Method and Apparatus for Volumetric Imaging

Abstract

Methods of volumetric imaging of a three-dimensional matrix of nucleic acids within a cell is provided. An automated apparatus for sequencing and volumetric imaging of a three-dimensional matrix of nucleic acids is provided.

1 . A method of analyzing a plurality of nucleic acids within a three dimensional polymerized matrix comprising
 amplifying the plurality of nucleic acids to produce amplicons within the matrix,   covalently bonding the amplicons to the matrix,   sequencing the plurality of amplicons using an optical sequencing method where the plurality of amplicons are labeled with a detectable label, and   volumetrically imaging the plurality of amplicons to produce three dimensional imaging data of the plurality of amplicons wherein light intensity data is processed into a three-dimensional volumetric image.   
     
     
         2 . The method of  claim 1  wherein the plurality of nucleic acids is contained within a biological sample and the matrix-forming material is introduced into the biological sample. 
     
     
         3 . The method of  claim 1  wherein the plurality of nucleic acids is contained within a cell and the matrix-forming material is introduced into the cell. 
     
     
         4 . The method of  claim 1  wherein the plurality of nucleic acids is contained within a tissue sample and the matrix-forming material is introduced into the tissue sample. 
     
     
         5 . The method of  claim 1  wherein the three dimensional imaging data identifies the relative position of the plurality of amplicons within the cell. 
     
     
         6 . The method of  claim 1  wherein the plurality of amplicons are sequenced using fluorescence in situ sequencing. 
     
     
         7 . The method of  claim 1  wherein the plurality of nucleic acids are volumetrically imaged using one or more of 3D structured illumination, selective planar illumination microscopy, light sheet microscopy, emission manipulation, volumetric imaging using pinhole confocal microscopy, volumetric imaging using aperture correlation confocal microscopy, volumetric imaging using volumetric reconstruction from slices, volumetric imaging using deconvolution microscopy, volumetric imaging using aberration-corrected multifocus microscopy, volumetric imaging using digital holographic microscopy. 
     
     
         8 . An automated sequencing and volumetric imaging device comprising
 a multi axis stage or positioning system including a sample holder for a three dimensional nucleic acid containing matrix,   a heating or cooling apparatus operationally connected to the stage, whereby the heating or cooling apparatus is programmable for time and temperature useful with thermo-cycling for amplification and sequencing,   a fluidics dispenser positioned to dispense one or more reagents into the sample holder wherein the fluidics dispenser is in fluid communication with one or more reservoirs for containing one or more reagents, whereby the fluidics dispenser is programmable for dispensing programmed volumes of liquid reagents to the sample holder,   a pump operationally connected to the fluidics dispenser whereby the pump forces or withdraws one or more regents from the one or more reservoirs through the fluidics dispenser,   an optical assembly including one or more optical axis,   one or more detectors positioned in light receiving communication with the sample holder, whereby the one or more detectors receives light intensity signals which are processed into a three-dimensional volumetric image of the nucleic acid sample,   one or more microprocessors with software for automating and controlling introduction of reagents into the sample holder, thermocycling of the sample holder, and image detection and acquisition.