Gradient light interference microscopy for 3D imaging of unlabeled specimens
A system and methods for quantitative optical phase imaging of a sample. First second replica field of an image field are generated, characterized by a respective optical phase, cross-polarized and shifted in a shift direction transverse to a normal to the surface of the sample. The replica fields are Fourier transformed, the second replica field is retarded by four successive phase shifts, and, after inverse Fourier transforming, the first and second replica fields pass through an analyzer polarizer and superposing the first and second replica fields on a detector array to create four successive detector signals. The four successive detector signals are solved to derive a gradient of the optical phase of the image field, which may be integrated to obtain a quantitative phase image.
1. A method for quantitative optical phase imaging of a sample characterized by a surface, the method comprising:
a. generating a first and a second replica field of an image field, each of the image field and the first and second replica fields characterized by a respective optical phase, the second replica field cross-polarized and shifted in a shift direction transverse to a normal to the surface of the sample by a transverse offset;
b. spatially Fourier transforming the first and second replica fields;
c. retarding the second replica field by four successive phase shifts;
d. inverse Fourier transforming the first and second replica fields;
e. passing the first and second replica field through an analyzer polarizer and superposing the first and second replica fields on a detector array to create four successive detector signals; and
f. solving the four successive detector signals to derive a gradient of the optical phase of the image field.
2. A method in accordance with claim 1 , wherein retarding the second replica field includes employing a spatial light modulator.
3. A method in accordance with claim 1 , wherein the four successive phase shifts are integral multiples of π/2.
4. A method in accordance with claim 1 , wherein the transverse offset is smaller than a diffraction spot characterizing an optical system whereby the first and second replica fields are obtained.
5. A method in accordance with claim 1 , further comprising integrating the gradient of the optical phase to obtain an image of the optical phase of the image field.
6. A method in accordance with claim 1 , wherein solving the four successive detector signals includes deriving a mutual intensity function between the first and second replica fields.
7. A system for quantitative optical phase imaging of a sample characterized by a surface, the system comprising:
a. a microscope for generating a first and a second replica field of an image field, each of the image field and the first and second replica fields characterized by an optical phase, the second replica field cross-polarized and shifted in a shift direction transverse to a normal to the surface of the sample by a transverse offset;
b. a first lens for spatially Fourier transforming the first and second replica fields;
c. a spatial light modulator for retarding the second replica field by four successive phase shifts;
d. a second lens for inverse Fourier transforming the first and second replica fields;
e. an analyzer polarizer aligned at 45° with respect to respective polarizations of the first and second replica fields;
f. a detector array for creating four successive detector signals; and
g. a processor for solving the four successive detector signals to derive a gradient of the optical phase of the image field.
8. A system in accordance with claim 7 , wherein the microscope includes a Wollaston prism for separating the first and second replica fields.
9. A module for receiving a first and a second replica field of an image field of a sample characterized by a surface and for quantitative optical phase imaging of the sample, wherein each of the image field and the first and second replica fields is characterized by an optical phase, the second replica field cross-polarized and shifted in a shift direction transverse to a normal to the surface of the sample by a transverse offset,
the module comprising:
a. a first lens for spatially Fourier transforming the first and second replica fields;
b. a spatial light modulator for retarding the second replica field by four successive phase shifts;
c. a second lens for inverse Fourier transforming the first and second replica fields; and
d. an analyzer polarizer aligned at 45° with respect to respective polarizations of the first and second replica fields.
10. A method for assessing viability of an embryo, the method comprising:
a. generating a first and a second replica field of an image field containing the embryo, each of the image field and the first and second replica fields characterized by a respective optical phase, the second replica field cross-polarized and shifted in a shift direction transverse to a normal to the surface of the sample by a transverse offset;
b. spatially Fourier transforming the first and second replica fields;
c. retarding the second replica field by four successive phase shifts;
d. inverse Fourier transforming the first and second replica fields;
e. passing the first and second replica field through an analyzer polarizer and superposing the first and second replica fields on a detector array to create four successive detector signals;
f. solving the four successive detector signals to derive a gradient of the optical phase of the image field; and
g. applying morphokinetic criteria to a function of the gradient of the optical phase to characterize embryo viability.