Patent Publication Number: US-9891422-B2

Title: Digital confocal optical profile microscopy

Description:
CROSS-REFERENCE TO RELATED APPLICATION(S) 
     This application claims priority to U.S. Provisional Patent Application No. 61/700,198, filed on Sep. 12, 2012. 
    
    
     GOVERNMENT INTERESTS 
     This invention was made with government support under DMR1004804 awarded by National Science Foundation. The government has certain rights in the invention. 
    
    
     BACKGROUND 
     Confocal microscopy is an optical technique commonly used to obtain detailed information about cells, tissues, microbial biofilms, and colloidal glasses. A confocal microscope typically includes an objective lens proximate a sample and a focusing lens proximate a screen with a pinhole. The confocal microscope collects in-focus light signals from the sample through the pinhole. As a result, out-of-focus signals are eliminated. As only light signals produced at and/or close to the focal plane of the objective lens can be detected, the image&#39;s optical resolution is much better than that of wide-field microscopes. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1A  is a partially schematic diagram of a digital confocal optical profile microscope in accordance with embodiments of the present technology. 
         FIG. 1B  is an example photograph collected by the digital confocal optical profile microscope in  FIG. 1A . 
         FIG. 2  is a schematic diagram illustrating details of a sample examined by the digital confocal optical profile microscope of  FIG. 1A . 
         FIG. 3  illustrates experimental and simulation contour plots of reflected light intensity in accordance with embodiments of the present technology. 
         FIG. 4  is a flow chart illustrating a process of analyzing image data in a digital confocal optical profile microscope in accordance with embodiments of the present technology. 
     
    
    
