Patent Publication Number: US-2023152230-A1

Title: Simultaneous detection of laser emission and fluorescence

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application claims the benefit of U.S. Provisional Application No. 63/017,899, filed on Apr. 30, 2020. The disclosure of the above application is incorporated herein by reference in its entirety. 
    
    
     GOVERNMENT CLAUSE 
     This invention was made with government support under ECCS1607250 awarded by the National Science Foundation. The government has certain rights in the invention. 
    
    
     FIELD 
     The present disclosure relates to method and systems for imaging tissue samples, for example, by detecting laser and fluorescence emissions simultaneously. 
     BACKGROUND 
     Both fluorescence and laser emission can be used for the detection of biomolecular interactions and imaging. For example, in fluorescence-based detection, biomaterials, such as biomolecules (e.g., DNA and proteins), cells, and tissues, are placed in a fluidic chamber or on a solid substrate. The fluorophores (such as, dyes and quantum dots) co-existing with the biomaterials are excited to produce fluorescence. The characteristics of fluorescence, such as intensity, polarization, lifetime, and the like, are detected so as to reflect the underlying biological processes and disease status. 
     In laser emission-based detection, biomaterials, such as biomolecules (e.g., DNA and proteins), cells, and tissues, are placed inside a laser cavity. Laser emission is realized when the fluorophores (such as, dyes and quantum dots) are excited above their lasing threshold. The characteristics of the laser emission (such as, lasing threshold, laser efficiency, and polarization) are detected so as to reflect the underlying biological processes or disease status. Laser emission and fluorescence are measured separately as a result of certain imaging constraints. For example, one imaging constraint is that mirrors used to define a laser cavity configured to measure laser emissions in the green spectrum blocks the green fluorescence, preventing simultaneous measurement of both laser emission and fluorescence. As such, typically for fluorescence emission detection, a top mirror forming the laser cavity is removed. 
     This section provides background information related to the present disclosure which is not necessarily prior art. 
     SUMMARY 
     This section provides a general summary of the disclosure, and is not a comprehensive disclosure of its full scope or all of its features. 
     In various aspects, the present disclosure provides an imaging system. The imaging system may include a laser cavity that is configured to receive a biological sample, where the biological sample is treated with a dye; an excitation light source that is configured to direct energy at the laser cavity so as to cause an emission from the biological sample, where the emission includes a laser emission at a first spectral band and a fluorescence emission at a second spectral band; a first detector that is configured to measure the laser emission generated by the biological sample; a second detector that is configured to measure the fluorescence emission generated by the biological sample; a splitter that is configured to direct the laser emission to the first detector and the fluorescence emission to the second detector; and a controller interfaced with the excitation light source, the first detector, and the second detector. 
     In one aspect, the laser cavity may be defined by a first mirror and a second mirror, and the biological sample may be disposed between the first mirror and the second mirror. 
     In one aspect, the first mirror may be arranged parallel to the second mirror. 
     In one aspect, a reflectivity of the first mirror may be greater than a first threshold so as to detect the laser emission, and a transmission of the first mirror may be above a second threshold so as to detect the fluorescence emission. 
     In one aspect, the splitter may be a dichroic mirror that is configured to separate the fluorescence emission and the laser emission included in the emission from the biological sample. 
     In one aspect, the first spectral band may be between about 524 nm and about 570 nm, and the second spectral band may be greater than about 590 nm. 
     In one aspect, the excitation light source may be configured to perform at least one of single-photon excitation and multi-photon excitation. 
     In one aspect, the imaging system may further include a motorized stage. The laser cavity may be disposed on the motorized stage. 
     In one aspect, the controller may further interface with the motorized stage. The controller may be configured to adjust a position of the laser cavity relative to the excitation light source using the motorized stage. 
     In one aspect, the controller may be configured to align a first location of the laser cavity with the excitation light source, and subsequently, to align a second location of the laser cavity with the excitation light source. 
