High-speed laser scanning microscopy platform for high-throughput automated 3d imaging and functional volumetric imaging

Abstract

A laser scanning system for capturing an image of a specimen is described herein. The laser scanning system includes a light source configured to emit a light beam for illuminating the specimen, a scanning unit including a plurality of reflectors for scanning the light beam along first and second axes, and a data acquisition unit configured to control acquisition of the image. The laser scanning system can include a control circuit configured to receive a reference clock signal for the first reflector and generate a synchronization clock signal based on the reference clock signal. The laser scanning system can include a synchronization controller configured to control the scanning unit and the data acquisition unit. The synchronization controller can be configured to receive the synchronization clock signal, receive a plurality of imaging parameters, and generate a plurality of control signals based on the synchronization clock signal and the imaging parameters.

1 . A laser scanning system for capturing an image of a specimen, comprising:
 a light source configured to emit a light beam for illuminating the specimen;   a scanning unit comprising a plurality of reflectors, wherein a first reflector scans the light beam along a first axis and a second reflector scans the light beam along a second axis;   a data acquisition unit configured to control acquisition of the image of the specimen;   a control circuit configured to receive a reference clock signal for the first reflector and generate a synchronization clock signal based on the reference clock signal; and   a synchronization controller configured to control the scanning unit and the data acquisition unit, the synchronization controller comprising a processor and memory operably coupled to the processor, the memory having computer-executable instructions stored thereon that, when executed by the processor, cause the synchronization controller to:   receive the synchronization clock signal;   receive a plurality of imaging parameters; and   generate a plurality of control signals based on the synchronization clock signal and the imaging parameters, wherein:
 a first control signal synchronizes operation of the first and second reflectors of the scanning unit, and 
 a second control signal synchronizes operation of the scanning unit and the data acquisition unit. 
   
     
     
         2 . The laser scanning system of  claim 1 , wherein the control circuit is further configured to:
 generate an enhanced reference clock signal from the reference clock signal; and   generate the synchronization clock signal from the enhanced reference clock signal.   
     
     
         3 . The laser scanning system of  claim 2 , wherein generating the enhanced reference clock signal comprises at least one of filtering, regulating the voltage of, suppressing fluctuation of, or supplying current to the reference clock signal. 
     
     
         4 . The laser scanning system of  claim 1 , wherein the control circuit comprises a frequency doubler circuit for generating the synchronization clock signal. 
     
     
         5 . The laser scanning system of  claim 1 , wherein the imaging parameters comprise at least one of a number of lines in the image, a number of pixels per line in the image, or a field of view. 
     
     
         6 . The laser scanning system of  claim 5 , wherein the field of view comprises a horizontal scan range and a vertical scan range. 
     
     
         7 . The laser scanning system of  claim 1 , wherein respective periods of the first and second control signals are approximately equal. 
     
     
         8 . The laser scanning system of  claim 5 , wherein a period of the first control signal is variable based on the number of lines in the image, and wherein a period of the second control signal is variable based on the number of lines in the image. 
     
     
         9 . (canceled) 
     
     
         10 . The laser scanning system of  claim 8 , wherein the respective periods of the first and second control signals is equal to a period of the synchronization clock signal times the number of lines in the image plus 1. 
     
     
         11 . The laser scanning system of  claim 5 , wherein an amplitude of the first control signal is variable based on the field of view. 
     
     
         12 . The laser scanning system of  claim 11 , wherein a level of the first control signal increases incrementally after each cycle of the synchronization clock signal to the amplitude before returning to a minimum level. 
     
     
         13 . The laser scanning system of  claim 1 , further comprising a translational device for supporting the specimen, the translational device being configured to move along a third axis, wherein the plurality of control signals include a third control signal for synchronizing operation of the scanning unit, the data acquisition unit, and the translational device. 
     
     
         14 . The laser scanning system of  claim 13 , wherein the imaging parameters comprise at least one of a translational device movement range or a number of translational device movement steps. 
     
     
         15 . The laser scanning system of  claim 13 , wherein the third control signal is synchronized with the first and second control signals. 
     
     
         16 . The laser scanning system of  claim 14 , wherein a period of the third control signal is variable based on the number of translational device movement steps. 
     
     
         17 . The laser scanning system of  claim 16 , wherein the period of the third control signal is equal to a period of the second control signal times the number of translational device movement steps. 
     
     
         18 . The laser scanning system of  claim 14 , wherein an amplitude of the third control signal is variable based on the translational device movement range. 
     
     
         19 . The laser scanning system of  claim 18 , wherein a level of the third control signal increases incrementally after each cycle of the second control signal to the amplitude before returning to a minimum level. 
     
     
         20 . The laser scanning system of  claim 5 , wherein the memory has further computer-executable instructions stored thereon that, when executed by the processor, cause the synchronization controller to generate a fourth control signal for controlling operation for the first reflector, and wherein an amplitude of the fourth control signal is variable based on the field of view. 
     
     
         21 . The laser scanning system of  claim 1 , wherein the laser scanning system is at least one of a confocal laser scanning microscope, a multi-photon laser scanning microscope, or an optical coherence tomography. 
     
     
         22 - 80 . (canceled)