Patent Publication Number: US-11392016-B2

Title: System and methods of fluorescence microscope calibration

Description:
RELATED APPLICATION 
     This application is a continuation of U.S. application Ser. No. 16/703,614, filed Dec. 4, 2019, which claims the benefit of U.S. Provisional Application No. 62/775,233, filed on Dec. 4, 2018. The entire teachings of the above applications are incorporated herein by reference. 
    
    
     GOVERNMENT SUPPORT 
     This invention was made with government support under Grant No. DA047733 awarded by the National Institute of Health, and Grant No. P60016170000198 awarded by the Worcester Science Foundation. The government has certain rights in the invention. 
    
    
     BACKGROUND 
     Fluorescence microscopy is a powerful investigative tool used throughout the fields of material sciences, biophysics, molecular biology, cell biology, medical diagnostics, and various pharmaceutical application to collect data from a target sample. When such a target sample (e.g., an organic or inorganic specimen) is exposed to light of a single or limited wavelength width (termed “excitation light”), certain chemical moieties or other compounds (e.g., a tryptophan amino acid, certain dyes, or fluorescence proteins) in the sample may emit light (termed “emission light”) in the form of fluorescence that may identify relevant structures or other properties of the target sample. 
     Typically, fluorescence microscopes deliver the excitation light to the target sample either through the same objective lens used to collect the emission light of the fluorescent molecules under observation, or through another mechanism. The excitation light used to illuminate the sample may emanate from a light source such as a laser light source or multi-wavelength light source. The excitation light may pass through one or more excitation filters designed to prevent all but a certain narrow range of light of a certain wavelength (e.g., an excitation wavelength) to pass through the filter. Once through the filter, the excitation light may be delivered to the sample by means of an objective and a dichroic mirror. The dichroic mirror is a specially designed optical filter that may reflect light of a certain excitation wavelength but permits the corresponding emission wavelength from the sample to pass through the dichroic mirror, or vice versa. The collected emission light then may be viewed by the naked eye or, more preferably, through the use of a detection device. 
     To provide the emission light as a usable image, the detection device, such as a CCD camera or the like, must be capable of imaging the emission light of target sample. While the use of various emission light detection devices is common, the detection characteristics of each device (and also the microscope) may differ. As a result, these differences in detection limits may make it difficult to compare the experimental results that may be collected using different microscopes or detection instruments between different laboratories or even through the use of a given instrument over time. 
     Another pressing issue when it comes to fluorescence microscopy is phototoxicity, that is, light-induced stress that may influence the behavior of the sample under observation. To minimize phototoxicity and to allow the assessment of the degree to which the effects of phototoxic may be present in the biological experiment that may be captured by the microscope image, a quantitative estimate of the excitation power (the amount of light used to excite the sample) is crucial. 
     SUMMARY 
     The embodiments of the invention described herein relate generally to microscopy and, more specifically, to a module for calibrating a fluorescence microscope and/or a fluorescence light detection device (e.g., a camera). 
     The described embodiments are directed to a system for, and method of, calibrating a fluorescence microscope and/or a detection device used to capture and image emission light from a target sample. The described embodiments may include a calibration apparatus configured to be attached to a fluorescence microscope and to measure the excitation characteristics of the microscope. These excitation characteristics may include, for example, the power and wavelength of the excitation light, detection of intensity-dependent variance, and back-aperture overfill of the objective. The described embodiments may facilitate measurement of the power of the excitation light delivered to the sample, or in some cases the back of the objective, and that measurement may then be reported along with the images obtained in the experiment. 
     The described embodiments of the apparatus may also facilitate the extraction of detector characteristics that allow model-based data analysis on the images acquired using the disclosed apparatus. For example, the described embodiments may generate different intensities of light that may permit measurement of variance maps for the detector that may help define a noise model of the detector. Noise models are camera-type-dependent but generally include, as a key parameter, the intensity and/or gain-dependent variance which, depending on the detector type, may be pixel-dependent (e.g., sCMOS cameras). Noise models may be used to improve data-analysis by enabling, for example, the use of statistics-based light detection and enhancement methods that may make the data obtained through the light detector more quantitative and reliable while reducing the degree of ambiguity introduced through manually set thresholds. Another example is the use in analyses that employ machine learning. Accurate detector models may be used to generate relevant training data for the analysis networks, making the networks more reliable, efficient and accurate. 
     Embodiments of the apparatus may be used also to calibrate a detection device by using the calibration apparatus to create a series of linearly increasing uniform intensity ramps of light. Images captured by the detection device of the intensity ramps may be then compared to other light data captured by the detection device to characterize the detection device. 
     Embodiments of the apparatus may generally include a main housing body comprising a control module, a sensor head, and a reflector assembly. An embodiment may comprise a main body housing that hosts a microcontroller configured to communicate with and/or control the sensor head. The sensor head may include a sensor board having one or more sensors (e.g., photodiodes, thermopile power detectors, light dependent resistors, photovoltaic detectors) one or more temperature sensors, one or more light sources (e.g., light emitting diodes, lamps, lasers) to emit light over a certain wavelength range, and one or more light sensors to measure emission light from the target sample. An adaptor may be disposed at an end of the apparatus for attachment to a microscope. Additionally, a receiving area positioned between the adapter and the sensor board may be configured to removably accept one or more of a converging lens, a diverging lens, a fixed-size iris, a variable-sized iris, or a light filter, as needed by the user. 