     In one aspect, the splitter may be a first splitter, and the imaging system may further include at least one of a beam expansion lens set, a mirror, a mirror scanning system, a scanning lens set, a second splitter, and an objective lens that is configured to direct the energy at the laser cavity. 
     In one aspect, the splitter may be a first splitter, and the imaging system may further include at least one of a second splitter, a tube lens, and an objective lends that is configured to direct the emission to the first splitter. 
     In various aspects, the present disclosure provides an imaging system. The imaging system may include a motorized stage; a laser cavity disposed on the motorized stage and configured to receive a biological sample, the biological sample is treated with a dye; an excitation light source configured to direct energy at the laser cavity causing an emission from the biological sample, where the emission includes a laser emission at a first spectral band and the excitation light source is configured to cause at least one of single-photon excitation and multi-photon excitation; a first detector that is configured to measure the laser emission generated by the biological sample; and a controller interfaced with the excitation light source, the first detector, and the motorized stage. The controller may be configured to adjust a position of the motorized stage based on a predetermined location within the laser cavity, and direct the excitation light source to direct energy to the predetermined location within the laser cavity to perform single-photon excitation and multi-photon excitation. 
     In one aspect, the imaging system may further include a second detector that is configured to measure a fluorescence emission at a second spectral band generated by the biological sample, where the emission includes the fluorescence emission, and the controller is further interfaced with the second detector. 
     In one aspect, the imaging system may further include a beam splitter that is configured to receive the emission and direct the laser emission to the first detector and direct the fluorescence emission to the second detector. 
     In one aspect, the predetermined location within the laser cavity may include an x location, a y location, and a z location. 
     In various aspects, the present disclosure provides and imaging system. The imaging system may include a motorized stage; a laser cavity disposed on the motorized stage and configured to receive a biological sample, where the laser cavity is defined by a first mirror and a second mirror and the biological sample is disposed between the first mirror and the second mirror, and the biological sample is treated with a dye; an excitation light source that is configured to direct energy at the laser cavity causing an emission from the biological sample, where the emission includes a laser emission at a first spectral band and a fluorescence emission at a second spectral band, and the excitation light source is configured to cause at least one of single-photon excitation and multi-photon excitation; a first detector that is configured to measure the laser emission generated by the biological sample; a second detector that is configured to measure the fluorescence emission generated by the biological sample; a beam splitter that is configured to direct the laser emission to the first detector and the fluorescence emission to the second detector; and a controller interfaced with the excitation light source, the first detector, the second detector, and the motorized stage. 
     In one aspect, a reflectivity of the first mirror may be greater than a first threshold so as to detect the laser emission, and a transmission of the first mirror may be above a second threshold so as to detect the fluorescence emission. 
     In one aspect, the beam splitter may be a dichroic mirror that is configured to separate the fluorescence emission and the laser emission included in the emission from the biological sample. 
     In one aspect, the controller may be configured to adjust a position of the motorized stage based on a predetermined location within the laser cavity; and direct the excitation light source to direct energy to the predetermined location within the laser cavity to perform single-photon excitation and multi-photon excitation. 
     Further areas of applicability will become apparent from the description provided herein. The description and specific examples in this summary are intended for purposes of illustration only and are not intended to limit the scope of the present disclosure. 
    
    
     
       DRAWINGS 
       The drawings described herein are for illustrative purposes only of selected embodiments and not all possible implementations, and are not intended to limit the scope of the present disclosure. 
         FIG.  1    is a diagram of a simultaneous detection imaging system. 
         FIG.  2    is a diagram of a side view of a laser cavity used in a simultaneous detection imaging system, such as the simultaneous detection imaging system as illustrated in  FIG.  1   . 
         FIG.  3    is graphical illustration of example transmission curves of laser cavity mirrors within a laser cavity in a simultaneous detection imaging system, such as the laser cavity as illustrated in  FIG.  2    and/or the simultaneous detection imaging system as illustrated in  FIG.  1   . 
