Multifocal imaging systems and method

Abstract

In the systems and methods of the present invention a multifocal multiphoton imaging system has a signal to noise ratio (SNR) that is reduced by over an order of magnitude at imaging depth equal to twice the mean free path scattering length of the specimen. An MMM system based on an area detector such as a multianode photomultiplier tube (MAPMT) that is optimized for high-speed tissue imaging. The specimen is raster-scanned with an array of excitation light beams. The emission photons from the array of excitation foci are collected simultaneously by a MAPMT and the signals from each anode are detected using high sensitivity, low noise single photon counting circuits. An image is formed by the temporal encoding of the integrated signal with a raster scanning pattern. A deconvolution procedure taking account of the spatial distribution and the raster temporal encoding of collected photons can be used to improve decay coefficient. We demonstrate MAPMT-based MMM can provide significantly better contrast than CCD-based existing systems.

1 . A multifocal imaging system comprising: 
 a multifocal optical device that provides a plurality of optical pathways;    a scanner that provides relative movement between the plurality of optical pathways and a material to be imaged;    an optical system that couples light from the optical device onto a region of interest of the material;    a detector array that detects light from a plurality of focal locations in the region of interest to generate image data, the detector array having a plurality of detector elements correlated with the focal locations; and    an image processor connected to the detector array.    
   
   
       2 . The system of  claim 1  wherein the scanner comprises a rotating mirror or a resonant mirror.  
   
   
       3 . The system of  claim 1  wherein each detector element has a collection area corresponding to a scattering distribution for each of the plurality of focal locations.  
   
   
       4 . The system of  claim 1  wherein the detector array detects a fluorescence signal from each focal location.  
   
   
       5 . The system of  claim 1  wherein the detector array comprises a multi-anode photomultiplier tube imaging detector having at least  64  detector elements.  
   
   
       6 . The system of  claim 1  further comprising a focusing lens system that adjusts a depth of focus within a sample in the range of 0 μm to 2000 μm.  
   
   
       7 . The system of  claim 1  wherein the detector comprises an array of photomultiplier elements.  
   
   
       8 . The system of  claim 1  further comprising a computer program that forms images including a deconvolution of pixel values with a scattering correction function.  
   
   
       9 . The system of  claim 1  further comprising a computer program that processes the image data.  
   
   
       10 . The system of  claim 9  wherein the program comprises a linear deconvolution process.  
   
   
       11 . The system of  claim 9  wherein the program comprises a non-linear deconvolution process.  
   
   
       12 . The system of  claim 9  wherein the program comprises a deconvolution process including scattering correction function.  
   
   
       13 . The system of  claim 12  wherein the image data comprises a three dimensional representation of a scanned region of interest, the representation having a plurality of pixel values, the scattering correction function including a plurality of adjacent pixel values for each pixel value of the representation.  
   
   
       14 . The system of  claim 13  wherein the plurality of adjacent pixel values comprises a weighting matrix that corrects for light scattering from tissue along adjacent optical pathways.  
   
   
       15 . The system of  claim 13  further comprising a holder for a material to be scanned.  
   
   
       16 . The system of  claim 1  wherein each of the plurality of optical pathways defines a plurality of focal locations in an imaging plane.  
   
   
       17 . The system of  claim 9  wherein the program comprises a normalization process.  
   
   
       18 . The system of  claim 1  wherein the multifocal optical device comprises a micro lens array.  
   
   
       19 . The system of  claim 16  wherein a distance between adjacent focal locations in the imaging plane is between 40 and 200 microns.  
   
   
       20 . The system of  claim 18  wherein the micro lens array has at least 64 lens elements.  
   
   
       21 . The system of  claim 1  wherein the detector array comprises a first detector array and a second detector array.  
   
   
       22 . The system of  claim 1  wherein the optical system comprises a moveable objective lens.  
   
   
       23 . The system of  claim 22  wherein the objective lens moves along an axis through the region of interest.  
   
   
       24 . The system of  claim 1  wherein the optical system comprises a first lens and a tube lens.  
   
   
       25 . The system of  claim 18  further comprising an iris defining an exit aperture of the micro lens array.  
   
   
       26 . The system of  claim 1  further comprising a confocal pinhole array adjacent to the detector array.  
   
