Ratiometry Application Guide
Basics of Ratiometry
There is a class of fluorochromes that are intended to measure changes in the intracellular milieu, known collectively as ratiometric dyes. These dyes, such as Fura-2, BCECF and beta-lactamase, are dual-excitation and/or dual-emission fluorochromes that have multiple absorption (excitation)/emission characteristics.
In order to accurately measure changes in fluorescence, the use of filter wheels is often imperative, since switching between single-band filter sets would entail a risk of shifting the sample, and subsequent misalignment of the final images. As an example, Fura-2 is often used to measure changes in intracellular calcium concentrations ([Ca2+]i). Fura-2 is a dual-excitation fluorochrome, and absorbs differently depending on its binding to Ca2+. When bound to Ca2+, Fura-2 exhibits an absorption peak at ~340 nm, whereas in a calcium-unbound form, it absorbs at a peak of ~380 nm.
Typical Fura-2 experiments involve the use of an excitation filter wheel to alternate between 340 and 380 excitation filters. Modern, fast-switching LED light sources are also a viable alternative for the use of filter wheels in many cases. Fluorescence measurements are taken by using a 470-550 nm bandpass emission filter mounted within the microscope. Post hoc image analysis is then used to compare 340/380 ratios (or calcium-bound vs. calcium-free levels of Fura-2), which give the user an indication of [Ca2+]i changes within the cells. One specialized ratiometric application is FRET (Förster Resonance Energy Transfer) microscopy.
Filter Requirements for Ratiometry
Excitation Filters
Like all fluorescence microscopy applications, filter selection should follow the fluorescence properties of the probe being used for both excitation and emission ranges. Given the increase in popularity of fast-switchable LED-based light sources, consideration must be paid to the spectral output of the light source being used to ensure that the ratiometric probe is excited appropriately, presuming it is a dual-excitation probe.
In cases like this, a dual-band excitation filter may be used, but in cases of true “white-light” sources (either arc lamps or LEDs), the former approach of using separate single-band excitation filters must be used.
Emission Filters
For dual-emission-based ratiometric probes, fluorescence emissions must be separated by either using separate single-band emission filters for each fluorescence peak or using an external beamsplitter device to separate the emissions into separate channels or portions of a detector chip.
In the latter case, a dual-band barrier/emission filter may be used, along with a dichroic mirror to separate the two bands, but many find the achieve better S/N using both a dual-band emission filter inside the filtercube (which also allows for visualization with the eye), as well as single-band emission filters downstream nearer to the detector.
Additional Resources
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Our applications team works with ratiometric setups across most major commercial platforms. Tell us your instrument and fluorochromes and we'll point you in the right direction.