         FIG.  4 A  is a graphical illustration of a fluorescence spectrum of an example dye. 
         FIG.  4 B  is a graphical illustration of a lasing spectrum of the example dye. 
         FIG.  5 A  is an image of a normal lung tissue captured during fluorescence-based detection. 
         FIG.  5 B  is an image of the normal lung tissue captured during lasing emission-based detection. 
         FIG.  5 C  is a heat map of the normal lung tissue captured using lasing emission-based detection, which illustrates the density of lasing spots. 
         FIG.  5 D  is an image of the normal lung tissue prepared using hematoxylin and eosin (“H&amp;E”) staining. 
         FIG.  6 A  is an image of a cancerous lung tissue captured during fluorescence-based detection. 
         FIG.  6 B  is an image of the cancerous lung tissue captured during lasing emission-based detection. 
         FIG.  6 C  is a heat map of the cancerous lung tissue captured using lasing emission-based detection, which illustrates the density of lasing spots. 
         FIG.  6 D  is an image of the cancerous lung tissue prepared using hematoxylin and eosin (“H&amp;E”) staining. 
         FIG.  7 A  is a graphical illustration shown in logarithmic scale of pump thresholds for various levels of cell differentiations in a cancerous lung tissue. 
         FIG.  7 B  is a graphical illustration shown in linear scale of pump thresholds for various levels of cell differentiations in a cancerous lung tissue. 
         FIG.  8 A  is a diagram of a side view of a laser cavity during regular single-photo excitation of tissue treated with dye. 
         FIG.  8 B  is a diagram of a side view of a laser cavity during multiphoton excitation of tissue treated with dye. 
     
    
    
     Corresponding reference numerals indicate corresponding parts throughout the several views of the drawings. 
     DETAILED DESCRIPTION 
     Example embodiments will now be described more fully with reference to the accompanying drawings. 
       FIG.  1    is a schematic illustration of a simultaneous detection imaging system  100 . The simultaneous detection imaging system  100  is configured to simultaneously perform fluorescence-based detection and lasing emission-based detection. The simultaneous detection imaging system  100  generally includes a controller  104 , a first detector  108 , a second detector  112 , a mirror scanning system  116 , a motorized stage  120 , a laser cavity  124 , an excitation light source  128 , a beam expansion lens set  132 , a mirror  136 , a scanning lens set  140 , a first beam splitter  144 , a tube lens  148 , a second beam splitter  152 , and an objective lens  156 . 
     The laser cavity  124  is configured to hold a tissue sample. In some example embodiment, such as described in further detail in  FIG.  2   , the laser cavity  124  may include a first reflection surface and a second reflection surface and the tissue sample may be located or sandwiched between the first reflection surface and the second reflection surface. In each instance, the laser cavity  124  may be is located on the motorized stage  120 . The motorized stage may be configured to move the tissue sample relative to the system  100  so to improve testing opportunities. In each instance, the excitation light source  128  is configured to illuminate a fluorophore in the tissue sample. For example, the excitation light source  128  may be configured to generate a pulsed laser, so as to interrogate a tissue or biological sample located in the laser cavity  124 . The pulsed laser generated by the excitation source  128  may be directed toward the laser cavity  124  by the beam expansion lens set  132 , the mirror  136 , the mirror scanning system  116 , the scanning lens set  140 , the second beam splitter  152 , and/or the objective lens  156 . The skilled artisan will recognize that, in various instances, other types of light sources may be similarly used in placed of the excitation light source  128 . 
     Prior to placement within the laser cavity  124 , the tissue sample may be treated with a dye (such as, YO-PRO® or fluorescein isothiocyanate (FITC)) so as to generate fluorescence and laser emissions in desired color wavelengths. The fluorophore (i.e., dye molecules) of the treated tissue absorb the pulsed laser and reflect an emission back through the objective lens  156  that is pass through the second beam splitter  152  in a direction toward the first beam splitter  144 . The second beam splitter  152  directs the emission to the first beam splitter  144  (and in turn to the first and second detectors  108  and  112 ) due to the change in wavelength of the emission (e.g., Stokes shift). 