     DETAILED DESCRIPTION 
     Various embodiments of digital confocal optical profile microscopy systems, devices, and associated methods of analysis are described below. Certain example digital confocal optical profile microscopy systems, devices, and methods are described below with particular components and operations for illustration purposes only. Other embodiments in accordance with the present technology may also include other suitable components and/or may operate at other suitable conditions. A person skilled in the relevant art will also understand that the technology may have additional embodiments, and that the technology may be practiced without several of the details of the embodiments described below with reference to  FIGS. 1A-4 . 
     As discussed above in the Background section, certain confocal microscopes can have higher optical resolutions than wide-field microscopes. However, such confocal microscopes have several drawbacks. For example, the pinhole in such confocal microscopes acts as an analog computer that applies a circular aperture function to light signals coming from the sample. The pinhole allows in-focus light signals to pass through but rejects out-of-focus signals, which may carry a great deal of information about the sample. As discussed in more detail below, several embodiments of the present technology monitor substantially the entire beam profiles of light signals from a sample. The monitored beam profiles can be modeled to yield additional and/or more accurate information about the sample than existing confocal microscopy. 
       FIG. 1A  is a partially schematic diagram of a digital confocal optical profile microscope  100  in accordance with embodiments of the present technology. As shown in  FIG. 1A , the digital confocal optical profile microscope  100  can include an illumination source  102 , a photo detector  104 , a focusing lens  106 , a beam splitter  108 , and an objective lens  110  operatively coupled to one another. In certain embodiments, the digital confocal optical profile microscope  100  can optionally include a stepper  103  configured to carry and move the sample  101  relative to the objective lens  110  along at least one of the x-, y-, or z-axis. The digital confocal optical profile microscope  100  can also include a controller  118  operatively coupled to the illumination source  102 , the photo detector  104 , and/or the optional stepper  103 . In other embodiments, the stepper  103  may be omitted, and the digital confocal optical profile microscope  100  can further include scanning mirrors and/or other suitable optical components configured to focus illumination onto the sample  101  at a specific (x,y) location. In further embodiments, the digital confocal optical profile microscope  100  may also include frames, ocular lenses, diaphragms, and/or other suitable mechanical/optical components. 
     The illumination source  102  can include a laser, a light emitting diode, a halogen lamp, a fluorescent lamp, and/or other suitable types of light source configured to produce an illumination beam  112 . Though not shown in  FIG. 1A , the illumination source  102  can also include a collector lens, a field diaphragm, a condenser diaphragm, a condenser lens, and/or other suitable optical components configured to influence a profile of the illumination beam  112 . For example, the illumination beam  112  may be manipulated to have a generally constant beam radius, as illustrated in  FIG. 1A . In other examples, the illumination beam  112  may be manipulated to have a conical, parabolic, point, and/or other suitable profiles. 
     The beam splitter  108  is positioned to receive the illumination beam  112  from the illumination source  102 . In certain embodiments, the beam splitter  108  is configured to direct the illumination beam  112  generally completely toward the sample  101  via the objective lens  110 . In other embodiments, the beam splitter  108  may direct a portion of the illumination beam  112  toward the photo detector  104  and/or toward other suitable components (not shown) of the digital confocal optical profile microscope  100 . The beam splitter  108  is also positioned to receive a reflected beam  114  from the sample  101  through the objective lens  110 . The beam splitter  108  is then configured to direct the received reflected beam  114  toward the photo detector  104  via the focusing lens  106 . 
     The beam splitter  108  can include a cube, plate, half-silvered mirror, dichroic mirrored prism, and/or other suitable components. In the illustrated embodiment, the focusing lens  106  and the photo detector  104  are shown in-line with the beam splitter  108  and the sample  101 . In other embodiments, the focusing lens  106  and the photo detector  104  may be slanted with respect to the sample  101 , and the beam splitter  108  may be configured to direct the reflected beam  114  along a suitable direction toward the focusing lens  106  and the photo detector  104 . Even though the digital confocal optical profile microscope  100  is shown in  FIG. 1A  as having the beam splitter  108  for directing lights, in other embodiments, the beam splitter  108  may be substituted with one or more mirrors and/or other suitable optical components (not shown). 
     As shown in  FIG. 1A , the objective lens  110  is positioned between the beam splitter  108  and the sample  101 , and the focusing lens  106  is positioned between the beam splitter  108  and the photo detector  104 . The objective lens  110  is configured to focus the illumination beam  112  onto the sample  101  as well as collect the reflected beam  114  from the sample  101 . The focusing lens  106  is configured to focus the reflected beam  114  into a signal beam  116 . The objective lens  110  and the focusing lens  106  can individually include a convex lens, a filter, and/or other suitable optical components. In one embodiment, the objective lens  110  and/or the focusing lens  106  may be stationary. In other embodiments, at least one of the objective lens  110  and the focusing lens  106  may be configured to move relative to each other and/or relative to the sample  101  along the z-axis. 
     In certain embodiments, the digital confocal optical profile microscope  100  does not include a physical pinhole between the photo detector  104  and the focusing lens  106 . Instead, the photo detector  104  is positioned proximate the focusing lens  106  for directly detecting the signal beam  116 . The photo detector  104  can include a charge coupled device (“CCD”), a complementary metal-oxide-semiconductor (“CMOS”) photo sensor, a photodiode, and/or other suitable photo detectors. The photo detector  104  can have a detection area generally equal to or larger than a cross-sectional area of the signal beam  116  at the photo detector  104 . For example, in the illustrated embodiment, the photo detector  104  has a detection area that is approximately five times larger than the cross-sectional area of the signal beam  116 . In other embodiments, the photo detector  104  can have other suitable detection areas. 
     In other embodiments, the digital confocal optical profile microscope  100  may include a detector stepper  123  (shown in phantom lines for clarity) operatively coupled to the photo detector  104 . The detector stepper  123  may be configured to move the photo detector  104  along at least one of the x-, y-, or z-axis such that the photo detector  104  is spaced apart from the focal plane of the focusing lens  106 . As a result, a fraction of the detection area of the photo detector  104  filled by the signal beam  116  may be increased to a target value (e.g., about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or about 1.0). In other embodiments, the detector stepper  123  may be omitted, and the photo detector  104  may be off focus by, for example, positioning the focusing lens  106  or via other suitable techniques. 