   
       27 . The system of  claim 1  further comprising a bandpass filter adjacent to the detector array.  
   
   
       28 . The system of  claim 1  further comprising a dichroic mirror that reflects light returning from the region of interest onto the detector array.  
   
   
       29 . The system of  claim 1  further comprising a first reflector positioned along an optical path between a light source and a micro lens array, the first reflector coupling light to a first detector.  
   
   
       30 . The system of  claim 1  further comprising a beam expander positioned between a light source and a micro lens array, a second reflector and a second detector.  
   
   
       31 . The system of  claim 21  further comprising a reflector that separates light returning from the region of interest onto a first optical path towards the first detector array and onto a second optical path towards a second detector array.  
   
   
       32 . The system of  claim 21  wherein the first detector array is a first photomultiplier array and the second detector is a second photomultiplier array.  
   
   
       33 . The system of  claim 15  wherein the holder is moveable in three orthogonal directions.  
   
   
       34 . The system of  claim 1  further comprising a light source.  
   
   
       35 . The system of  claim 1  wherein the light source comprises a laser.  
   
   
       36 . The system of  claim 34  further comprising a pulse compressor optically coupled to the light source.  
   
   
       37 . The system of  claim 34  further comprising an attenuator that adjusts light intensity.  
   
   
       38 . The system of  claim 18  further comprising a moveable micro lens array holder.  
   
   
       39 . The system of  claim 38  wherein the moveable micro lens array holder scans in three orthogonal directions.  
   
   
       40 . The system of  claim 1  further comprising a detector lens that focuses light returning from each focal location onto a corresponding detector element.  
   
   
       41 . The system of  claim 1  further comprising a controller connected to the scanner that controls scanning speed and resolution.  
   
   
       42 . The system of  claim 41  wherein the focal locations are separated from each other by at least 10 microns.  
   
   
       43 . The system of  claim 41  wherein the controller receives feedback control signals from a detector that monitors a light characteristics.  
   
   
       44 . The system of  claim 43  wherein the detector detects a reference beam and generates reference signals.  
   
   
       45 . The system of  claim 1  further comprising a reflector that reflects a portion of scanning light and a third detector that measures the scanning light.  
   
   
       46 . The system of  claim 1  wherein the detector array comprises a plurality of detector elements that detect light from focal locations that are separated from each other by more than 25 microns.  
   
   
       47 . The system of  claim 1  wherein the optical pathways each have a focal location within the region of interest, adjacent focal locations being separated by distance in a range between 0.2 and 20 times a mean free path of light illuminating in a tissue or material to be imaged.  
   
   
       48 . The system of  claim 47  wherein the distance between focal locations is correlated with a material to be scanned.  
   
   
       49 . The system of  claim 1  wherein the detector array comprises detector elements positioned at different focal distances to image at different depths within the region of interest.  
   
   
       50 . The system of  claim 1  wherein the multifocal optical device provides a plurality of optical pathways having different focal depths within the region of interest.  
   
   
       51 . The system of  claim 34  wherein the light source emits light at a wavelength such that at least two photons of the light that are incident at a focal location of a material within the region of interest are necessary induce a fluorescence emission from the material.  
   
   
       52 . The system of  claim 51  wherein the light source emits at a wavelength such that at least three photons of the light are incident at a focal location are necessary to induce fluorescence of the material.  
   
   
       53 . The system of  claim 1  wherein the multifocal optical device comprises a diffractive optical element.  
   
   
       54 . The system of  claim 1  wherein the multifocal optical element comprises a plurality of optical fibers.  
   
   
       55 . The system of  claim 1  further comprising a fiber optic device that couples a light source to the multifocal optical element.  
   
   
       56 . The system of  claim 55  wherein the fiber optic device comprises a coherent fiber optic bundle.  
   
   
       57 . The system of  claim 1  further comprising a fiber optic device that transmits light along an optical path between the region of interest and the detector array.  
   
   
       58 . The system of  claim 57  wherein the fiber optic device comprises a multichannel plate.  
   
   
       59 . The system of  claim 1  further comprising a spectral dispersing element that separates light returning from the region of interest into a plurality of wavelengths that are detected by the detector array.  
   
   
       60 . The system of  claim 59  wherein the spectral dispersing element comprises a transmission grating.  
   