     Other embodiments of the apparatus may include one or more multi-color light emitting diodes as a light source, one or more multi-color light sensors, one or more temperature sensors, and one or more optical power sensors, each disposed on the sensor board. The light sources and photodetectors may emit and detect narrow or broad light wavelength ranges according to a user specified application such that both intensity and wavelength may be detected and peaked (i.e. ‘single color’) as well as to generate broad-spectrum light (i.e., “temperature light”). 
     Embodiments of the apparatus may also include a reflector assembly positioned between the sensor board and light receiving area that may permit a user to direct incoming or outgoing light to a specific sensor or light source. The reflector assembly may include a reflective surfaces unit comprising one or more reflective surfaces disposed about the inside surface of the reflective surfaces unit, one or more reflective elements positioned on a movable mount (e.g., a mirror or prism) to direct the path of incoming or outgoing light, and a selector mechanism mechanically linked to the mount. Through the use of the selector mechanism, a user may rotate the mount in order to configure the angle of the reflective elements (e.g., a ratiometric beam splitter positioned in the path of incoming and outgoing light) to direct light to a reflective surface—which may be aligned with a sensor or light source—and the light is then reflected to a specific sensor or light source. In an example embodiment, the reflective surfaces are curved (e.g., a convex surface) and configured to direct the optimal amount of light to the sensors. 
     Embodiments of the apparatus may combine one or more of a light power sensor, a temperature sensor, a wavelength detection sensor (e.g., a red-green-blue RGB sensor), and a multi-color LED light source into a single, portable, microscope calibration module that may function as a power meter with an integrated calibrated light source. This may allow a user to switch back and forth—using the reflector assembly—between the use of specific sensors (e.g., temperature sensor and light sensor) as well the use of specific light sources without a time-consuming switching of the individual sensor positions and individual adjustment of the reflective element during use. 
     Some embodiments of the apparatus may measure incoming light over a wide range of wavelengths (e.g., 10 nm-1000 nm) and, usefully, may also create light of known intensity in the same wavelength range according to the type of light source used with the apparatus. The emitted light may be adjusted linearly, non-linearly, or a combination of linearly and non-linearly, over a wide range of the power spectrum (typically micro Watts to Watts). 
     Some embodiments of the apparatus may include an attachment mechanism (e.g., a thread adapter, friction-fit adaptor) to permit a user to attach and remove the apparatus easily from any known microscope system. Some embodiments of the invention may also include a temperature sensor so that calibration of the microscope or detection device may mitigate any effect on the light sensor caused by heat buildup from the light source. This may eliminate the need to cool the apparatus during use to stabilize the sensor. Embodiments of the apparatus may be used to estimate the excitation light wavelength using an integrated RGB sensor or a mini-spectrometer. Embodiments of the apparatus may be used to detect aperture-overfill of the objective. Other embodiments may include multiple aperture overfill detectors to determine the centroid over the overfilling beam for alignment purposes (e.g., through triangulation). Embodiments of the apparatus may be positioned either in place of an objective or positioned in the sample position to calibrate the microscope. 
     Embodiments of the apparatus may be operated remotely by a user via wireless connectivity (e.g., Bluetooth®, WiFi, Zigbee, 3G, 4G or 5G cellular protocols) through the use of a handheld or otherwise portable device (e.g., smartphone, hand-held tablet etc.). The apparatus may be also miniaturized and permanently integrated into a microscope. Embodiments of the apparatus may include an integrated quadrant detector to facilitate light power measurements that may be used to detect and correct alignment errors between the light source and objective. 
     Embodiments of the apparatus may be calibrated both for the detection of emission light and generation of excitation light against an integration sphere or other suitable methods connected in order to a power meter to measure light output and linearity of the light source. After such calibration, the apparatus may be then calibrated internally through a periodic comparison to a power meter reading of light source. 
     In one aspect, the invention may be an apparatus for calibrating a microscope, comprising a main body housing, a sensor head, and a microcontroller assembly. The main body housing may have an adapter configured to mechanically couple the main body housing to a microscope. The sensor head may be disposed within the main body housing. The sensor head may comprise (i) an optical power sensor configured to produce a power signal representative of an optical power magnitude of light applied to the optical power sensor, and (ii) an optical wavelength sensor configured to produce wavelength information associated with the light applied to the optical wavelength sensor. The microcontroller assembly may be in communication with the sensor head. The microcontroller assembly may be configured to generate an optical power magnitude value based on the power signal, and to adjust the optical power magnitude value according to the wavelength information. In some embodiments, the microcontroller assembly may be disposed within the main body housing. In other embodiments, the microcontroller assembly may be disposed outside of the main body housing (e.g., within the microscope system or remote to the microscope system and the main body housing) and communicate with the sensor head by a wired or wireless communication link. 
     The optical wavelength sensor may comprise a red-green-blue (RGB) optical sensor. The optical power magnitude value may be further adjusted according to a temperature of the optical power sensor. The microcontroller assembly may comprise a display. The microcontroller assembly may be further configured to produce a calibration result based on the optical power magnitude value and to display the calibration result on the display. The light applied to the optical power sensor may be excitation light that the microscope uses to illuminate a specimen. 
