed which shift their fluorescence excitation or emission spectrum upon binding the ions of interest. The design for single or dual wavelength intracellular ion measurements. VisiFluor supports e.g. FURA-2, BCECF, INDO-1 and other common ratiometric indicators.


For fluorescence measurements made at a single wavelength

The free ion concentration [I] is related to the fluorescence F by:

[I] = Kd(F-Fmin)/(Fmax-F)

Fmin and Fmax are respectively the fluorescence levels at zero and saturating ion concentrations, and Kd is the dissociation of the ion-indicator complex.


For fluorescence measurements made at dual wavelengths using ratiometric intracellular ion measurement indicators

The free ion concentration [I] is related to the fluorescence ratio R by the analogous equation: [I] = Kd.S.(R-Rmin)/(Rmax-R)

where S is a scaling factor given by the fluorescence at the denominator wavelength of R at zero ion concentration, divided by the fluorescence at a saturating ion concentration (see Grynkiewicz, Poenie and Tsien (1985) for derivation of this equation).


Time resolved FRET measurement

The donor emission as well as acceptor signal is recorded at every time point and a ratio is built (donor emission/FRET signal).

FRET value =  [ mean ratio (donor – acceptor) – mean ratio (donor only)] /mean ratio (donor only)

Donor-acceptor: cell line, expressing both donor and acceptor molecules

Donor only:cell line, expressing just the donor molecules

 

Threshold measurement: Apply  a grey level threshold  to each collected image to reduce the distracting effect of low level signal like background fluorescent and improve the accuracy of data collection by excluding the thersold region from ration claculation.

 

Event marks: the event mark function can be used  to store the injection time, changes in experiment conditions or applied triggers.

 

Logging of data to excel or text file: the data file stores the timestamp of each measurement and user defined selection of wavelength average intensity