   
       61 . The system of  claim 1  wherein the system comprises a light source connected to a probe with a fiber optic cable.  
   
   
       62 . The system of  claim 61  wherein the probe comprises a handle and a distal probe.  
   
   
       63 . The system of  claim 62  wherein the handle houses the multifocal optical element and the scanner and the distal probe houses the optical system.  
   
   
       64 . The system of  claim 63  wherein the distal probe is rigidly attached to the handle and further comprises a rigid center endoscope body.  
   
   
       65 . The system of  claim 63  wherein the optical system includes a distal lens.  
   
   
       66 . The system of  claim 63  wherein the optical system comprises a first lens, a second lens and a distal objective lens.  
   
   
       67 . The system of  claim 1  further comprising a second light source.  
   
   
       68 . The system of  claim 67  wherein the second light source provides a stationary light beam that is optically coupled to an output lens with a reflector.  
   
   
       69 . The system of  claim 63  wherein the handle further comprises the detector array.  
   
   
       70 . The system of  claim 69  wherein the detector array comprises array of photomultiplier tubes remotely connected to the image processor.  
   
   
       71 . The system of  claim 1  wherein the detector comprises a CMOS imaging device.  
   
   
       72 . The system of  claim 1  wherein the detector further comprising a binning charge coupled device (CCD) camera such that each binned pixel region has a light collection area corresponding to a scattering distribution from each focal location.  
   
   
       73 . The system of  claim 1  wherein the detector array comprises a plurality of avalanche photodiodes.  
   
   
       74 . The system of  claim 1  further comprising a laser light source including a picosecond laser or a femtosecond laser.  
   
   
       75 . The system of  claim 1  wherein the system has a resolution in the region of interest of about 0.1 microns to about 2.0 microns.  
   
   
       76 . The system of  claim 1  wherein the system images at least 5 frames per second, each frame having at least 256 by 256 pixels.  
   
   
       77 . The system of  claim 41  wherein the controller actuates illumination of different focal regions and controls detector readout in a time multiplexed process.  
   
   
       78 . The system of  claim 1  wherein the multifocal optical element is moveable by the controller.  
   
   
       79 . The system of  claim 1  further comprising a confocal light collection system  
   
   
       80 . The system of  claim 79  further comprising multiphoton light excitation.  
   
   
       81 . A method for multifocal imaging comprising: 
 illuminating a region of interest with light using a plurality of optical pathways;    providing relative movement between the plurality of optical pathways and the region of interest; and    detecting light from a plurality of focal locations in the region of interest to generate image data.    
   
   
       82 . The method of  claim 81  further comprising providing relative movement by scanning with a rotating mirror or a resonant mirror.  
   
   
       83 . The method of  claim 81  further comprising detecting with a detector array having a plurality of detector elements, each detector element having a collection area corresponding to a scattering distribution for each of a plurality of focal locations.  
   
   
       84 . The method of  claim 83  further comprising detecting a fluorescence signal from each focal location, the detector being connected to an image processor.  
   
   
       85 . The method of  claim 81  further comprising detecting with a multi-anode photomultiplier tube imaging detector having at least 64 detector elements.  
   
   
       86 . The method of  claim 81  further comprising providing a focusing lens system that adjusts a depth of focus within a sample in the range of 0 μm to 2000 μm.  
   
   
       87 . The method of  claim 81  wherein the detector comprises an array of photomultiplier elements.  
   
   
       88 . The method of  claim 81  further comprising forming images by a deconvoluting pixel values with a scattering correction function.  
   
   
       89 . The method of  claim 81  further comprising processing the image data with a computer program on an image processor.  
   
   
       90 . The method of  claim 89  further comprising processing with the program including a linear deconvolution process.  
   
   
       91 . The method of  claim 89  further comprising processing with the program including a non-linear deconvolution process.  
   
   
       92 . The method of  claim 89  further comprising processing with a deconvolution process including a scattering correction function.  
   
   
       93 . The method of  claim 92  further comprising processing image data including a three dimensional representation of a scanned region of interest, the representation having a plurality of pixel values, the scattering correction function including a plurality of adjacent pixel values for each pixel value of the representation.  
   
   
       94 . The method of  claim 93  further comprising using the plurality of adjacent pixel values as a weighting matrix that corrects for light scattering from tissue along adjacent optical pathways.  
   
