The Single-Molecule or Single-Particle Tracking function allows the observation of the motion of individual molecules/particles:

For video instructions on how to use the tracking function, check here.

Run Tracking

1. Click on Tracking from Tools to open the tracking window. The tracking algorithm has three input settings:
   
1. Exclusion Radius
   
2. Maximum Step Distance
   
3. Maximum Frame Gap
   
2. To initiate the tracking analysis, click on Run Tracking to start the tracking process. Spots are tracked as follows:
   
   
1. For each frame, spots are localized to give a list of X and Y coordinates.
2. Any spots with neighbors within the defined Exclusion Radius are discarded.
3. For each of the spots found in the previous frame (or frames, if Maximum Frame Gap > 0):
   
1. Find and store any spots in the current frame within the Maximum Step Distance between frames.
2. If more than one spot is found within the Maximum Step Distance between frames, store the closest spot as a match position.
   
4. For each match position found in step 3, if there are any other matches with the same spot in the current frame, only retain the match with the closest distance.
5. For each remaining match after step 4:
1. If the previous position is part of an existing track, add the new match position to that track.
2. Otherwise, create a new track comprising the first two positions found in this and the previous frame.
   
6. For unmatched positions found in previous frames, discard any which are more than the Maximum Frame Gap from the current frame.
   

Visualization Of Tracking

1. Select data for specific channels or a combination of channels that you would like to visualize in Viewing Options:
   
   
2. To change the color of localization and tracks, click on Advanced Viewing Options to set colors for each channel, in which the color map can be set to Channel, Diffusion Co., First Frame Index (the first frame index when the track starts to build), or Track Length:
   
   

1. 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.
   
2. 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:
   
   
3. 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

1. 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
   

2. 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
   

3. 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.
   

4. 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

http://book.bionumbers.org/what-are-the-time-scales-for-diffusion-in-cells/

If at any point you are having issues with the SPT process, please do not hesitate to contact the CX team.