Systems, methods, and apparatus for differential phase contrast microscopy by transobjective differential EPI-detection of forward scattered light

A microscope objective, comprising:

• a housing having a first end and a second end, the second end comprising mounting threads;
• a plurality of optical components defining an optical axis, the plurality of optical components comprising:
• an objective lens mounted at the first end, the objective lens configured to collect light from a sample placed in a field of view of the objective lens,
• wherein the plurality of optical components create a pupil plane at a first axial distance along the optical axis at which rays having the same angle of incidence on the objective lens from the within the field of view converge at the same radial distance from the optical axis;
• a first photodetector mounted within the housing at a second axial distance along the optical axis and offset from the optical axis by a first radial distance;
• and
• a second photodetector mounted within the housing at the second axial distance along the optical axis and offset from the optical axis by the first radial distance in a direction opposite from the first photodetector.

A detection apparatus for differential phase contrast microscopy by transobjective differential epi-detection of forward scattered light, comprising:

• a housing configured to be mechanically coupled to a scanning microscope such that the housing is disposed along an optical path of the scanning microscope;
• a substrate having a first surface and a second surface and an aperture defined by a through-hole from the first surface to the second surface, the substrate mounted within the housing such that, when the housing is mechanically coupled to the scanning microscope, the substrate is disposed along the optical path of the scanning microscope between an objective lens and an image plane associated with the objective lens;
• a first photodetector mechanically coupled to the first surface of the substrate and disposed at a first distance from a side of the aperture;
• and
• a second photodetector mechanically coupled to the first surface of the substrate and disposed at the first distance from an opposite side of the aperture from the first photodetector, such that second photodetector is separated from the first photodetector by the diameter of the aperture and twice the first distance.

A system for differential epi-detection of forward scattered light suitable for label free in vivo flow cytometry, comprising:

• a scanning microscope comprising:
• a first light source configured to emit light at a first wavelength;
• a second light source configured to emit light at a second wavelength;
• an optical train defining an optical path of the scanning microscope having an optical axis comprising:
• scanning components optically coupled to the light source and configured to scan a beam from the light source across a surface of a sample;
• and
• a microscope objective optically coupled to the scanning optical components;
• and
• a detector arranged to receive light emitted by the first light source and the second light source that has been directed into a sample via the microscope objective, forward scattered through the sample, and re-emitted from the sample on the same side as the microscope objective, the detector comprising:
• at least one pair of photodiodes optically coupled to detect forward scattered light emitted from the sample toward a first side of the microscope objective and a second side of the microscope objective that is opposite the first side;
• an amplifier electrically coupled to the detector, comprising:
• a differential amplifier configured to receive a first signal and a second signal from the at least one pair of photodiodes indicative of the intensity of light received at the first side of the microscope objective and the second side of the microscope objective at the first wavelength, respectively, and provide a signal indicative of a difference between the first signal and the second signal;
• and
• a sum amplifier configured to receive a third signal and a fourth signal from the at least one pair of photodiodes indicative of the intensity of light received at the first side of the microscope objective and the second side of the microscope objective at the second wavelength, respectively, and provide a signal indicative of a sum of the first signal and the second signal;
• and
• at least one hardware processor that is programmed to:
• cause the first light source to emit a first beam of light toward a sample via the optical train;
• cause the second light source to emit a second beam of light toward a sample via the optical train;
• cause the scanning components to scan the first beam of light and the second beam of light across the sample;
• receive, from the differential amplifier, a first plurality of output signals, each of the first plurality of output signals indicative of a structure of the sample at a location at which the first beam was focused;
• receive, from the sum amplifier, a second plurality of output signals, each of the second plurality of output signals indicative of an absorption by the sample at a location at which the second beam was focused;
• and
• generate image data indicative of the presence of blood cells and leukocytes in the sample based on the first plurality of output signals and the second plurality of output signals.