Functional imaging of cells with optical projection tomography

A method for 3D imaging of a biologic object in an optical tomography system comprising:

• (a) operating an optical tomography system with a processor, where the optical tomography system includes a microscope having an objective lens, a computer-controlled light source and a condenser lens assembly aligned along an optical axis, the optical tomography system also including a photodetector array for receiving light transmitted through the objective lens;
• (b) labeling a subcellular structure of the biological object by introducing at least one nanoparticle-biomarker;
• (c) positioning the labeled biological object relatively to a microscope objective to present varying angles of view;
• (d) illuminating the labeled biological object with radiation having wavelengths between 150 nm and 900 nm so that the at least one nanoparticle-biomarker is selected to emit or absorb radiation having wavelengths between 150 and 900 nm;
• (e) filtering a portion of the produced radiation using a set of filters to produce filtered signals within a set of wavelength bands;
• (f) sensing the filtered signals transmitted through the labeled biological object and the microscope objective within the set of wavelength bands to produce image data;
• (g) repeating steps (c) through (f) until a set of image data from varying angles of view has been produced;
• (h) forming a set of pseudo-projections of the biological object from the set of image data;
• (i) reconstructing the plurality of cross-sectional images to form a 3D image of the labeled biological object;
• (j) computing a mass for each of the set of pseudo-projections to generate a set of pseudo-projection mass values;
• (k) normalizing the set of pseudo-projection mass values to a common reference to produce a normalized set of pseudo-projection mass values;
• and
• (l) generating a 3D image reconstruction of the object corrected by the normalized set of pseudo-projection mass values.

A system for 3D imaging of cells in an optical tomography system comprising:

• at least one labeled biological object of interest;
• a delivery device positioned in an optical path relative to a microscope objective;
• at least one illumination source for producing light having a spectral bandwidth with wavelengths between 150 and 900 nm, wherein the illumination source employs plasmon resonance energy transfer (PRET), wherein the employment of PRET comprises:
• (a) using a dark-field microscopy configuration, wherein light scattered at high angles relative to the incident light is collected by the objective lens;
• and
• (b) incorporating into the labeled biological object at least one ensemble of metallic nanoparticles, each ensemble of metallic nanoparticles having at least one substantially identical labeling molecule attached to the surface of substantially each nanoparticle that is a member of the at least one ensemble of metallic nanoparticles;
• an objective lens being located to receive the light emanating from a labeled region of interest from a biological object;
• an axial translation mechanism coupled to translate the objective lens through a scanning range so as to extend the depth of field;
• a rotating mechanism for turning the object of interest to present a plurality of different views to the objective lens;
• a set of chromatic filters for selectively inserting into at least one optical path, wherein the set of filters produce filtered signals within a set of wavelength bands;
• at least one camera positioned to receive at least one wavelength of the light radiation emanating from the labeled biological object and the microscope objective;
• an image processor coupled to receive data from the at least one camera, where the image processor generates image data;
• and
• a reconstruction module coupled to the image processor, where the reconstruction module processes the image data to form a 3D image of the cell.