   
       95 . The method of  claim 81  further comprising providing a holder for a material to be scanned.  
   
   
       96 . The method of  claim 81  further comprising using each of the plurality of optical pathways to illuminate a plurality of focal locations in an imaging plane.  
   
   
       97 . The method of  claim 89  further comprising processing the image data with a normalization process.  
   
   
       98 . The method of  claim 81  further comprising forming the plurality of optical pathways with a micro lens array, a diffractive optical element or a plurality of optical fibers.  
   
   
       99 . The method of  claim 96  further comprising providing a distance between adjacent focal locations in the imaging plane between 40 and 200 microns.  
   
   
       100 . The system of  claim 98  further comprising providing a micro lens array having at least 64 lens elements.  
   
   
       101 . The method of  claim 81  further comprising providing a detector array having a first detector array and a second detector array.  
   
   
       102 . The method of  claim 81  further comprising providing an optical system having a moveable objective lens.  
   
   
       103 . The method of  claim 102  further comprising moving the objective lens along an axis through the region of interest.  
   
   
       104 . The method of  claim 81  further comprising providing an optical system having a first lens and a tube lens.  
   
   
       105 . The method of  claim 98  further comprising using an iris defining an exit aperture of the micro lens array.  
   
   
       106 . The method of  claim 81  further comprising obtaining a confocal image of a material with the detector array.  
   
   
       107 . The method of  claim 81  further comprising providing a bandpass filter adjacent to the detector array.  
   
   
       108 . The method of  claim 81  further comprising providing a dichroic mirror that reflects light returning from the region of interest onto the detector array.  
   
   
       109 . The method of  claim 81  further comprising providing a first reflector positioned along an optical path between a light source and the multifocal optical device, the first reflector coupling light to a first detector.  
   
   
       110 . The method of  claim 81  further comprising a beam expander positioned between a light source and the multifocal optical device, a second reflector and a second detector.  
   
   
       111 . The method of  claim 101  further comprising a reflector that separates light returning from the region of interest onto a first optical path towards the first detector array and onto a second optical path towards a second detector array.  
   
   
       112 . The method of  claim 101  further comprising providing the first detector array including a first photomultiplier array and providing the second detector including a second photomultiplier array.  
   
   
       113 . The system of  claim 81  further comprising providing optical pathways that have a focal location within the region of interest, adjacent focal locations being separated by distance in a range between 0.2 and 20 times a mean free path of the illuminating light of tissue or material being imaged.  
   
   
       114 . The method of  claim 81  further comprising providing a distance between adjacent focal locations that is correlated with a mean free path of light within a material to be scanned.  
   
   
       115 . The method of  claim 81  further comprising providing a detector elements positioned at different focal distances to image at different depths within the region of interest.  
   
   
       116 . The method of  claim 81  further comprising providing multifocal optical device having a plurality of optical pathways having different focal depths within the region of interest.  
   
   
       117 . The method of  claim 81  further comprising providing a light source that emits light at a wavelength such that at least two photons of the light that are incident at a focal location of a material within the region of interest are necessary induce a fluorescence emission from the material.  
   
   
       118 . The method of  claim 117  further comprising illuminating with light at a wavelength such that at least three photons of the light are incident at a focal location are necessary to induce fluorescence of the material.  
   
   
       119 . The method of  claim 81  further comprising providing a fiber optic device that couples a light source to the multifocal optical element.  
   
   
       120 . The method of  claim 81  further comprising providing a fiber optic device that transmits light along an optical path between the region of interest and the detector array.  
   
   
       121 . The method of  claim 81  further comprising applying a dye to a material to be imaged.  
   
   
       122 . The method of  claim 81  further comprising detecting a fluorescent protein in tissue.  
   
   
       123 . The method of  claim 81  further comprising detecting a genetically introduced fluorescent material.  
   
   
       124 . The method of  claim 81  further comprising detecting autofluorescence of a material.  
   
   
       125 . The met-hod of  claim 81  further comprising collecting time resolved spectroscopic data from the region of interest.  
   
   
       126 . The method of  claim 125  wherein the step of collecting time resolved data comprises collecting fluorescence lifetime data.  
   
   
       127 . The method of  claim 81  further comprising performing harmonic generation microscopy.  
   