     The sensor head further may further comprise a light source. The light source may be a broad-spectrum light source. The light source may be a multi-color light emitting diode. The optical power sensor may be a reflective surface, and the light source may be configured to direct light toward a detection device by directing the light toward the reflective surface, so that the light is reflected from the reflective surface and through a main aperture of the apparatus. 
     The sensor head may further comprise a temperature sensor and one or more light sensors. The apparatus may further comprise at least one of a converging lens, an iris, and light filter disposed within the main body housing. The iris may be may have a fixed aperture or a variable aperture. The microcontroller assembly may further generate an estimate of a wavelength of the light applied to the optical wavelength sensor based on the wavelength information. The microcontroller assembly may generate the optical power magnitude value based on the power signal, and adjust the optical power magnitude value according to the estimate of the wavelength of the light applied to the optical wavelength sensor. 
     The microcontroller assembly may further comprise a wireless transceiver configured to wirelessly communicate with external transceiver connected to a communications network. The communications network may be the Internet. The apparatus may further comprise a reflector assembly that comprises a reflective surfaces assembly including an interior surface defining a central opening, and one or more reflective surfaces radially distributed about the interior surface of the reflective surfaces assembly. The reflector assembly may further comprise one or more reflective elements attached to a mount and positioned within the central opening in a path of incoming or outgoing light. The reflector assembly may further comprise a selector mechanism mechanically linked to the mount such that movement of the selector mechanism rotates the mount to adjust an angle of the one or more reflective elements to direct the incoming or outgoing light to the one or more reflective surfaces, where the light is then reflected onto the one or more sensors. The one or more reflective elements may be a dichroic mirror or a prism. The one or more reflective surfaces may be a convex surface. 
     The apparatus may be coupled, using the adapter, to an objective mounting aperture of an objective turret of the microscope. The apparatus may further comprise an orientation sensor that produces an orientation signal representative of an orientation of the apparatus. The microcontroller assembly may initiate an excitation calibration procedure when the orientation signal indicates that the apparatus is in an active objective position. 
     In another aspect, the invention may be an apparatus for calibrating a microscope, comprising a main body housing, a sensor head, and a microcontroller assembly. The main body housing may have an adapter configured to mechanically couple the main body housing to a microscope. The sensor head may be disposed within the main body housing. The sensor head may comprise (i) an optical power sensor configured to produce a power signal representative of an optical power magnitude of light applied to the optical power sensor, (ii) an optical wavelength sensor configured to produce wavelength information associated with the light applied to the optical wavelength sensor, and (iii) a light source configured to direct light toward a detection device associated with the microscope. The microcontroller assembly may be disposed in the main body housing and in communication with the sensor head. The microcontroller assembly may be configured to generate an optical power magnitude value based on the power signal, and to adjust the optical power magnitude value according to the wavelength information. The microcontroller assembly may be further configured to calibrate the microscope and/or the detection device associated with the microscope. The calibration may comprise one or both of characterization of the detection device and characterization of the excitation light of the microscope. A component on the sensor head may have a reflective surface, and the light source may be configured to direct light toward a detection device by directing the light toward the reflective surface, thereby reflecting the light from the reflective surface and through a main aperture of the apparatus toward the detection device. 
     In another aspect, the invention may be a method of calibrating a detection device, comprising providing an apparatus comprising a main body housing, a sensor head, and a microcontroller assembly. The main body housing may have an adapter configured to mechanically couple the main body housing to a microscope. The sensor head may be disposed within the main body housing. The sensor head may comprise (i) an optical power sensor configured to produce a power signal representative of an optical power magnitude of light applied to the optical power sensor, (ii) an optical wavelength sensor configured to produce wavelength information associated with the light applied to the optical wavelength sensor, and (iii) a light source configured to direct light toward a detection device associated with the microscope. The microcontroller assembly may be in communication with the sensor head, and configured to generate an optical power magnitude value based on the power signal and adjusted according to the wavelength information. The method may further comprise attaching the apparatus to the microscope or the detection device, emitting light from the light source disposed on the sensor head, detecting the light by a light sensor disposed on the sensor head, calculating an intensity of light emitted by the light source, measuring an intensity of light detected by the detection device, comparing the intensity of light emitted by the calibration apparatus to the intensity of light detected by the microscope or light detector, and calibrating, by the apparatus, the microscope or detection device based upon a difference in intensity of light emitted by the apparatus and the intensity of light detected by the microscope or detection device. 
     Emitting light from the light source may further comprise generating a series of linearly increasing intensity ramps of light, and capturing, by the microscope or detection device, an image of each of the series of linearly increasing intensity ramps of light. 
     The method may further comprise comparing an intensity of light, detected by the microscope or the light detector in each of the series of linearly increasing intensity ramps of light, to an intensity of light emitted by the light source for each of the series of linearly increasing intensity ramps of light. The method may further comprise measuring the optical power magnitude value and storing the measured optical power magnitude value. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee. 
       The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments. 
         FIGS. 1A through 1M  illustrate an example embodiment of a system for calibrating a fluorescence microscope and associated emission light detection devices, according to the invention. 
