Methods for Counting Photons in Single-Molecule FRET TIRF-Microscopy

Intensity integration settings

iSMS implements two methods for calculating fluorescence intensities of individual molecules:

  • Aperture photometry uses an intensity mask (aperture) on the molecule and integrates the intensity within the mask. This is the default and recommended method.
  • 2D Gaussian PSF fitting fits a molecular 2D Gaussian PSF to each frame and uses the Gaussian volume as the total intensity of the molecule. Gaussian fitting is performed using either MLEwG (a maximum likelihood estimator) or GME (a least-squares fit).

The main disadvantages of using Gaussian PSF fitting is the inherent requirement on a good fit of each frame, which is often unreliable at low signal-to-noise ratios. Additionally, the fit depends on starting parameters and constraints. This means that Gaussian PSF does not work equally well on all molecules. Secondly, Gaussian PSF fitting is somewhat more slow compared to aperture photometry. Thirdly, the molecular PSF is not necessarily Gaussian shaped.

The main advantage of Gaussian PSF fitting is a lower fluctuation of background at high intensities and the ability to follow the Gaussian parameters in time, which is the technique exploited in TFM.

You can compare the performance of the different methods as described below.

Open the integration settings: 'Settings->Integration settings'.
In the settings dialog, choose integration method in the popup menu. Aperture photometry is the default and recommended method of choice.
When a Gaussian PSF method is chosen, set the optimization settings in the panel.

Compare fluorescence intensity integration methods

Tick the 'Compare methods' check box. This activates the lower part of the dialog. Select a molecule in the left list box (provided molecules exist in the current session). Select the two methods to compare in the two popup menus. The comparison is for the first 100 frames as defined in the edit box. To the right, you can see the fitted 2D Gaussian PSF at each frame and channel.

Set integration area interactively

If the integration aperture (mask) of a molecule does not fit perfectly on to the molecule you can interactively define the integration mask of the molecule in the FRET-pair window.

You can initially check the integration mask used for pixel integration by activating the 'Show/hide pixels used for integration' in the toolbar. This highlights the pixels used for calculating the fluorescence intensity in each channel.
To modify the integration mask, activate the 'Integration ranges' toggle button in the toolbar. This activates a ROI in the molecule images. Move and resize the ROI. To finish, press the toolbar toggle button again.
This creates a new integration mask of the molecule and the new intensity trace has been updated.
In the settings menu, select whether to use intensity after fluorophore has bleached as a measure of the background. If this setting is chosen, it can only be performed on the molecules that show bleaching.
This example shows how the background (black) looks when using a default background ring (top) and the intensity after bleaching (bottom) as background.