     In further embodiments, the digital confocal optical profile microscope  100  may be configured to operate under a confocal mode and/or a confocal profile mode. In such implementations, the two modes may be operated independently or concurrently (e.g., in parallel). For example, the digital confocal optical profile microscope  100  may include a retractable pinhole  121  (shown in phantom lines for clarity). Under the confocal mode, the retractable pinhole  121  may be positioned between the photo detector  104  and the focusing lens  106  to filter or at least reduce out-of-focus signals. Alternately, under the confocal profile mode, the retractable pinhole  121  may be removed from the optical path between the photo detector  104  and the focusing lens  106 . The photo detector  104  may then detect a profile of the signal beam  116  as described in more detail below. 
     In another example, the digital confocal optical profile microscope  100  may include an additional beam splitter  109  (shown in phantom lines for clarity) between the photo detector  104  and the focusing lens  106 . The additional beam splitter  109  may be configured to direct a first portion  116   a  of the signal beam  116  to the photo detector  104  and a second portion  116   b  of the signal beam  116  to a pinhole  111  in front of a photo counter  117  (e.g., a photomultiplier tube or spectrometer). The first and second portions  116   a  and  116   b  of the signal beam  116  may be generally equal or may have other suitable proportions in intensity. The pinhole  111  may be positioned at or near a focus plane of the focusing lens  106 . As a result, the photo counter  117  may detect only light signals produced at and/or close to the focal plane of the focusing lens  106 . Thus, an optical resolution of resulting images may be much better than that of wide-field microscopes, and the resulting images may be utilized in conjunction with the detected profiles by the photo detector  104  to derive useful information regarding the sample  101 . In further examples, the digital confocal optical profile microscope  100  may include mirrors, prisms, and/or other suitable configurations and/or components to be operated under the confocal mode and/or the confocal profile mode. In yet further examples, the additional beam splitter  109 , the pinhole  111 , and the photo counter  117  may be omitted. 
     As shown in  FIG. 1A , the controller  118  can include a processor  120  coupled to a memory  122  and an input/output component  124 . The processor  120  can include a microprocessor, a field-programmable gate array, and/or other suitable logic devices. The memory  122  can include volatile and/or nonvolatile computer readable media (e.g., ROM; RAM, magnetic disk storage media; optical storage media; flash memory devices, EEPROM, and/or other suitable non-transitory storage media) configured to store data received from, as well as instructions for, the processor  120 . The input/output component  124  can include a display, a touch screen, a keyboard, a track ball, a gauge or dial, and/or other suitable types of input/output devices configured to accept input from and/or provide output to an operator. 
     In certain embodiments, the controller  118  can include a computer operatively coupled to other components of the digital confocal optical profile microscope  100  via a hardwire communication link (e.g., a USB link, an Ethernet link, an RS232 link, etc.). In other embodiments, the controller  118  can include a logic processor operatively coupled to other components of the digital confocal optical profile microscope  100  via a wireless connection (e.g., a WIFI link, a Bluetooth link, etc.). In further embodiments, the controller  118  can include an application specific integrated circuit, a system-on-chip circuit, a programmable logic controller, and/or other suitable computing frameworks. 
     The operation of the digital confocal optical profile microscope  100  is described below with reference to embodiments without the optional beam splitter  109 , pinhole  111 , photo counter  117 , or the retractable pinhole  121 . However, one of ordinary skilled in the art would understand, other embodiments of the digital confocal optical profile microscope  100  with the foregoing components may be operated generally similarly as described below and/or with additional/different operations. 
     In operation, the controller  118  can cause the illumination source  102  to produce the illumination beam  112 . The beam splitter  108  receives and directs the illumination beam  112  to the objective lens  110 . The objective lens  110  then focuses the illumination beam  112  onto the sample  101 . In response, the sample  101  reflects at least a portion of the focused illumination beam  112  as the reflected beam  114 . The beam splitter  108  receives and directs the reflected beam  114  to the focusing lens  106 , which focuses the reflected beam  114  into the signal beam  116 . The photo detector  104  then detects and measures the signal beam  116  and provides an input signal to the input/output component  124  of the controller  118 . In certain embodiments, the controller  118  can cause the illumination source  102  to turn off the illumination beam  112 . Subsequently, the controller  118  can cause the optional stepper  103  to move the sample  101  along the x- and/or y-axis to a desired location. Then, the controller  118  may cause the illumination source  102  to turn on and the foregoing operations may be repeated until all desired locations have been traversed. 
     In one embodiment, the input signal corresponding to the signal beam  116  includes a digital photo of the signal beam  116 .  FIG. 1B  shows an example digital photo that may be captured by the photo detector  104 . As shown in  FIG. 1B , the captured signal beam  116  includes a bright center  115  and a plurality of fringes  113  along a radial direction as indicated by the arrow r. In contrast, conventional confocal microscopes can only produce a detected light intensity through a physical pinhole. As discussed in more detail below, based on the profile of the captured signal beam  116 , additional and/or more accurate information of the sample  101  may be obtained than conventional confocal microscopy. 
     Theoretical Background 
     Without being bound by theory, the following description is believed to provide a theoretical background for a better understanding of various aspects of the disclosed digital confocal microscopy. The applicants do not attest to the scientific truthfulness of the following description. 
     It is believed that when a light beam under-fills optical components, the light beam may be described by a spherical Gaussian beam. For example, referring to  FIGS. 1A and 2 , the illumination beam  112  is focused onto the sample  101  and a portion of which is reflected off an air/glass interface  132  located at z 1  to produce a first reflected Gaussian beam. The reflected beam  114  travels through the objective lens  110  and the focusing lens  106 , and impinges on the photo detector  104 . Assuming z 1c  equal to the distance from a detection plane of the photo detector  104  to a beam waist (w 0c ) and r equal to a radial distance from the beam axis, up to an arbitrary phase factor, the electric field amplitude at the photo detector  104  is given by 
                       E   ⁡     (   r   )       ∝       1     w   ⁡     (     z     1   ⁢   c       )         ⁢     exp   ⁡     [     -       r   2         w   2     ⁡     (     z     1   ⁢   c       )           ]       ⁢     exp   ⁡     [         -   ik     ⁢       r   2       2   ⁢     R   ⁡     (     z     1   ⁢   c       )             +     i   ⁢           ⁢     ζ   ⁡     (     z     1   ⁢   c       )           ]           ,           (   1   )               
where w(z 1c ) is a spot size, R(z 1c ) is a wavefront radius of curvature, and ζ (z 1c ) is the longitudinal phase delay. The foregoing quantities are given by
 