         FIGS. 2A through 2K  illustrate an alternative example embodiment of the apparatus shown in  FIGS. 1A-1M , according to the invention. 
         FIG. 3  illustrates an alternative example embodiment of the apparatus mounted in a microscope turret, according to the invention. 
         FIG. 4  shows an example calibration/characterization procedure, according to the invention. 
         FIG. 5  illustrates an example internal structure of a processing system that may be used to implement one or more of the embodiments described herein. 
     
    
    
     DETAILED DESCRIPTION 
     A description of example embodiments follows. 
     The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety. 
     The described embodiments are directed to a system for and method of calibrating a fluorescence microscope and associated emission light detection devices through the use of a calibration apparatus. Embodiments of the calibration apparatus may measure also the amount of light applied to the sample and/or objective and may monitor the light source for stability and power output. 
       FIGS. 1A through 1M  illustrate an example embodiment of a system for calibrating a fluorescence microscope and associated emission light detection devices according to the invention (referred to herein as the “system”). The system may comprise a calibration apparatus  102 .  FIG. 1A  illustrates a view of the calibration apparatus,  FIG. 1B  shows a cut-away view of the calibration apparatus  102 , and  FIG. 1C  shows an exploded view of the calibration apparatus  102 . The specific components of apparatus  102  described herein are presented as examples for descriptive purposes, and are not intended to be limiting. The underlying functionality of the components may be accomplished with alternative form factors and arrangements. 
     The calibration apparatus  102  may comprise at least one main body housing  104  having a proximal end  106  and a distal end  108 . The main body housing may comprise a first subsection  104   a  and a second subsection  104   b . The calibration apparatus  102  may further comprise a sensor/source assembly  109  (see, e.g.,  FIGS. 1D-1I ) and a control assembly  111 .  FIG. 1D  shows a sectional view of the assembled sensor/source assembly  109 , and  FIGS. 1G, 1H, and 1I  illustrate front, side and back views of the sensor/source assembly  109 , respectively. 
     Referring to  FIG. 1E , the sensor/source assembly  109  may comprise a reflective surfaces unit  110 , reflective element  132  that functions as a 50/50 splitter, a selector mechanism  130 , and an adapter  112 . The control assembly  111  may comprise a microcontroller assembly  114  having one or more microprocessors  116 , a sensor head  118 , and a display area  126 . The sensor head  118  may comprise a sensor circuit board  134  that may host a variety of light sensors and sources. 
     The main body housing  104  may be constructed of any lightweight plastic, polymer, metal, or in any combination that may permit the main body housing  104  to be sized and shaped to form a generally hollow tubular structure to house the various apparatus components disposed therein. The main body housing  104  may include one or more subsections (e.g.,  104   a ,  104   b ) or may be constructed of a single unitary main body housing. In an example embodiment, the main housing body  104  may be printed using a 3-D printer. Alternatively, the main body housing  104  may be constructed of a light weight metal such as anodized aluminum. 
     Referring to  FIG. 1J , the control assembly  111  may comprise the microcontroller assembly  114 , a main board  144  and a control board  146 . The control assembly  111  may provide various I/O connectors, such as a sensor board connector  148 , a photodiode and photodiode sensor connector  150 , and an external triggering source connector  152 . The control board  146  may comprise a display  126  (e.g., an OLED display), and input control resources such as buttons and switches  168 . The microcontroller assembly  114  may be in communication with the microprocessor  116  that, in turn, may interface with the sensor board  134 . The microcontroller assembly  114 , which in an example embodiment may be implemented by a device such as an Arduio or Adafruit microcontroller (e.g., a 3.3V Adafruit Feather controller) may include a microprocessor  116 , and components such as a wireless transceiver module  154  capable of wirelessly communicating with an external transceiver (e.g., Bluetooth®, WiFi, Zigbee, a cellular protocol such as 3G, 4G, or 5G, etc.), a USB connection interface  156 , and a battery connection interface  158 . The microcontroller assembly  114  may be configured to operate and control all the sensors and other components forming the sensor head  118 . 
     Referring again to  FIG. 1E , the sensor head  118  may include a sensor circuit board  134  that hosts a centrally located power sensor  138  and one or more other components radially distributed about the edge of the sensor circuit board  134 . The centrally located power sensor  138  may comprise an integrative sensor that facilitates accurate excitation power measurement. In an example embodiment, the power sensor  138  may be a photodiode. The radially distributed components may comprise one or more red-green-blue (RGB) light sensors  120  (or other such optical wavelength sensor), one or more temperature sources  122 , one or more multi-color light sources  124 , and one or more stray light sensors  125 . 
     The one or more red-green-blue (RGB) light sensor(s)  120  may be configured to estimate the excitation wavelength of incident light for wavelength-specific power measurement. The RGB sensor may comprise three integrated light sensors, each with different sensitivity to red, blue, or green light. The microcontroller assembly may receive detection values from each of these three components, and perform a fitting routine on the detection values that predicts the wavelength based on the RGB ratios. 
