Critical angle reflection imaging for quantification of molecular interactions
View Patent ↗This disclosure describes systems and methods for critical angle reflection (CAR) imaging to quantify molecular binding kinetics on a glass surface in some embodiments. CAR is a label-free method that measures the reflectivity change near a critical angle in response to molecular binding induced refractive index changes on the sensor surface. The sensitivity and dynamic range of CAR is tunable by varying the incident angle of light, which allows for optimizing the measurement for ligands with different sizes in both biomolecular and cell-based studies.
1 . A method of detecting molecular interactions, the method comprising:
contacting a liquid comprising a ligand with a first surface of a substrate functionalized with a receptor displayed on a surface of a cell that is disposed on the first surface of the substrate, wherein the substrate is optically transparent bare glass free of a coating and wherein a refractive index of the substrate exceeds a refractive index of the liquid;
introducing an incident light into an optical prism toward a second surface of the substrate at an incident angle with respect to a plane perpendicular to the first surface of the substrate, wherein the second surface is opposite the first surface and the incident angle is less than a critical angle defined as sin −1 (n a /n g ), wherein n g is the refractive index of the substrate and n a is the refractive index of the liquid;
obtaining one or more differential critical angle reflection (CAR) images of the cell from light reflected at an interface of the liquid and the first surface of the substrate; and
identifying one or more moving parabolic patterns within the cell in the differential CAR images and/or a cell membrane deformation of the cell and a surface refractive index change in the differential CAR images, thereby detecting the molecular interactions.
2 . The method of claim 1 , wherein the moving parabolic patterns are generated by motion of one or more organelles within the cell.
3 . The method of claim 1 , wherein the ligand, the receptor, or both comprise a molecule.
4 . The method of claim 3 , wherein the molecule comprises a nucleic acid or a protein.
5 . The method of claim 1 , further comprising tuning a sensitivity and dynamic range to selected levels by varying the incident angle.
6 . The method of claim 1 , wherein a sensitivity of the method increases as the incident angle approaches the critical angle.
7 . The method of claim 1 , further comprising monitoring interaction of the ligand and the receptor in real time.
8 . The method of claim 1 , wherein binding of the ligand by the receptor alters an effective refractive index of the substrate near the first surface of the substrate.
9 . The method of claim 1 , further comprising fluorescence imaging of the ligand, the receptor, or both.
10 . The method of claim 1 , wherein the incident light comprises p-polarized light, s-polarized light, non-polarized light, or circularly polarized light.
11 . An optical imaging system comprising:
an optically transparent substrate having a first surface and a second surface opposite the first surface, wherein the optically transparent substrate is bare glass free of a coating and wherein the first surface of the optically transparent substrate is functionalized with a receptor displayed on a surface of a cell that is disposed on the first surface of the optically transparent substrate;
an optical prism configured to be coupled to the second surface of the optically transparent substrate;
a light source configured to introduce collimated light into the optical prism at an incident angle with respect to a plane perpendicular to the second surface of the optically transparent substrate, wherein the incident angle is less than a critical angle defined as sin −1 (n a /n g ), wherein n g is a refractive index of the substrate and n a is a refractive index of the liquid, and wherein the light source is further configured to vary the incident angle to tune a sensitivity and dynamic ranges to selected levels;
a detector configured to obtain one or more differential critical angle reflection (CAR) images of the cell from light reflected from an interface of the first surface of the optically transparent substrate and a liquid in contact with the first surface of the optically transparent substrate; and
a processor configured to identify one or more moving parabolic patterns within the cell in the differential CAR images and/or a cell membrane deformation of the cell and a surface refractive index change in the differential CAR images when a liquid comprising a ligand is contacted with the first surface of the optically transparent substrate.
12 . The optical imaging system of claim 11 , wherein the moving parabolic patterns are generated by motion of one or more organelles within the cell.
13 . The optical imaging system of claim 11 , wherein the ligand, the receptor, or both comprise a molecule.
14 . The optical imaging system of claim 13 , wherein the molecule comprises a nucleic acid or a protein.
15 . The optical imaging system of claim 11 , wherein the collimated light comprises visible light or UV light; or, wherein the collimated light comprises p-polarized light, s-polarized light, non-polarized light, or circularly polarized light.