                       w   ⁡     (     z   c     )       =       w     0   ⁢   c       ⁢       1   +       (       z   c     /     z     0   ⁢   c         )     2             ,           (   2   )                   1     R   ⁡     (     z   c     )         =       z   c         z   c   2     +     z     0   ⁢   c     2           ,           (   3   )                 ζ   ⁡     (     z   c     )       =         tan     -   1       ⁡     (       z   c     /     z     0   ⁢   c         )       .             (   4   )               
z 0c  is the Rayleigh range,
 
 z   0c   =w   0c   2   k/ 2  (5)
 
where k=2π/λ and λ is the wavelength of light.
 
     As shown in  FIG. 2 , light can also reflect from a second glass/air interface  134 , located at z 2,actual  to produce a second reflected Gaussian beam. However, due to Snell&#39;s law, the light is bent to appear reflecting from an interface  130  at z 2 , given by
 
 z   2   −z   1 =( z   2,actual   −z   1 )/ n,   (6)
 
where n is the refractive index. For the purposes of modeling, z 2  was designated as the position of the second glass/air interface  130 . Light that reflects from the second glass/air interface  130  is described by Eq. (1), with the “1” subscripts replaced by “2”.
 
     It is believed that the electric field at the photo detector  104  is a superposition of the first and second reflected Gaussian beams. As shown in  FIG. 1A , the distances from the photo detector  104  to the beam waists are z 1c  and z 2c . From Eq. 1-4, the intensity of the combined first and second reflected Gaussian beams is given by 
                       I   ⁡     (   r   )       =           I   1       w   1   2       ⁢     exp   ⁡     (     -       2   ⁢     r   2         w   1   2         )         +         I   2       w   2   2       ⁢     exp   ⁡     (     -       2   ⁢     r   2         w   2   2         )         +         2   ⁢         I   1     ⁢     I   2               w   1     ⁢     w   2         ⁢     exp   ⁡     [     -       r   2     ⁡     (       1     w   1   2       +     1     w   2   2         )         ]       ⁢     cos   ⁡     (         κ   ⁢   r     2     +   δ     )             ,           (   7   )               
where
 