     The temperature source(s)  122  may comprise a temperature light emitting diode (LED) that emits broad-spectrum light for detector characterization. The multi-color light source(s)  124  may comprise a multi-color LED, which selectively emits light in a single color for wavelength-dependent detector characterization. The stray light sensor(s)  125  may comprise a light dependent resistor (LDR) sensor that measures stray light from aperture overfill and is used for self-calibration. In certain embodiments, the sensor circuit board  134  may be sized and shaped (e.g., circular) to fit the inside diameter of the main body housing  104 . 
     One or more temperature sensors (not shown), attached to or in communication with the sensor circuit board  134 , may be used to measure local temperature variations during calibration. For example, the multi-color light source(s)  124  may produce a small amount of heat during use, which may affect the accuracy of the information collected by the light sensor  120 . Alternatively, heat buildup in the apparatus  102  may also occur from the ambient temperature as well as from any incoming light. Measurement of any changes in the temperature of the apparatus  102  due to heat buildup may be used to compensate for any error of the light sensor  120  introduced by the heat. This may reduce or eliminate the need to regulate the temperature of the apparatus  102  during calibration. 
     Suitable light sensors  120  may include any photoelectric devices that may convert light energy into an electronic signal. Such light sensors  120  may be configured to detect light of certain wavelengths that may range from infrared light to ultraviolet light. In some embodiments, the light sensors  120  may be configured to detected electromagnetic radiation of a wavelength range defined by the used sensor from nm to micrometers, preferably from the near UV wavelength (350 nm) to the near infrared wavelength (900 nm). However, the use of multiphoton excitation may include wavelength of 1600 nm to 2400 nm. Embodiments may include a light sensor configured to detect light of a wavelengths of about 10 nm to about 1000 nm, and preferably about 200 nm to about 850 nm, and more preferably, about 350 nm to about 800 nm. The sensor head  118  may include several light sensors  120 , each of which may be configured to detect light of a different wavelength or the same wavelengths. Exemplary light sensors for use with the described embodiments may include photovoltaic sensors, photodiodes, and light dependent resistors. Certain embodiments may include one or more photodiodes configured to measure light intensity/power. 
     Certain embodiments may include one or more red, green, and blue (RGB) light sensors that may be sensitive to light wavelengths in the red, green, and blue wavelengths of incoming or outgoing light. As it is known that light wavelength effects the accuracy of photodiode measurements (e.g., a light power sensor), the RGB light sensors may detect the relative signal from each of red, green, and blue wavelengths and may be used to estimate the wavelength of the incoming or outgoing light rather than requiring the user to provide such data. An example embodiment of the RGB sensors may utilize the TCS3472 sensor manufactured by AMS AG. 
     The sensor head  118  and sensor circuit board  134  may also include additional sensors such as a leveling sensor, a quadrant detector (or other photodetector array) to determine whether the incoming light is ‘on axis’, a vibration detector to evaluate stability, or positional sensors to detect the selected optical configuration of the apparatus  102 . 
     The multi-color light source  124  (also referred to as “excitation light source”) may comprise any light source that is capable of emitting light in the desired wavelength as described herein, such as a laser, a light emitting diode, a multi-color light emitting diode, an incandescent bulb, or other white light or full light spectrum source. Available light sources may emit light in wavelengths from 10 nm to 2400 nm. In certain embodiments, the multi-color light source  124  may include one or more multi-color light emitting diodes, such as a red-green-blue light emitting diode and/or a broad-spectrum temperature light emitting diode configured to transmit light in wavelengths of about 10 nm to about 1000 nm, and preferably about 200 nm to about 850 nm, and more preferably, about 350 nm to about 800 nm. 
     Referring to  FIGS. 1K, 1L, and 1M , some embodiments may include also a reflective surfaces assembly  110  comprising a reflective surfaces housing  140 , one or more reflective surfaces  136 , a reflective element  132 , and a selector mechanism  130 . The reflective surfaces assembly  110  may be positioned between the sensor circuit board  134  and the receiving area  119 . Certain embodiments of the reflective surfaces assembly  110  may be constructed as a single unit sized and shaped to fit inside the main body housing  104  and may include one or more stationary reflective surfaces  136  disposed radially about a central opening  137  on an inner surface of the reflective surfaces housing  140 . When the reflective surfaces assembly  110  is in position in the main body housing, the reflective surfaces  136  positioned radially about the inner surface of the reflective surfaces housing  140  may be configured to align with each of the radially distributed light sensors  120  (one reflective surface per sensor) positioned on the sensor circuit board  134 . Each of the reflective surfaces  136  may include a shape and surface suited to direct light to and from a sensor. For example, a rough reflective surface may introduce some smoothing of the light distribution, or a curved surface may impact the beam size of the light at the spot it hits a sensor. In this way, the reflective surfaces  136  may be configured to control various properties of the sensors and light sources in the sensor head  118 . These properties may include, for example, the direction, diffusions, and convergence of reflective light towards and from a sensor or light source. The reflective surfaces  136  may be constructed of, for example, a polished metal, a mirror, or a coated reflective surface. 