     The emission traverses the tube lens  148  to reach the first beam splitter  144 . The first beam splitter  144  separates the emission into a laser emission and a fluorescence emission. The laser emission may be directed to the first detector  108 . The fluorescence emission may be directed to the second detector  112 . Because the lasing band and the fluorescence band are within separate wavelengths (as shown in  FIG.  3   ), the first beam splitter  144  can separate the two emissions to separately detect and image those emissions. The first beam splitter  144  and the second beam splitter  152  may include dichroic mirrors. The skilled artisan will recognize, however, that other types of beam splitters may be similarly used. The first detector  108  is configured to receive the laser emission from the emission beam reflected from the tissue sample that is being imaged. The first detector  108  provides the detected laser emission information to the controller  104 . The second detector  112  is configured to receive the fluorescence emission from the emission beam reflected from the tissue that is being imaged. More specifically, the second detector  112  measures the fluorescence emission from the fluorophore (e.g., dye molecule) with which the tissue sample was treated. The second detector  112  provides the detected fluorescence information to the controller  104 . The first detector  108  and the second detector  112  may both be cameras, or other imagers. The skilled artisan will recognize, however, that other types of detectors may be similarly used, including, for example, an avalanche photodiode, a spectrometer, or an avalanche photodiode and a spectrometer, and that the first detector  108  may be the same or different from the second detector  112 . 
     The controller  104  may be a computer or a mobile computing device. In an example embodiment, the controller  104  may be separately connected to a computer or computing device. The controller  104  may be implemented, for example, as a microcontroller. The skilled artisan will recognize that the logic for the control of the simultaneous detection imaging system  100  by the controller  104  may be implemented in hardware logic, software logic, or a combination of hardware and software logic. In this regard, the controller  104  can be or can include any of a digital signal processor (DSP), microprocessor, microcontroller, or other programmable device, which are programmed with software implementing the above-described methods. It should be understood that alternatively the controller  104  is or includes other logic devices, such as a Field Programmable Gate Array (FPGA), a complex programmable logic device (CPLD), or application specific integrated circuit (ASIC). When it is stated that the controller  104  performs a function or is configured to perform a function, it should be understood that the controller  104  is configured to do so with appropriate logic (such as, in software, logic devices, or a combination thereof). As described in further detail below, the controller  104  can be implemented in a variety of subsystems configured to adjust the position of the motorized stage  120  so as to direct the pulse laser at different locations within the laser cavity  124 . 
     In an example embodiment, the first detector  108  and the second detector  112  may be cameras that capture images of the laser emission and fluorescence emission, respectively, of the tissue sample. The images can be displayed on a user interface of the controller  104  or user interface of a computing device operably connected to the controller  104 . The motorized stage  120  can be controlled by the controller  104  to be translated or moved. 
     As discussed above, the first beam splitter  144  is configured to separate the laser emission and the fluorescence emission from the emission generated from the tissue sample. Additional information describing the setup of a similar imaging device, which detects only the laser emission, is described in International Publication No. WO 2018/125925, the disclosure of which is hereby incorporated by reference in its entirety. In each instance, as discussed above, the tissue is treated with a dye (such as, YO-PRO® or fluorescein isothiocyanate (FITC), which are within the range of a first wavelength (e.g., about 500 nm to about 550 nm) and depict a first color (e.g., green). Since the two surfaces (mirrors) of the laser cavity  124  are configured to block the first wavelengths, fluorescence emissions cannot be recovered using a traditional imaging configurations and methods. However, because the mirrors of the laser cavity  124  have a tail in a second wavelength (e.g., red wavelength), the fluorescence emission can be identified, detected, and separated at the same time as the laser emission. In some example embodiments, the fluorescence and laser emissions can be measured consecutively instead of simultaneously. 
