Oblique line scanner systems and methods for high throughput single molecule tracking in living cells
AssigneeEikon Therapeutics, Inc.
InventorsDaniel ANDERSON, David MCSWIGGEN, Jaclyn HO, Amine DRIOUCHI, Russell BERMAN, Brian MARGOLIN
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Abstract
High Throughput Single Molecule Tracking (htSMT) systems and methods are described wherein the htSMT workflows are adapted to characterize both known and novel pathway contributions to interaction networks in live cells, such as protein signaling interaction networks.
Claims32
A method of identifying a biological interaction between a compound and a target fluorescent protein in a live cell comprising:
- (a) contacting a sample comprising a population of live cells with the compound, wherein the live cells comprise target fluorescent protein;
- (b) tracking the movement of a plurality of individual target fluorescent proteins in a plurality of live cells in the sample, wherein said tracking comprises:
- (i) illuminating a field of view in a sample plane disposed within the sample with a light beam to cause fluorescence by a subset of the target fluorescent proteins in the live cells;
- and
- (ii) detecting the fluorescence from a plurality of the target fluorescent proteins in a detected field of view of the sample plane via a detector device, wherein the detected field of view has a size of about 150 μm to about 250 μm in a first dimension by about 100 μm to about 210 μm in a second dimension;
- and
- (c) determining a change in the movement of at a target fluorescent protein in the presence of the compound,
- wherein a change in the movement of the target fluorescent protein in the presence of the compound relative to the movement of the target fluorescent protein in the absence of the compound identifies a biological interaction between the compound and the target fluorescent protein.
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A method of determining a dose response of a compound that induces a change in the movement of a target fluorescent protein in a live cell comprising:
- (a) contacting a plurality of samples with the compound,
- (i) wherein each sample comprises a population of live cells;
- (ii) wherein the live cells comprise target fluorescent protein;
- and
- (iii) wherein the plurality of samples are contacted with distinct concentrations of the compound across a range of compound concentrations;
- (b) tracking the movement of individual target fluorescent proteins in a plurality of live cells of a sample, wherein said tracking comprises:
- (i) illuminating a field of view in a sample plane disposed within the sample with a light beam to cause fluorescence by at least a subset of the target fluorescent proteins in the live cells;
- and
- (ii) detecting the fluorescence from one or more of the target fluorescent proteins in a detected field of view of the sample plane via a detector device, wherein the detected field of view has a size of about 150 μm to about 250 μm in a first dimension by about 100 μm to about 210 μm in a second dimension;
- and
- (c) determining a change in the movement of a target fluorescent protein in the presence of the compound;
- and
- (d) repeating steps (b)-(c) for each of the plurality of samples across the range of compound concentrations,
- wherein a change in the movement of the target fluorescent protein in the presence of the compound across the concentration range indicates the dose response of the compound.
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A microscopy system configured to identify a biological interaction between a compound and a target fluorescent protein in a live cell comprising:
- (a) a stage for supporting a sample, wherein the sample comprises a population of live cells, and wherein the live cells comprise the target fluorescent protein;
- (b) a light source for emitting a light beam capable of inducing a light-based response from a plurality of the target fluorescent proteins in the sample;
- (c) an objective for focusing the light beam on the sample in the sample plane, wherein a subset of the target fluorescent proteins in the sample are disposed in a detected field of view of the sample plane and wherein the detected field of view has a size of about 150 μm to about 250 μm in a first dimension by about 100 μm to about 210 μm in a second dimension;
- (d) a detector device for monitoring the light-based response from the target fluorescent proteins in the presence of the compound;
- (e) a memory;
- and
- (f) a processor in communication with the memory and the detector device, wherein the processor is capable of determining the change in the movement of the target fluorescent protein in the presence of the compound relative to the absence of the compound.
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Dependencies are inferred from the claim text.