Method for tracking particles and life forms in three dimensions and in time
A method for tracking particles and life forms in three dimensions and in time. The present invention applies a numerical reconstruction approach to digital in-line holographic microscopy images in order to generate a time sequence hologram representing the trajectory of objects such as particles and life forms. By subtracting consecutive (in time) holograms of a particle sample volume and then adding the resulting differences, a final hologram is constructed that contains the time evolution of the object trajectory free from spurious background interference effects.
1. A method for tracking the trajectory in three-dimensions and in time of an object in a sample volume comprising the steps of:
a) recording a time-spaced sequence of digital in-line holograms of the sample volume, generating a sequence of N holograms;
b) in each successive pair of holograms in the sequence of N holograms, subtracting from a first hologram in the pair a second hologram in the pair to generate N/2 difference holograms;
c) summing the N/2 difference holograms to generate a summed hologram; and
d) numerically reconstructing images of the object at a plurality of depths into the sample volume representing the trajectory of the object from the summed hologram.
2. A method for tracking the trajectory in three-dimensions and in time of an object in a sample volume comprising the steps of:
a) recording a time-spaced sequence of digital in-line holograms of the sample volume, generating a sequence of N holograms;
b) subtracting a first hologram, selected from the sequence of N holograms, from each of the remaining holograms of the sequence of N holograms to generate N−1 difference holograms;
c) summing the N−1 difference holograms to generate a summed hologram; and
d) numerically reconstructing images of the object at a plurality of depths into the sample volume representing the trajectory of the object from the summed hologram.
3. A method for tracking the trajectory in three-dimensions and in time of an object in a sample volume comprising the steps of:
a) recording a time-spaced sequence of digital in-line holograms of the sample volume, generating a sequence of N holograms;
b) subtracting a first hologram, selected from the sequence of N holograms, from each of the remaining holograms of the sequence of N holograms to generate N−1 difference holograms;
c) numerically reconstructing an image of the object at a depth into the sample volume for each of the N−1 difference holograms, generating N−1 subject images; and
d) combining the N−1 subject images to generate a 3-D image representing the trajectory of the object.
4. A method for tracking the trajectory in three-dimensions and in time of a plurality of objects in a sample volume comprising the steps of:
a) recording a time-spaced sequence of digital in-line holograms of the sample volume;
b) generating a summed hologram by representing each hologram in the time-spaced sequence of digital in-line holograms as a series of pixels corresponding to the summed hologram and:
subtracting each pixel in the series of pixels from a corresponding pixel in the
summed hologram, for a hologram having an even ordinal number in the
sequence of digital in-line holograms; and
adding each pixel in the series of pixels from a corresponding pixel in the summed hologram, for a hologram having an odd ordinal number in the sequence of digital in-line holograms; and
c) numerically reconstructing images of the plurality of objects, at a plurality of depths into the sample volume, representing the trajectories of the objects, from the summed hologram.
5. The method of any one of claims 1 to 4 , wherein the step of reconstructing uses a Kirchhoff-Helmholtz transform to obtain image at a reconstruction plane for each of the plurality of depths in the sample volume.