     In other words, the simultaneous detection imaging system  100  enables the acquisition of fluorescence and laser emission from the same tissue sample, and also, from the same type of fluorophore or dye staining the sample. Usually, the fluorescence spectrum of a fluorophore is broad. For example, dyes such as YO-PRO® and fluorescein isothiocyanate (FITC) have a spectral band of about 50 nm. In contrast, the laser emissions from those fluorophores usually have a linewidth less than about 1 nm. The lasing lines are also confined within a narrow spectral range (for example, about 10 nm). The large spectral difference between fluorescence and laser emissions (as shown in  FIG.  3   ) allows the configuration of the laser cavity and the simultaneous detection imaging system  100  to spectrally separate the fluorescence and laser emissions by using the first beam splitter  144  to separate and direct the emissions to the respective detectors  108 ,  112 . 
     Simultaneous acquisition of fluorescence and laser emissions provides complementary information obtained by fluorescence and laser emissions, which in turn provides better understanding of biological processes and disease status represented in the tissue sample. In an example embodiment, the controller  104  generates two separate images, one for the fluorescence emission and one for the laser emission. Additionally or alternatively, the laser emission can be superimposed on the fluorescence emission to produce a single image. 
       FIG.  2    is an side-view illustration of a laser cavity  200 , as included in a simultaneous detection imaging system, such as the simultaneous detection imaging system  100  illustrated in  FIG.  1   . As illustrated, the laser cavity  200  includes a top or first mirror  204  and a bottom or second mirror  208 . A sample  212 , such as a tissue sample, is disposed between the first mirror  204  and the second mirror  208 . In an example embodiment, the laser cavity  200  represents a Fabry-Perot assembly. 
       FIG.  3    is a graphical illustration of example transmission curves for laser cavity mirrors within a simultaneous detection imaging system, such as the laser cavity  200  as illustrated in  FIG.  2    and the simultaneous detection imaging system  100  illustrated in  FIG.  1   . As illustrated, the x-axis in  FIG.  3    represents wavelength (nm), and the y-axis represents transmission (%). An exemplary transmission curve of the first mirror  204  is shown by a first curve  304 . An exemplary transmission curve of the first beam splitter  144  is shown by a second curve  308 . The fluorophore (i.e., dye molecules) can be excited through the first mirror  204  at a spectral window that has a high transmission (for example, about 475 nm). The laser emission from the fluorophore (i.e., dye molecules) can be achieved within the lasing band  314  (e.g., between about 525 nm and about 570 nm) that has a low mirror transmission (for example, high mirror reflectivity). Within this lasing band  314 , the laser emission can transmit through the first mirror  204 , while the fluorescence within this band is blocked. 
     However, the fluorescence outside the low transmission band (i.e., laser bead)  314  (for example, the wavelength above about 590 nm) can be transmit through the first mirror  204 . The first beam splitter  144  is used to separate the laser emission that is within the lasing band  314  and the fluorescence emission that is in the fluorescence band  318  (e.g., above about 590 nm), and directs the emissions to different cameras/detectors (e.g., the first detector  108  and second detector  112  as illustrated in  FIG.  1   ). 
       FIG.  4 A  is a graphical illustration of a fluorescence spectrum of an example dye (e.g., YO-PRO®), where the x-axis represents wavelengths (nm), and the y-axis represents normalized intensity. As illustrated in  FIG.  4 A , the full-width-at-half-maximum (“FWHM”) of the fluorescence is about 50 nm.  FIG.  4 B  is a graphical illustration of a lasing spectrum of the example dye (e.g., YO-PRO®), where the x-axis represents wavelengths (nm), and the y-axis represents normalized intensity. As illustrated in  FIG.  4 B , the laser emission band is about 10 nm, and the full-width-at-half-maximum (“FWHM”) of each lasing line is less than about 1 nm. 