                       w   1     ≡     w   ⁡     (     z     1   ⁢   c       )         ,           (   8   )                   w   2     ≡     w   ⁡     (     z     2   ⁢   c       )         ,           (   9   )                 κ   =       k   2     ⁡     [       1     R   ⁡     (     z     1   ⁢   c       )         -     1     R   ⁡     (     z     2   ⁢   c       )           ]         ,           (   10   )                 δ   =         tan     -   1       ⁡     (       z     2   ⁢   c       /     z     0   ⁢   c         )       -       tan     -   1       ⁡     (       z     1   ⁢   c       /     z     0   ⁢   c         )       +     δ   0         ,           (   11   )               
and δ 0  is the phase difference between the first and second reflected Gaussian beams due to different optical path lengths.
 
     Variables with the subscript c refer to respective values at the photo detector  104 , after the reflected beams have passed through the focusing lens  106 . The foregoing quantities may be related to beam parameters (z 0  and w 0 ) at the sample  101 . For example, if z denotes the position of the focal plane of the objective lens  110 , then: 
     
       
         
           
             
               
                 
                   
                     
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     Inserting these expressions into Eq. 8-11, and using Eq. 5 to relate the beam waist to the Rayleigh range, yields 
                       w     1   ,   2       =       W   0     ⁢       1   +     4   ⁢         (       z     1   ,   2       -   z     )     2       z   0   2                 ,           (   15   )                 κ   =         2   ⁢     z   0         W   0   2       ⁡     [           z   1     -   z         4   ⁢       (       z   1     -   z     )     2       +     z   0   2         -         z   2     -   z         4   ⁢       (       z   2     -   z     )     2       +     z   0   2           ]         ,           (   16   )                 δ   =         tan     -   1       ⁡     (     2   ·         z   2     -   z       z   0         )       -       tan     -   1       ⁡     (     2   ·         z   1     -   z       z   0         )       +     δ   0         ,           (   17   )               
where W 0 =(f 2 /f 1 )w 0 .
 
