System level calibration
Disclosed herein are systems and methods of calibrating a microscope or an imaging system prior to acquiring image data of a sample. In some embodiments, a method is disclosed including the steps of (a) running a power output calibration module to calibrate an imaging apparatus for repeatability; (b) running an image intensity calibration module to calibrate the imaging apparatus for reproducibility and to mitigate differences in detection efficiency between channels; (c) collecting image data from a microscope or imaging system; (d) optionally running an unmixing module to unmix the collected image data into individual image channel images; and (e) optionally running a contrast agent intensity correction module to calibrate for differences in brightness between different contrast agents.
1. A method of imaging a biological sample stained with one or more stains, each stain identifying a particular target within the biological sample, comprising:
setting a first power output of a light source for a first channel such that a first predetermined power value is reached;
adjusting a first exposure time of a camera to yield a first target intensity at the first predetermined power value for the first channel;
acquiring a first image of the biological sample in a first channel based on the adjusted first exposure time; and
adjusting pixel data in the first acquired image based on a first derived image correction factor specific to one of the one or more stains within the first channel, wherein the first derived image correction factor is computed by (i) normalizing brightness values for a plurality of stains; and (ii) subtracting the normalized brightness value for the first stain from the integer 1.
2. The method of claim 1 , wherein the first power output level for the light source is derived from preexisting calibration data for the first channel.
3. The method of claim 2 , wherein the preexisting calibration data is a calibration curve of power output versus measured power.
4. The method of claim 1 , wherein the first predetermined power value is specific to a particular assay.
5. The method of claim 4 , wherein the first predetermined power value is the same for all channels.
6. The method of claim 1 , wherein the first target intensity is a predetermined percentage of a standardized target intensity of a calibration sample for the first channel.
7. The method of claim 6 , wherein the predetermined percentage ranges from about 20% to about 500% of the standardized target intensity for the first channel.
8. The method of claim 6 , wherein the standardized target intensity for the first channel is derived by (i) determining calibration intensity values of the calibration standard for each of a plurality of image channels; (ii) normalizing the calibration intensity values of the calibration standard for each of the plurality of image channels to a highest intensity value of each of the normalized calibration intensity values; (iii) deriving a ratio of normalized intensity across the plurality of image channels; (iv) assigning a target intensity value for the brightest image channel in the plurality of image channels; and (v) calculating the standard intensity value for the remaining image channels.
9. The method of claim 1 , wherein the pixel data within the first image is adjusted by multiplying each pixel value within the first image by the first image correction factor.
10. A system comprising one or more processors and a memory coupled to the one or more processors, the memory to store computer-executable instructions that, when executed by the one or more processors, cause the one or more processors to perform operations comprising:
setting a first power output of a light source for a first channel such that a first predetermined power value is reached;
adjusting a first exposure time of a camera to yield a first target intensity at the first predetermined power value for the first channel;
acquiring a first image of the biological sample in a first channel based on the adjusted first exposure time; and
adjusting pixel data in the first acquired image based on a first derived image correction factor specific to one of the one or more stains within the first channel, wherein the first derived image correction factor is computed by (i) normalizing brightness values for a plurality of stains; and (ii) subtracting the normalized brightness value for the first stain from the integer 1.
11. The system of claim 10 , wherein the first power output level for the light source is derived from preexisting calibration data for the first channel.
12. The system of claim 11 , wherein the preexisting calibration data is a calibration curve of power output versus measured power.
13. The system of claim 10 wherein the first predetermined power value is specific to a particular assay.
14. The system of claim 13 , wherein the first predetermined power value is the same for all channels.
15. The system of claim 10 , wherein the first target intensity is a predetermined percentage of a standardized target intensity of a calibration sample for the first channel.
16. The system of claim 15 , wherein the standardized target intensity for the first channel is derived by (i) determining calibration intensity values of the calibration standard for each of a plurality of image channels; (ii) normalizing the calibration intensity values of the calibration standard for each of the plurality of image channels to a highest intensity value of each of the normalized calibration intensity values; (iii) deriving a ratio of normalized intensity across the plurality of image channels; (iv) assigning a target intensity value for the brightest image channel in the plurality of image channels; and (v) calculating the standard intensity value for the remaining image channels.
17. The system of claim 10 , wherein the pixel data within the first image is adjusted by multiplying each pixel value within the first image by the first image correction factor.
18. A system comprising one or more processors and a memory coupled to the one or more processors, the memory to store computer-executable instructions that, when executed by the one or more processors, cause the one or more processors to perform operations comprising: setting a first power output of a light source for a first channel such that a predetermined power value is reached; adjusting a first exposure time of a camera to yield a first target intensity at the predetermined power value for the first channel; acquiring a first image of a biological sample at a first channel based on the adjusted exposure time, wherein the biological sample is stained with one or more stains, each stain selectively identifying a particular target within the biological sample; and adjusting pixel data in the first acquired image based on a first derived image correction factor specific to one of the one or more stains within the first channel, wherein the first power output level for the light source is derived from preexisting calibration data for the first channel, wherein the preexisting calibration data for the first channel is a calibration curve of power output versus measured power.
19. The system of claim 18 , wherein the pixel data within the first image is adjusted by multiplying each pixel value within the first image by the first image correction factor.
20. The system of claim 18 , wherein the first target intensity is a predetermined percentage of a standardized target intensity of a calibration sample for the first channel.