Systems and methods for actively mitigating vibrations
Various embodiments of the present disclosure disclose methods and systems for actively monitoring an opto-fluidic instrument for vibrational disturbances and correcting images obtained during the detected disturbances. In various embodiments, an optical imaging system may acquire Z-stack images of samples supported by an XY-stage. Vibrations can cause individual stacks to be sheared, i.e., to not be co-located with respect to neighboring Z-stacks. In various embodiments, an offset between the measured positions and the expected positions of the sheared stacks may be computed, and a determination as to whether to correct or reacquire the Z-stack images may be made based at least in part on the computed offset.
1. A method, comprising:
acquiring, using an optical imaging system, a first plurality of Z-stack images of a sample supported by an XY-stage, wherein the first plurality of Z-stack images are images of a respective plurality of two-dimensional (2D) slices of the sample;
measuring, using a position sensor coupled to the XY-stage, a position of one of the plurality of 2D slices, wherein one of the first plurality of Z-stack images is an image of the one of the plurality of 2D slices;
computing, using a processor coupled to the position sensor, a position offset between the measured position and a nominal position, of the one of the plurality of 2D slices; and
determining whether to adjust, using the processor, the position information associated with the one of the first plurality of Z-stack images, or acquire, using the optical imaging system, a second plurality of Z-stack images of the sample, based at least in part on a comparison of the position offset and a threshold offset.
2. The method of claim 1 , wherein the optical imaging system includes an epifluorescence microscope.
3. The method of claim 1 , wherein the acquiring the first plurality of Z-stack images includes moving the sample in X-direction or Y-direction using the XY-stage during the acquiring of the first plurality of Z-stack images by the optical imaging system.
4. The method of claim 1 , wherein the acquiring the first plurality of Z-stack images occurs at a rate ranging from about 10 Hz to about 20 Hz.
5. The method of claim 1 , wherein the measuring the position of one of the plurality of 2D slices occurs at a rate ranging from about 500 Hz to about 10 kHz.
6. The method of claim 1 , wherein successive 2D slices of the plurality of 2D slices are separated from each other by a distance ranging from about 500 nm to about 1000 nm.
7. The method of claim 1 , wherein the optical imaging system is coupled to a Z-stage configured to move an objective lens of the optical imaging system in the Z-direction.
8. The method of claim 7 , wherein the acquiring the first plurality of Z-stack images includes moving, using the Z-stage, the objective lens of the optical imaging system in the Z-direction during the acquiring of the first plurality of Z-stack images by the optical imaging system.
9. The method of claim 7 , wherein:
the position of one of the plurality of 2D slices includes an X-Y position of the one of the plurality of 2D slices; and
the position sensor is coupled to the XY-stage and is configured to measure the X-Y position of the one of the plurality of 2D slices.
10. The method of claim 1 , wherein the plurality of 2D slices correspond to focal planes of an objective lens of the optical imaging system.
11. The method of claim 1 , wherein the determining includes determining to adjust the position information when the position offset is less than a threshold offset.
12. The method of claim 1 , wherein the determining includes determining to acquire the second plurality of Z-stack images when the position offset is equal to or greater than the threshold offset.
13. The method of claim 1 , wherein the position of the one of the plurality of 2D slices includes a plurality of positions of the one of the plurality of 2D slices and the position offset includes a plurality of position offsets between the plurality of positions and the nominal position of the one of the plurality of 2D slices, the method further comprising:
computing, using the processor, a standard deviation of the plurality of positions offsets.
14. The method of claim 13 , wherein the determining includes determining to adjust the position information when the computed standard deviation is less than a threshold standard deviation and the position offset is less than a threshold offset.
15. The method of claim 14 , wherein:
the measured position includes an x-direction measured position value (“MPV-x”), and a y-direction measured position value (“MPV-y”), and the nominal position includes an x-direction nominal position value (“NPV-x”), and a y-direction nominal position value (“NPV-y”);
the position offset includes an x-direction offset (“OFF-x”) between MPV-x and NPV-x, and a y-direction offset (“OFF-y”) between MPV-y and NPV-y;
the threshold offset includes an x-direction threshold offset (“TO-x”) and a y-direction threshold offset (“TO-y”); and
the position offset being less than the threshold offset includes the OFF-x and the OFF-y being less than the TO-x and the TO-y, respectively.
16. The method of claim 14 , wherein:
the measured position includes an x-direction measured position value (“MPV-x”) and a y-direction measured position value (“MPV-y”), and the nominal position includes an x-direction nominal position value (“NPV-x”) and a y-direction nominal position value (“NPV-y”);
the position offset includes an x-direction offset (“OFF-x”) between MPV-x and NPV-x and a y-direction offset (“OFF-y”) between MPV-y and NPV-y; and
the position offset being less than or greater than the threshold offset includes ((OFF-x) 2 +(OFF-y) 2 ) 0.5 being less than the threshold offset.
17. The method of claim 14 , wherein:
the measured position includes an x-direction measured position value (“MPV-x”) and a y-direction measured position value (“MPV-y”), and the nominal position includes an x-direction nominal position value (“NPV-x”) and a y-direction nominal position value (“NPV-y”);
the position offset includes an x-direction offset (“OFF-x”) between MPV-x and NPV-x and a y-direction offset (“OFF-y”) between MPV-y and NPV-y;
the one of the first plurality of Z-stack images depicts a fluorescent object; and
the adjusting the position information includes shifting an x-direction position of the fluorescent object by the OFF-x and/or a y-direction position of the fluorescent object by the OFF-y.
18. The method of claim 13 , wherein the determining includes determining to acquire the second plurality of Z-stack images when the computed standard deviation is equal to or greater than a threshold standard deviation and/or the position offset is equal to or greater than a threshold offset.
19. The method of claim 18 , wherein:
the measured position includes an x-direction measured position value (“MPV-x”), and a y-direction measured position value (“MPV-y”), and the nominal position includes an x-direction nominal position value (“NPV-x”), and a y-direction nominal position value (“NPV-y”);
the position offset includes an x-direction offset (“OFF-x”) between MPV-x and NPV-x, and a y-direction offset (“OFF-y”) between MPV-y and NPV-y;
the threshold offset includes an x-direction threshold offset (“TO-x”) and a y-direction threshold offset (“TO-y”); and
the position offset being equal to or greater than the threshold offset includes the OFF-x and the OFF-y, or a z-direction offset (“OFF-z”) being equal to or greater than the TO-x, the TO-y, or a z-direction threshold offset (“TO-z”), respectively.
20. The method of claim 18 , wherein:
the measured position includes an x-direction measured position value (“MPV-x”) and a y-direction measured position value (“MPV-y”), and the nominal position includes an x-direction nominal position value (“NPV-x”) and a y-direction nominal position value (“NPV-y”);
the position offset includes an x-direction offset (“OFF-x”) between MPV-x and NPV-x and a y-direction offset (“OFF-y”) between MPV-y and NPV-y; and
the position offset being less than or greater than the threshold offset includes ((OFF-x) 2 +(OFF-y) 2 ) 0.5 being equal to or greater than the threshold offset.