Molecules

Molecules is the second panel of module Simulation. Access the panel content by pressing Bottom arrow . The panel closes automatically after other panels open or after pressing Top arrow .

Use this panel to define the molecule sample in terms of size, positions and of molecule and dye properties.

Molecule positions

Use this interface to define the number and positions of molecules in the simulated field of view.

The number of single molecules to simulate, noted N, is shown in (b).

Molecule positions in the simulated field of view can be aquired in two was:

Randomly generated by activating (a),
Imported from an external ASCII file by activating (c).

Random positions

To generate random molecule positions, activate the option (a) and type in (b) the number of single molecules N to be simulated.

Import positions

To import molecule positions from an external ASCII file, activate the option (c) and select the appropriate file in the browser. After successful import, the file name is displayed in (d) and the number of molecule derived from the file is shown in (b).

For the positions to be correctly imported, certain rules must be followed:

the file must be structured in two or four columns, with x- and y-positions written in odd and even columns respectively.
if the file contains left- or right-channel coordinates only, coordinates in the other channel are automatically calculated by adding or subtracting one channel width to x-positions.
imported coordinates can me dismissed by reactivating the option (a)

Coordinates can also be imported from a pre-set parameter file; see Pre-set parameters for more details.

default: N = 100 molecules with randomly generated positions.

Pre-set parameters

Use this interface to import pre-defined parameters for individual molecules from an external MATLAB binary file (.mat).

Pre-set parameters can be imported from an external .mat file by activating (a). In that case:

the file must contain a MATLAB structure with at least one of the following fields:

field name	description	data type
`FRET`	FRET_j values and deviations wFRET_j for a J-state model; see State configuration for more details about the meaning of these values	J-by-2-by-Ndouble
`trans_rates`	transition rate matrix restricted to 2 states k_jj’	J-by-J-by-N double
`trans_prob`	transition probability matrix w_jj’ being in state j (normalized row-wise)	J-by-J-by-N double
`ini_prob`	initial state probabilities	N-by-J double
`gamma`	γ factors and deviations wγ; see Donor emission for more details about the meaning of these values	N-by-2 double
`tot_intensity`	intensities I_tot,em and deviations wI_tot,em; see Donor emission for more details about the meaning of these values	N-by-2 double
`coordinates`	x- and y- molecule coordinates in donor and/or acceptor channel	N-by-2 or -4 double
`psf_width`	PSF standard deviations w_det,D and/or w_det,A in donor and/or acceptor channel respectively	N-by-1 or -2 double

if parameter trans_prob is absent, uniform transition probabilities are defined
if parameters ini_prob is absent, initial state probabilities are calculated from the unrestricted transition rate matrix
if parameter coordinates contains coordinates in only one channel, x-positions are automatically translated to obtain coordinates in the other channel.
if parameter psf_width is of dimension N-by-1, PSF widths are applied to both channels.
loaded pre-sets can me removed by deactivating (a)

After successful import:

imported file name is displayed in (b)
the field used to edit the number of molecules in Molecule positions will be locked as it is set by any preset parameter
the field used to edit the number of states in State configuration will be locked if FRET or trans_rates are defined
the fields used to edit the FRET_j and wFRET_j values in State configuration will be locked if FRET is defined
the fields used to edit the Transition rates will be locked if trans_rates is defined
the fields used to edit the I_tot,em and wI_tot,em values in Donor emission will be locked if tot_intensity is defined
the fields used to edit the γ and wγ values in Donor emission will be locked if gamma is defined
the fields used to edit the w_det,D and w_det,A values in Point spread functions will be locked if psf_width is defined

Fields will be rendered editable when removing the pre-set file.

A *.m template file is provided with the source code at:

MASH-FRET/tools/createSimPrm.m

default: no parameter file is loaded, all parameters are set via the GUI.

State configuration

Use this interface to define the number of states and corresponding FRET values.

The state configuration is described by a number J of states set in (a), and the corresponding FRET_j values set in (c) after selecting state [j] in list (b), with [j] the state index.