     Eq. 7 and Eq. 15-17 can be used to iteratively model image data of the signal beam  116  as a function of r and z based on the captured image until a substantial match is obtained. The adjustable parameters can include at least one of I 1 , I 2 , W 0 , z 0 , z 1 , z 2 , or δ 0 . From the modeling, positions of the interfaces, z 1  and z 2  may be extracted. The difference between z 2  and z 1  yields d/n, where d is the thickness of the sample  101  and n is the refractive index in Eq. (6). A process of analyzing image data of digital confocal optical profile microscope  100  is described in more detail below with reference to  FIG. 4 . 
     Experimental Setup 
     A confocal optical profile microscope generally similar to the configuration of the digital confocal optical profile microscope  100  shown in  FIG. 1A  was constructed. A continuous wave laser (Coherent Verdi, 532 nm wavelength, TEM 00  single mode, 2.25 mm diameter) was used as the illumination source. The laser beam was focused by an objective lens (Zeiss LD Plan-Neofluar, 20×, f 1 =10 mm) onto a sample mounted on a manual xyz stage (Thorlabs). Reflected beam was focused by a focusing lens (f 2 =250 mm) onto a CCD camera (Imaging Source DMK 21BU04, 640×480 pixel array, 8 bit dynamic range) as the photo detector. An exposure time of 1/16 second was used. In this experiment, the sample was a glass microscope slide and images of the reflected beam were collected as a function of depth (z). 
     Experiment Procedures and Results 
     Using the manual xyz stage, the sample was translated in the −z direction in steps of 25.4 μm, equivalent to increasing a focal plane position z in 25.4 μm steps. A total of 39 images were captured; i.e., z varied from 0 to 25.4×38=965 μm. Each image was converted to a plot of average intensity versus r. The 39 intensity-versus-r plots are represented as a contour plot in  FIG. 3 . Spots of high intensity occur where the objective lens focuses light onto a glass/air interface. Fringes arise due to interference between the two reflected beams. 
     The data were simulated according to the model discussed above. As shown in  FIG. 3 , the simulation captures the major features of the experimental data. From the model, values of z 1 =118 μm and z 2 =795 μm were obtained, resulting in d/n=677 μm. For BK7 glass, n=1.5196 at a wavelength of 532 nm. The thickness of the glass slide was therefore 677×n=1029 μm, which was in agreement with that measured by a mechanical micrometer, to within ±1 μm. 
       FIG. 4  is a flow chart illustrating a process  200  of analyzing image data in a digital confocal optical profile microscope in accordance with embodiments of the present technology. Even though the process  200  is described below with reference to the digital confocal optical profile microscope  100  of  FIG. 1 , one skilled in the art will recognize that several embodiments of the process  200  can also be implemented in other systems with similar or different system configurations. 
     As shown in  FIG. 4 , the process  200  includes acquiring sensor image at stage  202 . In one embodiment, the sensor image (e.g., the example photograph shown in  FIG. 1B ) may be captured with a CCD, CMOS, photodiode, and/or other suitable photo sensors. In other embodiments, the sensor image may be acquired manually and/or via other suitable techniques. Another stage of the process  200  can include modeling image data as described above with reference to the Theoretical Background section. For example, in certain embodiments, at least one of I 1 , I 2 , W 0 , z 0 , z 1 , z 2 , or δ 0  may be adjusted based on Eq. 7 and 15-17. In other embodiments, the image data may be otherwise suitably modeled. 
     The process  200  then includes comparing the modeled data to the acquired sensor image at stage  206  and determining if a match is found. In one embodiment, the modeled image data is deemed to match the captured image when the size of the center  115  ( FIG. 1B ) substantially matches (e.g., within a predetermined threshold). In another embodiment, the modeled image data is deemed to match the captured image when the size and/or spacing of the fringes  113  ( FIG. 1B ) substantially matches. If a match is not found, the process  200  reverts to modeling image data at stage  204  to further adjust at least one of I 1 , I 2 , W 0 , z 0 , z 1 , z 2 , or δ 0 . If a match is found, the process  200  continues to deriving sample parameters (e.g., positions of interfaces, z 1  and z 2 ) at stage  210 . The process  200  then includes a decision stage  210  to determine if the process should continue. If yes, then the process  200  reverts to acquiring sensor image at stage  202 ; otherwise, the process ends. 
     Even though the process  200  is described above as being at least partially iterative, in other embodiments, parameters of the sample may be derived directly without iteration. For example, the acquired sensor image may be analyzed to derive an area of the center  115  ( FIG. 1B ), a spacing of the fringes  113  ( FIG. 1B ), and/or other suitable parameters of the sensor image. Subsequently, the derived parameters of the sensor image may be input into Eq. 7, 15-17, or other suitable combinations thereof to derive the parameters of the sample. In further examples, the parameters of the sample may be derived directly in other suitable fashion. 
     Several embodiments of the digital confocal optical profile microscope  100  and associated process  200  can produce additional and/or more accurate information about the sample  101  than conventional devices by capturing substantially the entire profile of the signal beam  116 . For example, conventional devices with a physical pinhole cannot easily distinguish between light absorption by the sample  101  and a distance of the sample  101  relative to the objective lens  110 . If the sample  101  absorbs a portion of the illumination beam  112 , the sample  101  would appear to be farther away from the objective lens  110  because the measured light intensity at the pinhole would be low. In contrast, embodiments of the digital confocal optical profile microscope  100  capture not only the light intensity at the center of the signal beam  116 , but also other characteristics of the signal beam  116 . For example, it is believed that the area of the center  115  and the spacing of the fringes  113  are related to a position and thickness of the sample  101 , respectively. As a result, more accurate information of the sample  101  may be obtained. 
     From the foregoing, it will be appreciated that specific embodiments of the disclosure have been described herein for purposes of illustration, but that various modifications may be made without deviating from the disclosure. In addition, many of the elements of one embodiment may be combined with other embodiments in addition to or in lieu of the elements of the other embodiments. Accordingly, the technology is not limited except as by the appended claims.