       FIG.  5 A  is an image of a normal lung tissue captured using fluorescence-based detection. The detected fluorescence emission of the normal tissue sample can compared to a detected fluorescent emission of a tissue sample in a disease state (e.g., cancerous lung tissue) to identify differences between normal state and disease state tissues. For example,  FIG.  6 A  is an image of a cancerous lung tissue captured during fluorescence-based detection. 
       FIG.  5 B  is an image of the normal lung tissue captured using lasing emission-based detection. The detected laser emission (i.e., LEM image) of the normal tissue sample can be compared to a detected laser emission (i.e., LEM image) of a tissue sample in a disease state (e.g., cancerous lung tissue) to identify differences between normal state and disease state tissues. For example,  FIG.  6 B  is an image of the cancerous lung tissue captured during lasing emission-based detection. 
       FIG.  5 C  is a heat map of the normal lung tissue captured using lasing emission-based detection. The heat map of the normal tissue sample can be compared to a heat map of a tissue sample in a disease sate (e.g., cancerous lung tissue) to identify differences between normal state and disease state tissues. For example,  FIG.  6 C  is a heat map of the cancerous lung tissue captured using lasing emission-based detection, which illustrates the density of lasing spots. 
       FIG.  5 D  is an image of the normal lung tissue prepared using hematoxylin and eosin (“H&amp;E”) staining. The hematoxylin and eosin image (i.e., H&amp;E image) of the normal tissue sample can be compared to the hematoxylin and eosin image (i.e., H&amp;E image) of a disease state (e.g., cancerous lung tissue) to identify differences between normal state and disease state tissues. For example,  FIG.  6 D  is an image of the cancerous lung tissue prepared using hematoxylin and eosin (“H&amp;E”) staining. 
     The image of the normal lung tissue captured using fluorescence-based detection, as illustrated in  FIG.  5 A , and the image of the normal lung tissue captured using lasing emission-based detection, as illustrated in  FIG.  5 B , may be detected and captured simultaneously or consecutively. Further, in certain variations, the two images (i.e.,  FIGS.  5 A and  5 B ) may be combined into a single image with different emission colors indicating the emission as fluorescence or lasing. 
     Similarly, the image of the cancerous lung tissue captured using fluorescence-based detection, as illustrated in  FIG.  6 A , and the image of the cancerous lung tissue captured using lasing emission-based detection, as illustrated in  FIG.  6 B , may be detected and captured simultaneously or consecutively. Further, in certain variations, the two images (i.e.,  FIGS.  6 A and  6 B ) may be combined into a single image with different emission colors indicating the emission as fluorescence or lasing. Similar comparisons may be completed for  FIGS.  5 C- 5 D and/or  6 C- 6 D . 
       FIGS.  7 A and  7 B  are graphical illustrations of pump thresholds for various levels of cell differentiations in a cancerous lung tissue. Pump thresholds refer to a minimum excitation powder needed to achieve non-zero median density of lasing spots per tissue sample (e.g., the normal lung tissue sample, the cancerous lung tissue sample). The illustration in  FIG.  7 A  is in logarithmic scale. The illustration in  FIG.  7 B  is in linear scale. As illustrated, the pump threshold is different for different levels of differentiated samples (e.g., cancerous lung tissues) and normal samples (e.g., normal lung tissues). 
     In another aspect of the present disclosure, the simultaneous detection imaging system  100  of  FIG.  1    can direct the interrogating lasing beam (e.g., pulsed laser) that is illuminating or exciting a fluorophore of the dye to a specific location of the laser cavity  124  by using multiphoton excitation. Described with respect to  FIG.  1   , the simultaneous detection imaging system  100  can perform multiphoton excitation to detect a laser emission. For example,  FIG.  8 A  is a diagram of regular single-photon excitation of the tissue sample  750  in the laser cavity  124 , showing a side view of the laser cavity  124  including the top or first mirror  204  and the bottom or second mirror  208  (as introduced in  FIG.  2   ). In standard single-photon excitation, the excitation light source  128  excites a single photon within an excitation volume  710 , which is then absorbed by dye molecules  704  and  708  within the excitation volume  710 . The dye molecules  704  and  708  emit a photon, which (by definition) is weaker than the absorbed photon, and the simultaneous detection imaging system  100  detects the lasing and fluorescence emission reflected from the tissue sample  750 . However, single-photon excitation, as shown by the excitation volume  710 , generally excites an area of the laser cavity  124  and is not narrowly directed to a specific location of the laser cavity  124 . That is, since the excitation volume  710  is a large area, the simultaneous detection imaging system  100  is unable to discover information about a specific x-y location of dye molecules along the z-direction. In addition, the image resolution in the x-y plane is low. 