If needed, sample heterogeneity can be introduce by attributing a strictly positive deviation wFRET_j (d) to the FRET_j value. In this case, a random FRET value is drawn for each molecule, using a Gaussian distribution with mean FRET_j and standard deviation wFRET_j.

default:

J = 2
for state 1: FRET₁ = 0, wFRET₁ = 0
for state 2: FRET₂ = 1, wFRET₂ = 0

Transition rates

Use this interface to define the rate coefficients that govern state transitions, noted k_jj’.

Transition rates are given in second^-1 and are organized in a matrix, where the cell (row j, column j’) concerns the unidirectional transition from state j to state j’.

A rate set to zero defines a forbidden transition.

Heterogeneous transition kinetics are characterized by multiple rate coefficients and can be simulated by using degenerated states, i.e., using states with same FRET_j value but different transition rate coefficients. For an example, please refer to the literature¹.

default: 0.1 second^-1.

Note: For more practicability, the transition rate matrix is limited to five states. To simulate a system with more than five states, please refer to Simulate more than five states

References

S. Schmid, T. Hugel, Efficient use of single molecule time traces to resolve kinetic rates, models and uncertainties, J. Chem. Phys. 2017, DOI: 10.1063/1.5006604

Donor emission

Sets the donor emission intensity in absence of acceptor, noted I_tot,em.

Intensity I_tot,em is set in (a) and is given in units defined in Intensity units.

If needed, sample heterogeneity can be introduced by attributing a strictly positive deviation wI_tot,em (b). In this case, a random intensity value is drawn for each molecule, using a Gaussian distribution with mean I_tot,em and standard deviation wI_tot,em.

Differences in donor and acceptor quantum yields and detection efficiencies can be modulated by setting the factor γ in (c). Donor emission is affected according to the relation:

$I_{\textup{D,em}}\left ( t\right ) = \frac{I_{\textup{D,em,0}}} {\gamma}$

with I_D,em,0 the original donor fluorescence intensity in presence of acceptor, and I_D,em the gamma-modified version.

Thus, introducing a gamma factor lower or higher than 1 place the donor intensity on a different scale than acceptor intensity.

Similarly to the intensity, sample heterogeneity in gamma factor can be introduced by setting a strictly positive deviation wγ in (d).

Intensity units

Intensity units of I_tot,em and wI_tot,em can be set in photon counts (pc) or camera offset-free image counts (ic) when the option in (e) is activated or inactivated respectively. This choice also affects the units of background intensities set in Background.

Photon counts μ_pc and camera offset-free image counts μ_ic are linked by the relation:

$\mu_{\textup{ic}} = \mu_{\textup{pc}} \times \eta \times K$

with camera characteristics η and K, the detection efficiency and overall gain respectively.

Camera offset-free image counts are used here because experimental I_tot,em value can be obtained by averaging the sum of donor and acceptor intensity-time traces in Trace processing after background correction, which includes subtraction of camera offset.

If one of the characteristics is not defined within the chosen camera noise model, the following default values are used:

η = 1 ec/pc
K = 1 ic/ec

See Camera SNR characteristics for more information.

Only photon counts μ_pc are registered in memory. Image counts are recalculated every time another camera noise model with different η and K is selected.

default:

I_tot,em = 36 pc
wI_tot,em = 0 pc
γ = 1
wγ = 0;

Cross-talks

Use this interface to set the direct excitation and bleedthrough coefficients.

Cross-talks bias the measured fluorescence intensities because of instrumental imperfections.

The donor and acceptor direct excitation coefficients dE_D and dE_A are the fractions of signal collected when illuminated by the pair’s excitation wavelength. They can be set in (a) and (b) respectively. Here, setting dE_D is senseless as simulations are limited to continuous- (donor-) wavelength excitation.

The donor and acceptor bleedthrough coefficients bt_D and bt_A are the fractions of signal leaking in the pair’s channel. They can be set in (c) and (d) respectively.

default: default coefficients are based on our experimental setup:

dE_D = 0
dE_A = 0.02
bt_D = 0.07
bt_A = 0

Photobleaching

Defines the time spent before photochemical destruction of the donor fluorophore.

Photochemical destruction of the donor, i.e., photobleaching, can be activated by activating the option in (a). In that case, intensity-time traces are interrupted at random photobleaching time by a sudden and irreversible extinction of donor emission.

Photobleaching times are randomly drawn for each molecule from an exponential distribution with a decay constant set by (b).

default: no photobleaching, the observation time is set by video length L.