     A reflective element  132  may be attached to a rotatable mount (not explicitly shown) and positioned within the central opening  137  of the reflective surfaces housing  140  such that the reflective element  132  may be in the path of light traveling through the central axis of the apparatus. The reflective element  132  may include one or more mirrors (e.g., dichroic mirror), prisms, glass, or a coated surface (e.g., a metal coated surface such as glass coated with a transparent film of aluminum deposited on the surface using vaporized aluminum or a dichroic material). The reflective element  132 , positioned in the light path, may be configured to direct incoming or outgoing beams of light to and from a reflective surface  136  and then to a specific sensor or light source  120 ,  122 ,  124 . For example, a reflector element  132  may be configured to direct incoming light to an RGB sensor  120  by rotating the mount in order to adjust the angle of the reflector element  132  such that the incoming light may be directed off one of the reflective surfaces  136  and into the RGB sensor  120 . Certain embodiments may include a reflector element  132  that is a ratiometric  50 / 50  beam splitter attached to the rotatable mount and positioned within the central opening  137  of the reflective surfaces unit  110 . The ratiometric beam splitter  132  may direct half of the light to one of the sensors on the periphery of the sensor board using its respective reflective surface and while permitting half the light to pass through the beam splitter and impact a power meter (e.g., a photodiode) positioned centrally on the sensor board  134 . In some embodiments, the reflector element  132  may use a ratiometric beam splitter that implements ratios other than 50/50, depending on the associated sensors and the specific application of the apparatus  102 . 
     Referring to  FIG. 1F , selector mechanism  130  (e.g., a selector knob) may be connected to the rotatable mount. The selector mechanism  130  may be configured to rotate the mount and the reflector mechanism  130  to control the angle of the reflector element  132  through a mechanical linkage. Preferably, the selector mechanism  130  and mechanical linkage may be configured to direct a rotating beam of light  160  to and from a certain reflective surface  136  and then to a sensor  120 ,  122 ,  124 . For example, the selector mechanism  130  may travel a certain distance in a slot  164  in the main housing body corresponding to a first position that rotates the mount and simultaneously adjusts the reflector element such that the beam of light is directed to a first reflective surface and then from the first reflective surface  136  to a first sensor  120 ,  122 ,  124 . The selector element  130  may then be moved to a second position that rotates the mount and adjusts the reflective element such that the light beam is directed to a second reflective surface (not shown) and then on to a second sensor (not shown). This rotation is indicated by dashed arrows  166   a ,  166   b . This process may be repeated for each sensor/reflective surface pair in the apparatus. In some embodiments, the ratiometric beam splitter may transmit a portion of the light emitted by a peripheral light source on the sensor board (the remaining portion exits). The transmitted portion of the light may then be reflected by the opposing reflective element onto the peripheral sensor on the opposite side of the sensor board. This allows the device to self-characterize using opposing peripheral sensors. Likewise, turning the ratiometric splitter holder by 180 degrees may allow any peripheral light source on the sensor board to reflect a known fraction of the light onto the power sensor placed centrally on the sensor board, allowing the power to be measured. 
     Certain embodiments may also include a receiving area  119  (e.g., one or more slots) at the proximal end  106  or distal end  108  of the main body housing  104  configured to receive one or more converging lenses, light filters, diffuser elements, or fixed-sized irises that may be used to control, for example, the amount/intensity of incoming light or wavelength of light. Preferably, the one or more converging lenses, light filters, or fixed-size irises are removable and selectable by the user. 
     In certain embodiments, one or more control buttons or switches  168  may be disposed along the main housing body  104  and in communication with the microcontroller assembly  114  comprising the microprocessor(s)  116  to control the function of the apparatus  102 . The apparatus  102  may include further an adapter  112  disposed at the proximal end  106  or the distal end  108  of the main body housing  104  for attachment to a microscope or viewing device. The adapter  112  may include, for example, a threaded adaptor, a friction fit adaptor (i.e., snap on), a clamp-on adaptor, a magnetic attachment adapter, or other such adapter known in the art that is suitable for connecting the calibration apparatus  102  with a microscope. In some embodiments, the apparatus may be fixedly attached to the microscope or viewing device. 
     Certain embodiments may include a display screen  126  disposed on or at least partially within the main body housing  104 , and may be in communication with the microcontroller assembly  114 . The display screen  126  may be configured to allow users to view, for example, various instructions, calibration measurement outcomes, device and apparatus settings, and status of the apparatus  102 . 
     Certain embodiments may be used to calibrate one or more fluorescence microscopes such that collected data from one fluorescence microscope may be comparable quantitatively to data collected from another fluorescence microscope. Additionally, certain embodiments may be used to ensure the repeatability of data collection for a given fluorescence microscope over time. 
     Calibration of a fluorescence microscope may be achieved using an embodiment of an apparatus  102  to measure excitation power levels at discrete instrument settings (e.g., the settings of the microscope used in in the collection of data for certain experiments). For example, the microscope objective may be removed, and the apparatus  102  attached to the microscope in place of the objective using the adapter located at the proximal end  106  of the main body housing  104 . Once in position, the apparatus  102  may measure the amount of light the microscope system delivers to the backside of the objective. The apparatus  102  may include a receiving area  119  (e.g., slots) in the housing body  104  to receive one or more irises  128  of fixed-size that may be matched to the diameter of the back opening of the objective to limit the area of the sample impacted by excitation light. In this way, an iris  128  limits the sensitivity of the power sensor to the amount of excitation light that would be transmitted by the objective into the sample. Alternatively, for low numerical aperture objectives, the apparatus may be positioned in sample holder during calibration. 
