- The rendering of localization can also be set to either Fixed or Precision in Advanced Viewing Options. For Fixed, the default value of Gaussian blur is 3.5 nm.
Filters can be applied for tracking, in which track colors will take into account the filters to change the LUT when tracks are visualized as diffusion coefficient or track length:
Select an ROI or the track ID for visualization:
Histogram Plots
Tracking analysis automatically plots the data for the diffusion coefficient, track length, and stokes diameter as histograms. Options are available to replot the data by changing the bin size and the maximum value of the X axis for each histogram type. The Y axis represents the frequency of each bin.
Stokes Diameter or Particle Sizing
For particle sizing, Temperature is automatically taken from the metadata. For viscosity, the default value for water is set in mPa.s and option to enter the desired value is also available by unchecking the Calculate water Viscosity. The histogram can be exported as a CSV file by right-clicking and Copy To Clipboard:
Particle Concentration
The information panel has channel-specific measurements for particle sizes and concentration. The concentration is the average number of localizations per frame divided by the assumed volume of the field of view:
Export Tracks
Tracking data can be exported as a CSV file with all the filters applied:
CSV export will have information regarding channels, frames, track ID, steps, position (X, Y, and Z), photon counts, diffusion coefficient, and total displacement:
Technical Part
Mean squared distance:
For a track with positions pi at frame indices fi
The mean squared distance is calculated as follows:
msd=1n−1∑i=2n‖pi−pi−1‖fi−fi−1
In the (usual) case where all positions are taken from adjacent frames, this reduces to:
msd=1n−1∑i=2n‖pi−pi−1‖
Diffusion coefficient
We apply a correction for the non-zero exposure time from Savin and Doyle (2005):
Given exposure and time between adjacent frames Δt we calculate:
ϵ=1−te3Δt
For 2D:
D=msd4Δtϵ
Stokes Diameter:
Given temperature T and viscosity μ, we calculate the Stokes diameter d:
d=BzT3πμD
Where Bz is the Boltzmann constant
Bz=1.380649×10−23
Diameter Statistics:
The Nanoimager software provides various statistics on the Stokes diameter, such as mean, mode, standard deviation, and percentile values.
The mean, standard deviation, and percentiles are calculated from unfiltered Stokes diameters less than 10,000 nm. The 10,000 nm limit is an arbitrary value to exclude non-diffusing spots.
The mean is the arithmetic mean. The standard deviation is the population standard deviation.
The mode is calculated from the currently displayed histogram (e.g. dependent on bin width and lower/upper limits) and is the mid-point of the tallest bin. If two bins are identical in height, the bin for the smaller diameter will be used.
All tracks have equal weighting.
Concentration:
The concentration is the average number of localizations per frame divided by the assumed volume of the field of view.
The volume of the field of view = area (50 x 80 um) x depth (1 µm)
The 1 µm depth is hard coded as most of the localisations are within this range.
Reference
One can refer to the list of published diffusion coefficients of a range of biomolecules from Cell Biology by the numbers below
If at any point you are having issues with the SPT process, please do not hesitate to contact the CX team.