   
       128 . The method of  claim 81  further comprising detecting Raman scattered data from each of the focal locations.  
   
   
       129 . The method of  claim 128  further comprising performing a coherent anti-Stokes Raman scattering measurement of a material.  
   
   
       130 . The method of  claim 81  further comprising collecting a multiphoton quantum data image from the region of interest.  
   
   
       131 . The method of  claim 81  further comprising collecting a surface plasmon image from the region of interest.  
   
   
       132 . The method of  claim 81  further comprising performing stimulated emission depletion microscopy of a material.  
   
   
       133 . The method of  claim 81  further comprising providing a probe having a handle and a probe element connected to the handle and illuminating a tissue region of a subject with the probe to collect data.  
   
   
       134 . The method of  claim 133  further comprising inserting the probe element within the body of a mammalian subject to collect image data of tissue within the subject.  
   
   
       135 . The method of  claim 133  further comprising inserting the probe element within a body cavity or lumen of a subject.  
   
   
       136 . The method of  claim 133  further comprising providing a control circuit, a detector array, a multifocal optical element and an optical scanner within the handle.  
   
   
       137 . The method of  claim 133  further comprising coupling a light source to the handle with a fiber optic cable.  
   
   
       138 . The method of  claim 136  further comprising connecting the control circuit to an external image processor.  
   
   
       139 . The method of  claim 133  wherein the probe element comprises an endoscope body.  
   
   
       140 . The method of  claim 139  wherein the endoscope body has a length of at least 5 cm.  
   
   
       141 . The method of  claim 81  further comprising forming a plurality of beams that simultaneously provide focal locations at a plurality of depths within a material to be scanning, and scanning the material at the plurality of depths simultaneously to provide a three dimensional image data set.  
   
   
       142 . The method of  claim 81  further comprising performing time multiplexed illumination of focal locations.  
   
   
       143 . The method of  claim 142  further comprising using a controller to actuate a light source to provide the time multiplexed illumination.  
   
   
       144 . The method of  claim 142  further comprising selecting a pulse separation and pulse width parameters.  
   
   
       145 . The method of  claim 142  further comprising detecting the focal locations with a single detections channels.  
   
   
       146 . The method of  claim 81  further comprising forming an image of a mammalian organ.  
   
   
       147 . The method of  claim 81  further comprising determining whether tissue cells are cancerous.  
   
   
       148 . The method of  claim 81  further comprising forming an image of vascular tissue.  
   
   
       149 . The method of  claim 81  further comprising sectioning a portion of tissue such as brain tissue.  
   
   
       150 . The method of  claim 81  further comprising measuring a response to a therapeutic agent in tissue.  
   
   
       151 . A multifocal light detecting system comprising: 
 a multifocal optical device that provides a plurality of light beams;    an optical system that couples light from the optical device onto a region of interest of the material;    a detector device that detects light from a plurality of focal locations in the region of interest to generate data; and    a processor connected to the detector.    
   
   
       152 . The system of  claim 151  further comprising a scanner such as a rotating mirror or a resonant mirror.  
   
   
       153 . The system of  claim 151  wherein the detector has a collection area corresponding to a scattering distribution for each of the plurality of focal locations.  
   
   
       154 . The system of  claim 151  wherein the detector detects time resolved data for deconvoultions.  
   
   
       155 . The system of  claim 151  further comprising a computer program that process time resolved data in combination with spectroscopic data to distinguish components of tissue.  
   
   
       156 . A method for multifocal light detection comprising: 
 illuminating a region of interest with light using a plurality of optical pathways; and    detecting light from a plurality of focal locations in the region of interest to generate data.    
   
   
       157 . The method of  claim 156  further comprising providing relative movement between the pathways and a material by scanning with a rotating mirror or a resonant mirror.  
   
   
       158 . The method of  claim 156  further comprising detecting with a detector array having a plurality of detector elements, each detector element having a collection area corresponding to a scattering distribution for each of a plurality of focal locations and collecting time realized data and fluorescence data.  
   
   
       159 . The method of  claim 156  further comprising a detection array of photomultiplier elements.  
   
   
       160 . The method of  claim 156  further comprising providing a light source that emits light at a wavelength such that at least two photons of the light that are incident at a focal location of a material within the region of interest are necessary induce a fluorescence emission from the material.