     Activation of the light source  124  positioned in the sensor head  118  may simulate the emission light detected in an experiment of interest. The emission light signal may be either of a broad wavelength range—in which case the emission filters inside the microscope may be used to restrict the color detected—or the emission light signal may be of a narrow range of wavelengths similar to the emission spectrum of various dyes or fluorophores. When the emission light signal is of a narrow range of wavelengths, the emission filters may act to restrict the color, resulting in less background signal. Measuring the signal detected by the microscope for a known and fixed input signal from the apparatus may allow a user to track performance and alignment of the microscope excitation light source over time. 
     Additionally, the described embodiments may be used to detect overfill of the back-aperture. Overfill of an objective generally may indicate the microscope may be poorly aligned. In such scenarios, insets—made of a light diffusing material (e.g., glass, acrylic) having a central ring configured to match the size of the back aperture of the objective may be inserted into the slots to replace an iris. Once in position, the insets may absorb light that may typically pass through the objective but may scatter light to the periphery that is not blocked by the central obscuration. A light sensor  120  in the apparatus  102  may then detect the scattered light that would otherwise be blocked by the objective indicating the overfill. 
     Alternatively, when using certain aperture objectives and/or air spaced objectives with the microscope, the apparatus  102  also may be configured, for example, to have the form of a cover glass, a tissue culture dish used for imaging (typically 35 mm, glass bottom dish) or a 12 well plate (96 well, 384 well have the same outer form factor) and may be placed in the position of the sample. In this way, embodiments may be “formed” or otherwise configured to be used in devices other than microscopes such as, for example, in robotic devices to calibrate on-board imaging devices. 
     In some embodiments, the apparatus  102  may be configured in the form of a microscope objective that may be attached to the objective turret of the microscope in an unused objective mounting aperture (or in the available aperture after removal of an objective) for microscope calibration (e.g., excitation characterization and/or detection device characterization). The objective form factor of the apparatus  102  may be varied such that it fits within given space constrains, or simply minimized to be as small as possible. In this way, the apparatus  102  may be installed and stored on the turret like an unused objective during operation of the microscope. Doing so may eliminate the need to repeatedly remove and unmount the apparatus, during which an objective may be damaged. The apparatus  102  in the form of a microscope objective also may be moved into place through the operation of an automated turret during an experiment, to capture reference data to monitor performance of the microscope or detector over time, and, specifically, during lengthy experiments. 
       FIGS. 2A through 2K  illustrate an alternative example embodiment of the apparatus  102  shown in  FIG. 1A . The apparatus  202  of this example embodiment is substantially smaller than apparatus  102 , and is configured to be mounted onto a microscope turret  302  in an empty objective slot, as shown in  FIG. 3 . 
     The basic operation of apparatus  202  corresponds to the operation described herein of apparatus  102 , with certain differences due to the smaller size and different form factor of apparatus  202 . Revisions in the apparatus  202  embodiment are concerned with improvements of the physical footprint and ease-of-use. For example, the footprint of apparatus  202  is reduced with respect to apparatus  102  to the size of an oversized objective. This allows the apparatus  202  to remain in the objective turret  302  while using the microscope, removing the need to install and remove the apparatus  202  for microscope characterization. 
     The microcontroller assembly of apparatus  202  may include a wireless transceiver (e.g., Bluetooth®, WiFi, Zigbee, a cellular protocol such as 3G, 4G, or 5G) to facilitate cloud connectivity, so that, for example, apparatus  202  may be operated and/or updated over the Internet or other communication network. Such connectivity may also enable automated online storage of measurement results and wireless communication with the microscope computer. In the apparatus  202 , the number of optical and mechanical components is reduced as compared to apparatus  102 , to reduce size and cost. The apparatus  202  has more overfill detectors as compared to apparatus  102  so that the beamcenter may be determined through triangulation, which allows the apparatus  202  to track excitation alignment. The display of the apparatus  202  has been simplified, as compared to the apparatus  102 , to an 8×8 matrix. The apparatus  202  includes an indicator ring of 16 RGB LEDs to provide state signaling to the user. 
     A navigation ring drives a rotary encoder with click function, allowing the user to control the apparatus  202  by navigating basic menus and changing settings. The cloud connectivity, however, removes the requirement for most physical interactions between the user and the apparatus  202 . The housing may include two more buttons and an RGB LED status indicator on the side to set up, reset and monitor the microcontroller and its connection to the cloud. 
     The primary optical path of the apparatus  202  has been redesigned and compressed, as compared to the apparatus  102 . The back-reflection of the surface of the silicon photodiode (used to measure the excitation power) may also be used to guide and steer the outgoing light. To do so, the photodiode is placed on an adjustable gimbal plane under an angle, allowing the output of the broad-spectrum LED light-source (corresponding to temperature source  122  of apparatus  102 ) to be reflected out of the main aperture by means of the photodiode back-reflection. In doing so, the photodiode can accurately measure the light output of the apparatus  202 , enabling self-calibration and stabilization. The gimbal mount of the photodiode allows tip and tilt adjustment of the outgoing beam, whereas a small convex lens with adjustable distance to the broad-spectrum LED can adjust the focus. Together, these adjustment opportunities allow the apparatus  202  output to be tuned to fit the particular optical and geometrical properties of the associated microscope. 