       FIG.  8 B  is a diagram of multiphoton excitation of a tissue sample  752  treated with dye in the laser cavity  124 , showing a side view of the laser cavity  124  including the top or first mirror  204  and the bottom or second mirror  208  (as introduced in  FIG.  2   ). The tissue sample  752  is placed in the laser cavity  124  between the first mirror  204  and the second mirror  208 . The tissue sample  752  includes dye molecules  712  and  716 . As shown in  FIG.  8 B , multiphoton excitation occurs when the excitation light source  128  directs an excitation volume  720  at a specific location x, y, and z in the laser cavity  124 . The excitation volume  720  excites multiple photons and one dye molecule (for example, dye molecule  716 ) absorbs the multiple excited photons, in most cases two or three photons. 
     In this way, the photon emitted from the dye molecule  716  that absorbs the multiple photons (i.e., the laser emission) is stronger than single-photon excitation due to the multiphoton excitation and absorption. Further, the excitation volume  720  provides the ability to penetrate the laser cavity  124  at greater depths along the z-axis as well as more specific locations along the z-axis. In other words, multiphoton excitation provides the ability to direct the excitation light source  128  at a specific location of the laser cavity  124  and penetrate the laser cavity  124  at varying depths along the z-axis to excite multiple photons for absorption at specific locations in the laser cavity  124  to generate the laser emission, which has not previously been possible. To excite multiple photons, the excitation light source  128  is a high powered, pulsed laser with a high spatial and temporal density of photons to have two (or more) photons reach the specific location in the laser cavity  124  at the same time. 
     While  FIG.  8 B  shows a single dye molecule being excited, dye molecule  716 , in practice, only a small portion of the dye molecules in the z-direction are excited at the x and y location of the laser cavity  124  as a result of the small excitation volume  720 . By adjusting the excitation volume  720  or excitation location, the dye distribution along the z-direction can be mapped at the x and y location. In addition, due to the smaller excitation area in the x-y plane, the image resolution using multiphoton excitation is higher than using single-photon excitation. That is, the simultaneous detection imaging system  100  of  FIG.  1    can excite a specific x, y, z location within the laser cavity to excite a small portion of dye molecules at the location. The simultaneous detection imaging system  100  is further configured to perform multiphoton excitation at each location within the laser cavity (in the x, y, and z directions) to detect the laser emission of the small portion of dye molecules at each location. 
     In combination, the simultaneous detection imaging system  100  can excite a specific location within the laser cavity while detecting both fluorescence and laser emissions of the small portion of dye molecules at the specific location. In an example embodiment, the controller  104  includes location information that relate each location in the laser cavity  124  to a position of the motorized stage  120 . 
