referring now to fig3 , multi region - of - interest ( mroi ) imaging is a resonant - galvo - galvo ( rgg ) scanning approach that extends widely used rg raster scanning and gg random - access scanning approaches ( fig3 a ) for in vivo 2plsm to allow two innovative new capabilities : random - access imaging , rather than point or contour random - access scanning 10 - 12 , to allow robustly motion correctable optical recordings across a wide fov multi - region cellular resolution imaging with greater compatibility to existing microscopes and greater scalability , to beyond 2 regions , than existing approaches 6 , 13 rgg scanning may also be used in other key applications in brain science . previous random - access laser scanning approaches described to date have employed galvo or acousto - optic deflector 12 , 14 - 17 scanning pairs . galvo scan bandwidths are limited to ˜ 1 khz line rates . for this reason , random - access optical recordings with gg scanner pairs have employed complex scan trajectory based on parametric 10 , 18 or heuristic 11 optimization of scanner trajectory to maximize the recording rate and the number of sites recorded . however , such approaches are only suited for motion - free in vitro applications ; locking such complex scans during in vivo imaging would require highly complex registration and control heuristics . acousto - optic deflectors ( aods ) allow extremely fast ( inertia - free ) scans suited for multi - area scanning , but are not currently suited for use with wide - fov optics due to their limited scanner étendue 19 , defined as the product of the optical aperture and angular range ( fig3 b ). étendue is an invariant quantity preserved by any subsequent optics imaging the scanner to the objective back entrance aperture . current wide fov objectives ( nikon cfi 75 16 ×) have back entrance apertures ˜ 20 mm and entrance angular ranges of +/− 4 °. filling the aperture ( to achieve full resolution ) and angular range ( to reach full extent of the fov ) would require a scanner étendue of 160 mm - degrees ; with even larger values required for emergent wider fov objectives 6 . aods suited for 2plsm are currently limited to only & lt ; 50 mm - degrees and thus cannot fully utilize even today &# 39 ; s wide - fov objectives 15 . referring now to fig1 , the multi - roi imaging strategy overcomes these limitations by combining the 16 khz line rate of resonant scanning ( cambridge technology crs 8 ) with 2d random - access gg scanning to allow random - access imaging ( fig1 b ). galvo scanners used for 2plsm can reach large étendue values of 240 mm - degrees ( e . g . the cambridge technology 6215 6 mm , +/− 20 ° scanner ). moreover , their limited scan speed does not greatly limit mroi imaging : the gg transit time ( 100 - 500 μs ) is negligible , to first order , between each rectangular roi in comparison to each roi imaging period . the photon efficiency of targeting the laser scan only to rois can be flexibly allocated to achieve gains in imaging speed , pixel integration time , and / or image resolution , compared to the reference full fov raster scan ( table 1 ). two very recent reports have described new technologies to address the challenge identified by this proposal : simultaneous cellular - resolution imaging of multiple separated cortical regions . one technology circumvents the limited fov of standard microscope systems by using two independent microendoscopic paths 13 . another technology , trepan2p , employs two parallel gg scanner pathways , together with temporal multiplexing , to achieve targeted two - area imaging 6 . compared to mroi imaging , both of these techniques are considerably more complex to implement and align ; moreover , neither readily scales to beyond two regions . moreover trepan2p is not readily extensible to 10 × faster resonant ( rg ) raster imaging , so that its frame rates are slower than mroi imaging ( table 1 ) despite truly simultaneous dual region imaging . mroi imaging hardware is , in contrast , readily inserted as a plug - and - play upgrade to existing 2plsm systems , reaching at least & gt ; 1 mm diameters as provided here ( aspect 1 ). notably , the same core rgg - scanning technology , combining random and raster - access scanning , can be applied to other key applications in brain science . for instance , mroi imaging achieves fast , photon - efficient imaging for subcellular scale applications , such as comprehensive mapping of synaptic input to a single neuron . rgg scanning may also be applied to multi - site photostimulation applications using optogenetics 20 , which has been recently made compatible with two - photon excitation using area scanning approaches targeted to neural cell bodies 21 , 22 . finally , rgg scanning is applicable to neural imaging and photostimulation applications in other brain areas [ ] and other model organisms such as the fly [ ] and zebrafish [ ] that are accessible by 2plsm . these uses broaden the impact of this research . referring now to fig4 , vidrio technologies flagship scanimage product