Methods of calibrating an imaging system using calibration beads
View Patent ↗When utilized in a flow imaging instrument, calibration beads provide a known data source that can be employed in various self-diagnostic, calibration and quality metric applications for the both the optical system of the flow imaging instrument, as well as the flow cell of the flow imaging instrument. Such data can be used to determine point spread functions associated with an imaging system, to determine a sensitivity of an imaging system, and to determine a focal point of the imaging system. Imagery collected from calibration beads can be used to determine core size and stability and TDI/flow speed synchronization. Calibration beads can be beneficially employed to enable stable system operation, even when very low sample concentration, or very small sample sizes are to be analyzed.
1. A set of calibration objects configured to support the operation of a flow imaging instrument, the flow imaging instrument being configured to image objects entrained in a flow of fluid passing through a field of view, using a time-delay-integration (TDI) detector, comprising a plurality of calibration objects, wherein each calibration object is sufficiently large to enhance the determination of:
(a) a velocity of the flow of fluid passing through the field of view;
(b) whether the flow of fluid is aligned with a desired axis within the field of view;
(c) whether the TDI detector is synchronized to the flow of fluid; and
(d) whether the flow imaging system is properly focused.
2. The set of calibration objects of claim 1 , wherein each calibration object in the set is between about 1 micron in diameter and about 10 microns in diameter.
3. The set of calibration objects of claim 1 , wherein a plurality of calibration objects in the set are fluorescently labeled, to facilitate calibration of the flow imaging instrument.
4. A set of calibration beads configured to support the operation of a flow imaging instrument, the flow imaging instrument being configured to image objects entrained in a flow of fluid passing through a field of view, using a time-delay-integration (TDI) detector, comprising:
(a) a plurality of calibration beads of a first type, wherein each calibration bead of the first type is sufficiently large to enhance the determination of the velocity of the flow of fluid passing through the field of view; and
(b) a plurality of calibration beads of at least one additional type, each calibration bead of said at least one additional type having at least one characteristic that is usable to support at least one of the following diagnostic functions:
(i) determining a point spread function associated with optical components of the flow imaging system; and
(ii) calibrating the flow imaging system.
5. The set of calibration beads of claim 4 , wherein calibration beads of the first type range from about 1 micron in diameter to about 10 microns in diameter.
6. The set of calibration beads of claim 4 , wherein at least some calibration beads of the at least one additional type have a second size selected to enhance a determination of the point spread function.
7. The set of calibration beads of claim 6 , wherein the calibration beads of the second size are relatively smaller than the calibration beads of the first type.
8. The set of calibration beads of claim 6 , wherein the calibration beads of the second size are selected as a function of optical characteristics of the flow imaging system and a pixel size of the TDI detector, such that images of the calibration beads of the second size that are incident on the TDI detector are substantially confined to a single pixel.
9. The set of calibration beads of claim 6 , wherein the calibration beads of the second size range from about 200 nanometers in diameter to about 500 nanometers in diameter.
10. The set of calibration beads of claim 4 , wherein at least some calibration beads of the at least one additional type are labeled with known fluorescent labels, such that fluorescent data collected by the flow imaging system is compared to the known fluorescent labels to calibrate the flow imaging system.
11. The set of calibration beads of claim 4 , wherein the set includes a sufficient quantity of calibration beads of the first type, such that based on a flow rate characteristic of the flow imaging system and a desired operating period, at least one calibration bead of the first type will be imaged by the TDI detector per update cycle when the set of calibration beads is introduced into the flow imaging instrument at the flow rate that is characteristic of the flow imaging system and for the desired operating period.
12. A set of calibration beads configured to support operation of a flow imaging instrument, the flow imaging instrument being configured to image objects entrained in a flow of fluid passing through a field of view using a time-delay-integration (TDI) detector, comprising:
(a) a plurality of calibration beads of a first size selected to enhance a diagnostic function; and
(b) a plurality of calibration beads of a second size that is relatively larger than the first size, the second size having been selected to enhance a determination of a velocity of the flow of fluid.
13. The set of calibration beads of claim 12 , wherein the first size falls within a range from about 200 nanometers to about 500 nanometers in diameter.
14. The set of calibration beads of claim 12 , wherein the second size falls within a range from about 1 micron to about 10 microns in diameter.
15. The set of calibration beads of claim 12 , wherein the calibration beads of the first size are selected as a function of optical characteristics of the flow imaging system and a pixel size of the TDI detector, such that images of individual calibration beads of the first size incident on the TDI detector are each substantially confined to a single pixel.
16. The set of calibration beads of claim 12 , wherein the calibration beads of the first size are useful for determining a point spread function associated with optical components of the flow imaging system.
17. The set of calibration beads of claim 12 , further comprising a plurality of calibration beads labeled with known fluorescent labels, such that fluorescent data collected by the flow imaging system can be compared to the known fluorescent labels to determine if the flow imaging system is producing reproducible results.
18. The set of calibration beads of claim 12 , wherein the set includes a sufficient quantity of the calibration beads of the second size, such that based on a flow rate characteristic of the flow imaging system and a desired operating period, at least one calibration bead of the second size will be imaged by the TDI detector per update cycle when the set of calibration beads is introduced into the flow imaging instrument at the flow rate characteristic of the imaging system, and for the desired operating period.
19. A set of calibration beads configured to support operation of a flow imaging instrument, the flow imaging instrument being configured to image objects entrained in a flow of fluid passing through a field of view using a time-delay-integration (TDI) detector, comprising:
(a) a plurality of calibration beads of a first type, wherein each calibration bead of the first type has a size selected to enhance a determination of the velocity of the flow of fluid passing through the field of view; and
(b) a plurality of calibration beads of at least one additional type, each calibration bead of an additional type having at least one characteristic that can be employed to support a diagnostic function.
20. The set of calibration beads of claim 19 , wherein calibration beads of the first type range from about 1 micron in diameter to about 10 microns in diameter.
21. The set of calibration beads of claim 19 , wherein at least some calibration beads of the at least one additional type are selected to have specified size based on a function of optical characteristics of the flow imaging system and a pixel size of the TDI detector, such that images of individual calibration beads of the specified size incident on the TDI detector are substantially confined to a single pixel, images confined to a single pixel facilitating determining a point spread function associated with optical components of the flow imaging system.
22. The set of calibration beads of claim 19 , wherein at least some calibration beads of the at least one additional type are labeled with known fluorescent labels, such that fluorescent data collected by the flow imaging system can be compared to the known fluorescent labels to determine if the flow imaging system is producing reproducible results.