One of the main
strengths of TPM is its ability to image thick tissues specimens.
This gives us the ability to perform image cytometry in thick
3D samples such as tissues. Below is a composite image of a ex
vivo human skin sample 1 cm which has been imaged down to a depth
of 70 microns. Thus it is possible to perform image cytometry
on cells while they are still in their intact state, preserving
many of their biochemical and mechanical inputs, and most importantly
their native 3D morphology and its relation to the 3D architecture
of the tissue. This provides a wealth of information about tissue
biophysics and biology on macroscopic samples that has not been
available before.
We are currently
extending the capability of the instrument by increasing the scanning
speed to make it comparable to processing rates found in flow
cytometry, and combing histological sectioning to allow us to
evaluate specimens that have an axial extent greater than the
standard 200 - 500 micron limit in two-photon microscopy.
An important avenue that we are also pursing is the visualization
and image analysis tools necessary to study these types of datasets.
A typical dataset can generate tens of gigabytes of data, far
too much for a human operator to manually classify. It becomes
necessary to use automated segmentation procedures to classify
the cell population into various sub-populations of biological
interest.
We are currently working on developing the computer algorithms
for studying these datasets.