Tomographic bright field imaging (TBFI)
Disclosed herein is a method of tomographic bright field imaging (TBFI): an optical imaging technique that enables the measurement of cellular refractive index and dry mass density using a standard transillumination optical microscope and software embodying said method.
1. A method of imaging a sample illuminated by a light source of a transillumination microscope, the method of imaging a sample illuminated by a light source of a transillumination microscope comprising:
focusing on a first z-plane of the sample;
focusing on a second z-plane of the sample;
measuring the intensity of the light from the light source, said light passing through the sample at the first z-plane and the second z-plane, thereby creating a set of 2-D measurements of the sample;
combining the set of 2-D measurements to form a 3-D image cube;
processing the image cube using a TBFI algorithm, said TFBI algorithm comprising a transport of intensity equation (TIE) and an Eikonal equation.
2. The method of imaging a sample illuminated by a light source of a transillumination microscope of claim 1 wherein the TFBI algorithm further comprises a first convolution theorem and wherein the processing results in a 3-D image cube of phase values.
3. The method of imaging a sample illuminated by a light source of a transillumination microscope of claim 1 wherein the TFBI algorithm further comprises a relation to a refractive index of the sample and a second convolution theorem.
4. The method of imaging a sample illuminated by a light source of a transillumination microscope of claim 3 wherein the processing results in a 3-D image cube of refractive index values.
5. The method of imaging a sample illuminated by a light source of a transillumination microscope of claim 3 wherein the TFBI algorithm further comprises a calibration model and wherein the processing results in a 3-D image cube of mass density profile values.
6. The method of imaging a sample illuminated by a light source of a transillumination microscope of claim 1 further comprising post processing of the 3-D image cube by defining a border of the sample in the 3-D image cube.
7. The method of imaging a sample illuminated by a light source of a transillumination microscope of claim 6 wherein post processing of the 3-D image cube further comprises determining the volume of the sample, determining the dry mass density of the sample, and/or determining the mass of the sample.
8. The method of imaging a sample illuminated by a light source of a transillumination microscope of claim 7 wherein the dry mass density of the sample is calculated using an equation of n(r ⊥ ,z)=n H 2 O [α(λ)C(r ⊥ ,z)+1], where α(λ) [dL/g] is a specific refractive increment of cell solids.
9. The method of imaging a sample illuminated by a light source of a transillumination microscope of claim 7 wherein the mass of the sample is calculated using an equation of M=∫ D C(r ⊥ ,z)X(r ⊥ ,z)dr ⊥ dz.
10. The method of imaging a sample illuminated by a light source of a transillumination microscope of claim 7 wherein the mass of the sample is calculated using an equation of V=∫ D X (r ⊥ ,z)dr ⊥ dz.