Adapter for microscopic imaging

Abstract

Disclosed herein are adapters configured to be optically coupled to a plurality of microscopes, said adapter comprising: a) a first microscope interface configured to optically couple a first microscope to an optical element in optical communication with an optical probe; b) a second microscope interface configured to optically couple a second microscope to the optical element in optical communication with the optical probe; and c) an optical arrangement configured to direct light collected from a sample with aid of the optical probe to (1) the first microscope and second microscope simultaneously, or (2) the first microscope or second microscope selectively.

1 .- 129 . (canceled) 
     
     
         130 . An adapter configured to be optically coupled to a first microscope and a second microscope, said adapter comprising:
 a first objective lens configured to be optically coupled to the first microscope;   a second objective lens configured to be optically coupled to an optical probe; and   an optical arrangement configured to direct light collected from a sample with aid of the optical probe and the second objective lens to (i) the first objective lens and (ii) an interface configured to optically couple the second microscope.   
     
     
         131 . The adapter of  claim 130 , wherein the first objective lens is an infinity corrected lens. 
     
     
         132 . The adapter of  claim 130 , wherein the first objective lens and the second objective lens have at least one different optical property. 
     
     
         133 . The adapter of  claim 130 , wherein the first objective lens and the second objective lens have different optical axes. 
     
     
         134 . The adapter of  claim 130 , wherein the first objective lens and the second objective lens have non-parallel optical axes. 
     
     
         135 . The adapter of  claim 130 , wherein the optical arrangement is configured to direct light collected from the sample to (i) the first objective lens and (ii) the interface (1) simultaneously, or (2) selectively at different times. 
     
     
         136 . The adapter of  claim 130 , wherein the first objective lens and the second objective lens are supported by a housing of the adapter. 
     
     
         137 . The adapter of  claim 136 , wherein the housing has a maximum dimension of 20 centimeters (cm). 
     
     
         138 . The adapter of  claim 136 , wherein a collective weight of the housing and the optical arrangement is less than or equal to about 0.5 kilograms. 
     
     
         139 . The adapter of  claim 130 , wherein the first objective lens has one or more properties selected from the group consisting of: a diameter of about 5 cm or less, a numerical aperture of 0.95 or less, and a working distance of 20 millimeters (mm) or less. 
     
     
         140 . The adapter of  claim 130 , wherein the second objective lens has one or more properties selected from the group consisting of: a diameter of about 5 cm or less, a numerical aperture of 0.95 or less, and a working distance of 20 mm or less. 
     
     
         141 . The adapter of  claim 130 , wherein the optical probe and the second objective lens are in optical alignment. 
     
     
         142 . The adapter of  claim 130 , wherein the optical probe is attachable and separable from the adapter. 
     
     
         143 . The adapter of  claim 130 , wherein the optical probe comprises a GRIN lens. 
     
     
         144 . The adapter of  claim 130 , wherein the optical arrangement comprises an optical element in optical communication with the first objective lens and the interface. 
     
     
         145 . The adapter of  claim 144 , wherein the optical element is a mirror configured to rotate about an axis or a beamsplitter. 
     
     
         146 . The adapter of  claim 130 , wherein the first microscope and the second microscope are configured to generate images based on the light collected from the sample, and wherein the adapter is configured to cause an image generated by the first microscope and an image generated by the second microscope to align. 
     
     
         147 . The adapter of  claim 130 , wherein the first microscope is a one-photon microscope and the second microscope is a two-photon microscope, and wherein the adapter causes an image generated by the first microscope and an image generated by the second microscope that is different from the image generated by the first microscope to align. 
     
     
         148 . The adapter of  claim 147 , wherein the image generated by the first microscope and the image generated by the second microscope are simultaneously displayed and overlapping. 
     
     
         149 . The adapter of  claim 130 , further comprising a compensator to correct for beam shift and improve a positional accuracy of a stimulation light beam as the stimulation light beam impinges on a target region within a field-of-view of the first microscope or the second microscope. 
     
     
         150 . The adapter of  claim 149 , wherein the compensator is a fixed component of the adapter and is oriented at a substantially 45° angle relative to an axis of the stimulation light beam. 
     
     
         151 . The adapter of  claim 149 , wherein the compensator is installed in one position of a multi-position mirror holder which further comprises a dichroic reflector in a different position. 
     
     
         152 . The adapter of  claim 151 , wherein the multi-position mirror holder is a rotary mirror wheel or a linear slider. 
     
     
         153 . A method for selectively exciting optogenetically-modified neurons in a tissue sample, the method comprising:
 a) providing the adapter of  claim 130 , wherein the first microscope is a one-photon microscope, the second microscope is a two-photon microscope, and the optical probe is in optical communication with the tissue sample; and   b) using the two-photon microscope to deliver a train of temporally focused laser pulses to selectively excite individual optogenetically-modified neurons, or sub-cellular compartments thereof.   
     
     
         154 . The method of  claim 153 , wherein the first microscope is a one-photon epifluorescence microscope. 
     
     
         155 . The method of  claim 153 , wherein the first microscope is a miniature microscope having a weight of 4 grams or less. 
     
     
         156 . The method of  claim 153 , wherein the first microscope is a miniature microscope having a volume of 500 mm 3  or less. 
     
     
         157 . The method of  claim 153 , further comprising using real-time bandpass filtering of a series of images captured by the one-photon microscope to facilitate focusing. 
     
     
         158 . A method for enhancing the accuracy of alignment of images captured by a one-photon microscope and a two-photon microscope, the method comprising:
 a) providing the adapter of  claim 130 , wherein the first microscope is a one-photon microscope, and the second microscope is a two-photon microscope;   b) projecting a series of images captured by the one-photon microscope into a single image;   c) applying a bandpass filter to the projected image created in step (b) to remove low frequency background and high frequency noise;   d) identifying a subset of images selected from a z-stack of two-photon optical image slices that overlap with a focal depth of the one-photon image by:
 (i) generating a moving projection of two-photon optical image slices, wherein the number of two-photon optical image slices included in the projection is determined by dividing the focal depth of the one-photon image by a thickness of a two-photon optical image slice, and wherein the starting optical image slice for a subset of the two-photon optical slices included in the moving projection is incremented by a value of one for each sequential projection; 
 (ii) applying the same bandpass filter as used in step (c) to each of the two-photon projections created in step (d)(i); and 
 (iii) calculating the cross-correlation between the filtered one-photon image of step (c) with each of the filtered two-photon projection images of step (d)(ii) to identify that which is best correlated with the one-photon image; 
   (e) applying an elastic registration algorithm to the filtered one-photon image of step (c) and the filtered two-photon projection image identified in step (d)(iii) to generate a set of coordinate transformations; and   (f) applying the coordinate transformation to the filtered one-photon image of step (c) or the filtered two-photon projection image identified in step (d)(iii) to align the images.   
     
     
         159 . The method of  claim 158 , further comprising using real-time bandpass filtering of a series of images captured by the one-photon microscope to facilitate focusing. 
     
     
         160 . The method of  claim 159 , wherein the elastic registration algorithm is a vector-spline regularization algorithm.