Bleedthrough Correction Process Description

Bleedthrough Correction Process Description:

"Bleedthrough" occurs when the fluorophore from one channel contributes to the intensity in another channel, due to spectral overlap or imperfect filtering. With single-chain (intramolecular) biosensors, the localization of the fluorophores in each channel is always identical. Therefore, the bleedthrough contribution is automatically canceled out during the ratioing step. However, with dual-chain (intermolecular) biosensors, the localization is almost always at least slightly different for the two fluorophores, which means that bleedthrough must be corrected. This is achieved by determining the bleedthrough coefficients in a separate experiment where each half of the sensor is imaged independently. These coefficients are then used to correct the actual fluorophore images. The coefficients can be calculated from these bleedthrough experiments using the Bleedthrough Coefficient Calculation tool in the “Tools” menu on the “Control Panel – u-probe” (see the user's manual for more details).

Figure 1. Left: due to overlapping emission spectra, CFP fluorophore emission is collected by the YFP filter set (bleedthrough). Right: due to overlapping excitation spectra, YFP fluorophore is partially excited by the CFP excitation (crosstalk).

Parameter Descriptions:

Input Channel:

This allows you to select the channel for bleedthrough correction. This should be the activity channel (usually FRET), which will be the numerator in the ratio.

Coefficients:

This table allows you to input the correction coefficients determined previously. The column specifies the coefficients to use for bleedthrough correction. You can specify a coefficient for each valid channel. Please refer below for more information on bleedthrough coefficients.

FRET Ratio Calculation:

The FRET ratio is calculated as follows:

$$ \text{Ratio} = \frac{(1 \times DA - \alpha \times DD - \beta \times AA)}{DD} $$

where:

• DA: Donor_excitation Acceptor_emission
• DD: Donor_excitation Donor_emission
• AA: Acceptor_excitation Acceptor_emission
• α and β are correction coefficients for bleedthrough. Note that the coefficient for DA is always 1.

Before running FRET, a control experiment is needed to determine α, and another control experiment is needed to determine β. These coefficients can be calculated from the bleedthrough control experiments using the Bleedthrough Coefficient Calculation tool in the “Tools” menu on the “Control Panel – u-probe” (see the user's manual for more details).