High speed scanning systems for super resolution imaging
Disclosed herein is a high throughput optical scanning system to generate super resolution images and methods of use. The optical scanning device and methods of use provided herein can allow high throughput scanning of a continuously moving object with a high resolution despite fluctuations in stage velocity. This can aid in high throughput scanning of a substrate, such as a biological chip comprising fluorophores. Also provided herein are improved optical relay systems and scanning optics.
1. A method of imaging a moving substrate, wherein said moving substrate comprises a plurality of fields, comprising:
a) disposing said substrate on a stage, wherein said stage moves along a defined path adjacent to a light source;
b) receiving, by a central processing unit, positional information from an encoder, wherein said central processing unit performs a plurality of calculations of a plurality of positions for said stage;
c) receiving, by a scanning mirror coupled to an axis adjacent to said defined path, said plurality of calculations of said plurality of positions for said stage;
d) imaging at least a field of said plurality of fields with one or more cameras, thereby generating a plurality of images; and
e) using an imaging algorithm to process said plurality of images to identify a position of an analyte of a plurality of analytes displaced on said substrate or a relative position of an analyte displaced on said substrate with respect to another analyte displaced on said substrate;
wherein said plurality of analytes are disposed on said substrate at a density such that a minimum effective pitch between an analyte of said plurality of analytes and another analyte of said plurality of analytes is less than λ/(2*NA), wherein λ is a wavelength of light used in said defined path and ‘NA’ is a numerical aperture of said one or more cameras.
2. The method of claim 1 , wherein (b)-(d) are performed synchronously.
3. The method of claim 1 , wherein a plurality of movements of said scanning mirror is generated from said plurality of calculations of said central processing unit.
4. The method of claim 1 , wherein said plurality of calculations is a function of a change in velocity of said stage.
5. The method of claim 1 , wherein (b) further comprises providing, by said central processing unit, said plurality of calculations in a calibration waveform to said scanning mirror.
6. The method of claim 5 , wherein (b) further comprises configuring said central processing unit to:
generate one or more correction values for said position of said analyte of said plurality of analytes displaced on said substrate or said relative position of said analyte displaced on said substrate with respect to another analyte displaced on said substrate; and
apply said one or more correction values to said calibration waveform.
7. The method of claim 1 , wherein (c) further comprises moving said scanning mirror to an original position upon completing a scan of a field of said plurality of fields and scanning a subsequent field of said plurality of fields.
8. The method of claim 1 , wherein (b) further comprises generating, by said encoder, a signal period from a plurality of encoder counts.
9. The method of claim 8 , wherein said encoder counts are positioned at a distance of 0.05 micrometers to 30 micrometers.
10. The method of claim 8 , wherein said encoder generates said signal period from said plurality of encoder counts at a signal period of up to 512 nanometers (nm).
11. The method of claim 1 , wherein an image of said plurality of images comprises a blur of less than 10 nm to 40 nm.
12. The method of claim 11 , wherein said blur is generated at a frame rate of 20 frames per second to 150 frames per second.
13. The method of claim 5 , wherein each point of said calibration waveform comprises an associated control value for said one or more cameras, said light source, or a combination thereof.
14. The method of claim 5 , wherein said calibration waveform is determined from a pre-calculated table.
15. The method of claim 1 , wherein said plurality of fields comprises at least 20 fields.
16. The method of claim 15 , wherein said substrate comprises a plurality of lanes, and wherein a lane of said plurality of lanes comprises a plurality of columns.
17. The method of claim 1 , wherein said plurality of calculations comprises instantaneous calculations.
18. The method of claim 1 , wherein said light source comprises a laser or a plurality of lasers.
19. A system comprising:
a) a light source;
b) one or more cameras operably coupled to an objective lens to capture a plurality of fields;
c) a stage, wherein, when in use, said stage holds a substrate and moves along a defined path adjacent to said light source;
d) an encoder mechanically operably coupled to said stage;
e) a central processing unit, wherein, when in use, said central processing unit receives positional information about said stage from said encoder and performs a plurality of calculations of a plurality of positions of said stage;
f) a scanning mirror coupled to an axis adjacent to said defined path, wherein said scanning mirror is operably coupled to said central processing unit to receive said plurality of calculations of said plurality of positions of said stage; and
g) an image processor, wherein, when in use, said image processor uses an imaging algorithm to process a plurality of images to identify a position of an analyte of a plurality of analytes displaced on said substrate or a relative position of an analyte displaced on said substrate with respect to another analyte displaced on said substrate, wherein said plurality of analytes are disposed on said substrate at a density such that a minimum effective pitch between an analyte of said plurality of analytes and another analyte of said plurality of analytes is less than λ/(2*NA), and wherein λ is a wavelength of light used in an optical path and ‘NA’ is a numerical aperture of said one or more cameras.