has been widely used for single cortical region ( column ) in vivo imaging applications in the mammalian brain [ . . . ], including recent applications using resonant scanned volume imaging [ . . . ] ( fig4 a ). one of vidrio technologies &# 39 ; customers ( hhmi / janelia research campus ) has constructed a custom optical system that includes a resonant scanner and galvo scanner pair in sequence . vidrio has developed alpha - level software for this customer demonstrating the concept of mroi imaging ( fig4 b ). recently this was combined with swept axial scanning to allow mapping of activity along multiple adjacent dendritic segments ( fig4 c ). referring now to fig5 , a compact scanner assembly is designed and fabricated to mount and align the scanner surfaces ( mirrors ) of one resonant and two galvo scanners ( fig5 ). a . scan at or near diffraction limit across 1 mm diameter with current objectives a . minimize pupil shift effect limiting power throughput at large angles the nikon cfi 75 16 × is a representative recent wide - fov objective with full entrance aperture and angular range values of 18 mm and 9 degrees , respectively ( 162 mm - degrees of étendue ). full aperture & amp ; angle imaging through this objective achieves diffraction - limited imaging across a reported field of 1 . 8 mm diameter , but requires the use of additional custom relay optics 6 ( see risks & amp ; alternatives ). we provide the use of a more compact assembly to achieve ease of integration with existing microscope systems , while still achieving & gt ; 1 mm fov . referring again to fig5 , a scanner assembly of 5 mm aperture size and 20 degree angular range ( 100 mm - degrees ) is built from standard components ( crs 8 resonant and 6215h / 6 mm galvo scanners ; cambridge technology ). these are selected and ordered such that each successive mirror is larger than the previous mirror : resonant x ( 5 mm ), galvo x ( 6 mm ), galvo y ( 6 mm elongated ). this , along with maximal close packing , will ensure the full angular range of each scanner is incident on each successive scanner ( fig5 ). close packing of the scanner furthermore minimizes “ pupil shift ” caused by the axial displacement between the scanner surfaces . in 2plsm systems , scanners are imaged to the objective entrance by telescope optics of magnification m , in order to maximize filling of the objective and thereby the resulting optical resolution 25 . axial displacement is also imaged to the objective entrance , causing beam to be shifted on the entrance pupil ( i . e . pupil shift ) at large scan angles , reducing power throughput . the axial displacement at objective is more strongly magnified by m 2 ( e . g . m = 4 × to magnify 5 mm scanner to 20 mm objective aperture results in 16 × axial displacement magnification ). thus minimizing scanner separation is important . despite the pupil - shift effect , virtually all existing 2plsm systems already use closely spaced rg or gg scanner pairs . our provided three scanner design adds minimal amount since the thin - shafted resonant scanner is placed very closely to the subsequent x galvo ( fig5 ). additionally , this configuration allows resonant scanner to extend vertically away from the optical axis . consequently , the lateral footprint of assembly remains nearly unchanged from a standard gg scanner pair , allowing for ease of integration into existing 2plsm optical trains . an initial design of the rgg scanner block is developed using mechanical engineering skills on staff . this design refines the design , adds a soundproofed enclosure , and adds cable connector blocks . the prototype assemblies are tested using a collimated laser diode ( cps180 ; thorlabs ), expanded to a 5 mm beam , at the entrance , and a simple photodiode at the output . the gg pair is positioned to central , intermediate , and full deflection angles across the +/− 10 ° range in each dimension . at each 2d angular coordinate , the output beam diameter and relative power is measured , to ensure that a 5 mm diameter and & gt ; 50 % transmission ( relative to central angle ) is achieved throughout the 20 ° angular range . the current design adds pupil shift beyond current 2plsm scopes which may lead to significant power dropoff towards edges of the fov , possibly falling to & lt ; 50 % transmission relative to central angle . a more optimal optical design for the rgg scanner assembly eliminates pupil shift via afocal relay optics between scanners 26 . such optics are further optimized , together with the microscope scan optics , to avoid off - axis optical aberrations that otherwise prevent the full utilization of étendue beyond 100 mm - degrees . optimizing to support & gt ; 150 mm - degrees maximizes the fov utilization of both existing ( e . g . nikon cfi 75 16 ×) and emerging wide - fov objectives 6 . this design is not a compact upgrade path for existing 2plsm systems , but is used for new mroi 2plsm systems . referring now to fig4 , a software prototype is developed for mroi volume imaging using arbitrary rgg scanning hardware ( including compact configurations such as in aspect 1 ). this software is built on the backbone of vidrio &# 39 ; s scanimage production hardware and software for resonant raster scanning 2plsm ( fig4 a ). using this software prototype , mroi optical recordings in two or more separated cortical volumes may be shown in customer laboratories . software will generate & amp ; acquire small raster scans at each roi and then reposition the gg pair to each subsequent roi with command for shortest transit time possible , computed from a two - parameter model sufficient to describe galvo control systems : acceleration and max velocity 10 . the backbone scanimage platform uses field - programmable gate array ( fpga ) hardware to process high - speed input light data at each resonant scanner period into image lines . for mroi optical recordings , the imaging and non - imaging ( inter - roi transit ) times will be discretized into integer sets of the 8 khz resonant scanner periods , with transit periods being discarded at the fpga level . because the pupil - shift effect and other factors limits power transmission as a function of scan angle , power normalization is implemented whereby power is increased towards the edges of the fov . this normalization is superimposed atop power normalization for each resonant scanned roi , to compensate for the variable dwell time at each pixel . referring now to fig6 , users are provided the ability to select rois in two - dimensions from a reference image or image stack recently collected via resonant or galvo raster scanning ( latter having a larger angular range ). for three - dimensional volume imaging , an axial sweep scan command signal is generated for analog control of focus , e . g . using either a piezoelectric actuator or an electrically tuned lens ( optotune ). the axial sweep is synchronized to a user - specified integer number z of axial planes over a specified range , each containing a set of 2 or more selected rois , with an ordering & amp ; image directionality to optimize overall galvo transit time ( fig6 ). volume imaging rates are f /( z + 1 ), where f is the 2d mroi frame rate , with one frame period discarded to allow for the axial scanner flyback . for instance , imaging of 3 rois of 4 × zoom through 8 axial planes is achieved at ˜ 8 hz with 64 × 64 pixel imaging ( table 1 ). an mroi imaging prototype system is tested . mroi volume imaging of neural ensemble activity in 2 or more distinct cortical columns is shown at & gt ; 4 hz and & gt ; 100 neurons per ensemble . for 1 mm diameter fov ( aspect 1 ), specifying roi zoom factors of 4 ×( 250 μm diameter ) and a set of 8 planes ( e . g . 30 μm spacing ) would be expected to contain & gt ; 100 cells in even sparsely labeled subjects , with expected rates of ˜ 8 hz and ˜ 4 hz for 3 and 4 rois , respectively . for each mroi volume imaging dataset , a comparison acquisition is obtained using conventional raster volume imaging . the mroi advantage is determined by computing the neuronal volume imaging figure - of - merit m : where t vol is the imaging period for the full volume , snr i is the signal - to - noise ratio assessed at each roi , and n roi is the number of rois selected for mroi imaging . for an roi zoom factor of 4 ×, snr increases of up to 4 × per roi and aggregate frame speed increases up to 2 × would nominally achieve m = 16 regardless of scenario chosen ( table 1 ), with real values of m & gt ; 5 after accounting for inter - roi transit times and possible sublinear scaling of snr with pixel integration time . a stepped axial scanning mode was considered using a fast stepping electrically tuned lens 27 . this would avoid the tilting of the image volume that occurs with the swept axial scan provided . but given best current specifications ( optotune ; 7 ms axial step time ), this approach was determined to discard too much time for each set of axial planes . increasing dwell time at each pixel does not linearly augment the snr owing to higher order photophysical effects in 2plsm 28 . this limits the mroi figure - of - merit m achieved , but substantial gains in speed and / or signal quality are nonetheless achieved . 1 . mountcastle v b . modality and topographic properties of single neurons of cat &# 39 ; s somatic sensory cortex . j neurophysiol . 1957 ; 20 ( 4 ): 408 - 434 . http :// www . ncbi . nlm . nih . gov / pubmed / 13439410 . accessed jan . 27 , 2015 . 2 . mountcastle v b . the columnar organization of the neocortex . brain . 1997 ; 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78 ( 4 ): 2159 - 2162 . doi : 10 . 