     In this way, the controller  104  can automatically adjust the position of the motorized stage  120  to capture or detect the lasing and fluorescence emission at each location (small portion of dye molecules) within the laser cavity  124 . That is, the controller  104  is configured to automatically move the motorized stage  120  so that the excitation light source  128  is directed at each location in the laser cavity  124 . The controller  104  iterates through each location, moving the motorized stage  120  and detecting the laser emission at each location. The motorized stage  120  may also be manually moved via user input through the controller  104 . The location information to instruct where to move the motorized stage  120  to can be stored on a memory associated with the controller  104 . Further, in the automatic implementation described above, the controller  104  instructs the storing of the detected emissions for each location to map the emissions of the entire tissue sample in the laser cavity  124 . Additional information describing the single and multiphoton excitation are described in Zipfel, W. R., Williams, R. M., &amp; Webb, W. W., Nonlinear magic: multiphoton microscopy in the biosciences,  Nature Biotechnology  (2003) and Ustione, A. &amp; Piston, D. W., A simple introduction to multiphoton microscopy,  Journal of Microscopy  (2011), the disclosures of which is hereby incorporated by reference in their entirety. 
     In certain variations, the present disclosure provides method for capturing both a laser emission at a first spectral band and a fluorescence emission at a second spectral band from a sample. The may include adding a dye (such as, YO-PRO® or fluorescein isothiocyanate (FITC)) to a tissue sample and disposing the tissue sample including the die in a laser cavity, such as detailed above. In certain variations, the method may include obtaining the tissue sample. 
     The techniques described herein or portions thereof may be implemented by one or more computer programs executed by one or more processors. The computer programs include processor-executable instructions that are stored on a non-transitory tangible computer readable medium. The computer programs may also include stored data. Non-limiting examples of the non-transitory tangible computer readable medium are nonvolatile memory, magnetic storage, and optical storage. 
     Some portions of the above description present the techniques described herein in terms of algorithms and symbolic representations of operations on information. These algorithmic descriptions and representations are the means used by those skilled in the data processing arts to most effectively convey the substance of their work to others skilled in the art. These operations, while described functionally or logically, are understood to be implemented by computer programs. Furthermore, it has also proven convenient at times to refer to these arrangements of operations as modules or by functional names, without loss of generality. 
     Unless specifically stated otherwise as apparent from the above discussion, it is appreciated that throughout the description, discussions utilizing terms such as “processing” or “computing” or “calculating” or “determining” or “displaying” or the like, refer to the action and processes of a computer system, or similar electronic computing device, that manipulates and transforms data represented as physical (electronic) quantities within the computer system memories or registers or other such information storage, transmission or display devices. 
     Certain aspects of the described techniques include process steps and instructions described herein in the form of an algorithm. It should be noted that the described process steps and instructions could be embodied in software, firmware or hardware, and when embodied in software, could be downloaded to reside on and be operated from different platforms used by real time network operating systems. 
     The present disclosure also relates to an apparatus for performing the operations herein. This apparatus may be specially constructed for the required purposes, or it may comprise a computer selectively activated or reconfigured by a computer program stored on a computer readable medium that can be accessed by the computer. Such a computer program may be stored in a tangible computer readable storage medium, such as, but is not limited to, any type of disk including floppy disks, optical disks, CD-ROMs, magnetic-optical disks, read-only memories (ROMs), random access memories (RAMs), EPROMs, EEPROMs, magnetic or optical cards, application specific integrated circuits (ASICs), or any type of media suitable for storing electronic instructions, and each coupled to a computer system bus. Furthermore, the computers referred to in the specification may include a single processor or may be architectures employing multiple processor designs for increased computing capability. 
     The algorithms and operations presented herein are not inherently related to any particular computer or other apparatus. Various systems may also be used with programs in accordance with the teachings herein, or it may prove convenient to construct more specialized apparatuses to perform the required method steps. The required structure for a variety of these systems will be apparent to those of skill in the art, along with equivalent variations. In addition, the present disclosure is not described with reference to any particular programming language. It is appreciated that a variety of programming languages may be used to implement the teachings of the present disclosure as described herein. 
     The foregoing description of the embodiments has been provided for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosure. Individual elements or features of a particular embodiment are generally not limited to that particular embodiment, but, where applicable, are interchangeable and can be used in a selected embodiment, even if not specifically shown or described. The same may also be varied in many ways. Such variations are not to be regarded as a departure from the disclosure, and all such modifications are intended to be included within the scope of the disclosure.