1016 / s0006 - 3495 ( 00 ) 76762 - 2 . the references recited herein are incorporated herein in their entirety , particularly as they relate to teaching the level of ordinary skill in this art and for any disclosure necessary for the commoner understanding of the subject matter of the claimed invention . it will be clear to a person of ordinary skill in the art that the above embodiments may be altered or that insubstantial changes may be made without departing from the scope of the invention . accordingly , the scope of the invention is determined by the scope of the following claims and their equitable equivalents . the above detailed description includes references to the accompanying drawings , which form a part of the detailed description . the drawings show , by way of illustration , specific embodiments in which the invention can be practiced . these embodiments are also referred to herein as “ examples .” all publications , patents , and patent documents referred to in this document are incorporated by reference herein in their entirety , as though individually incorporated by reference . in the event of inconsistent usages between this document and those documents so incorporated by reference , the usage in the incorporated reference ( s ) should be considered supplementary to that of this document ; for irreconcilable inconsistencies , the usage in this document controls . in this document , the terms “ a ” or “ an ” are used , as is common in patent documents , to include one or more than one , independent of any other instances or usages of “ at least one ” or “ one or more .” in this document , the term “ or ” is used to refer to a nonexclusive or , such that “ a or b ” includes “ a but not b ,” “ b but not a ,” and “ a and b ,” unless otherwise indicated . in the appended claims , the terms “ including ” and “ in which ” are used as the plain - english equivalents of the respective terms “ comprising ” and “ wherein .” also , in the following claims , the terms “ including ” and “ comprising ” are open - ended , that is , a system , device , article , or process that includes elements in addition to those listed after such a term in a claim are still deemed to fall within the scope of that claim . moreover , in the following claims , the terms “ first ,” “ second ,” and “ third ,” etc . are used merely as labels , and are not intended to impose numerical requirements on their objects . method examples described herein can be machine - implemented or computer implemented at least in part . some examples can include a tangible computer - readable medium or machine - readable medium encoded with instructions operable to configure an electronic device to perform methods as described in the above examples . an implementation of such methods can include code , such as microcode , assembly language code , a higher - level language code , or the like . such code can include computer readable instructions for performing various methods . the code may form portions of computer program products . further , the code may be tangibly stored on one or more volatile or non - volatile computer - readable media during execution or at other times . these computer - readable media may include , but are not limited to , hard disks , removable magnetic disks , removable optical disks ( e . g ., compact disks and digital video disks ), magnetic cassettes , memory cards or sticks , random access memories ( rams ), read only memories ( roms ), and the like . the above description is intended to be illustrative , and not restrictive . for example , the above - described examples ( or one or more aspects thereof ) may be used in combination with each other . other embodiments can be used , such as by one of ordinary skill in the art upon reviewing the above description . the abstract is provided to comply with 37 c . f . r . § 1 . 72 ( b ), to allow the reader to quickly ascertain the nature of the technical disclosure . it is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims . also , in the above detailed description , various features may be grouped together to streamline the disclosure . this should not be interpreted as intending that an unclaimed disclosed feature is essential to any claim . rather , inventive subject matter may lie in less than all features of a particular disclosed embodiment . thus , the following claims are hereby incorporated into the detailed description , with each claim standing on its own as a separate embodiment . the scope of the invention should be determined with reference to the appended claims , along with the full scope of equivalents to which such claims are entitled .
provided herein are apparatuses and methods using a compact scanner assembly for multi region of interest imaging and targeting of an intact tissue sample . the compact scanner assembly for mroi imaging , comprises a set of three electromagnetically actuated scanning mirrors , the first comprising a resonant scanner driven at its resonant frequency , the second comprising a galvanometer scanner having a mirror , the third comprising a galvanometer scanner having a mirror , wherein the galvanometer scanners and are driven by a lower bandwidth control signal specifying an angle for mirror and , wherein the scanners are arranged sequentially as , wherein the set of three scanning mirrors are within a single scanner assembly , wherein the compact scanner assembly for mroi combines raster scanning with random - access scanning to target multiple rois for targeting and imaging .