     By using the photodiode as the main reflective element, the need for a complex optical selector (i.e., selector mechanism  130  of apparatus  102 ) is removed, which greatly reduces the number of optical and mechanical components needed to facilitate the use of various sensors and light sources in concert. It also makes it more straightforward to incorporate sensors near the aperture—a fact that is exploited by the incorporation of three overfill detectors behind a diffuser with an aperture that matches the objective back-aperture of the intended objective. The placement of these overfill detectors allows the centroid of the beam to be estimated through triangulation, so that the apparatus  202  operates as a useful alignment tool, as well as allowing the excitation alignment to be tracked. 
     The apparatus  202  drastically reduces the need for user interaction. The apparatus  202  may have an orientation sensor (not explicitly shown, although the tilt sensor  238  may be used instead of or in addition to the orientation sensor), which detects when apparatus  202  is moved to the active objective position (i.e., by rotating the objective turret). Detecting when the apparatus is in the active objective position may initiate the excitation calibration procedure, the results of which may be automatically uploaded to a cloud database. The user may be informed of completion, at which point the apparatus  202  may be rotated out of the active objective position and the user can resume their experiments—all in a matter of seconds, without the need for user input or record-keeping on the part of the user. 
     The apparatus  202  includes a threaded base assembly (TBA) located at the bottom of the tool. The TBA is clamped into the base of the apparatus  202 , allowing the apparatus to be exchanged for use with different microscopes. The free rotation of the TBA with respect to the apparatus also allows the rotational orientation of the apparatus, with respect to the microscope user, to be adjusted, making it easier to operate the device and read out the display. 
     The TBA may include all microscope-specific components of the apparatus  202 , allowing most of the apparatus  202  to remain untouched when configuring the device for a specific microscope or objective. The TBA may include an outer thread that mounts the objective into the turret or side-port of the microscope. The minimum diameter is designed for RMS objectives, so the TBA can be made for most common objective/port thread (i.e., M25, M32, C-mount, SM1). The TBA may include a diffuser with a circular aperture that matches the back-aperture of the microscope objective. This facilitates overfill detection and allows misalignment to be detected and tracked. The TBA may include an optional lens that can accommodate exotic microscope configurations (e.g., high excitation beam diameter, or very long or complex emission paths). The TBA may include an optional neutral density or color filter to accommodate high-power or wide-spectrum excitation sources. Users that would like to use one apparatus  202  with multiple microscopes or very distinct objectives or modalities can exchange the TBA in a matter of minutes, or install multiple apparatus  202  into empty objective positions or side-ports. 
       FIGS. 2I, 2J, and 2K  depict cross-section views of apparatus  202 , which reveal the positioning of the main optical components. A small focusing lens  252  allows the defocus and scaling broad spectrum light source on the detector, whereas the gimbal mount  254  that positions the photodiode  256  facilitates tip and tilt of the outgoing calibration beam  258 . The threaded bottom assembly  250  (TBA) is clamped onto the bottom of the apparatus  202  and can be exchanged to make the apparatus  202  compatible with a variety of microscopes and objectives. 
     The apparatus  202   FIG. 2A  shows the apparatus  202  completely assembled, and  FIG. 2B  shows the apparatus  202  with the housing  204  and navigation ring  206  removed.  FIG. 2C  shows the apparatus  202  with the housing  204  removed but with the navigation ring  206  in place, along with the power sensor  208  and a mounting thread  210  for attaching the apparatus  202  to a microscope turret  302 .  FIG. 2D  shows an alternative view of the apparatus  202  fully assembled, showing set/reset buttons  212 , a matrix display  214 , and a status LED  216 .  FIG. 2E  shows the apparatus  202  with the navigation ring  206  removed, showing the indicator ring  218 , charging indicators  220 , a room light sensor  222 , and a USB connector  224  for charging and serial data communication.  FIG. 2F  illustrates the apparatus  202  with the housing  204  and the navigation ring  206  removed, showing the diffuser aperture  226 , the optical axis  228 , and the focus adjust  230 .  FIG. 2G  illustrates the apparatus  202  with the housing  204  and the navigation ring  206  removed, showing the tip/tilt adjust  232 , an optional lens  234 , and a space  236  for a battery.  FIG. 2H  illustrates the apparatus  202  with the housing  204  and the navigation ring  206  removed, showing the tilt sensor  238 , the microcontroller assembly  240 , and a piezo speaker  242 . 
     The described embodiments may also be used to calibrate a detection device, such as a CCD camera, configured to capture and image emission light. With reference to  FIG. 4  the apparatus may calibrate the detection device by creating a series of intensity ramps of a light signal (that is, a series of light signals with a known, linearly increasing power intensity), which may then be captured in a series of images using the microscope and the detection device. These measurements may be compared to emission light data (e.g., images) previously captured, to determine the amount/intensity of light signal received by the detection device. The user may then adjust, based on the comparison of emission light data of the intensity ramps and experimental data, the various detection device settings to optimize emission light capture. 
     At the start of the example calibration/characterization procedure  400  shown in  FIG. 4 , optical image settings are determined  402 , the microscope objective is removed  404 , and the apparatus  102 ,  202  is mounted  106  in the removed objective&#39;s location in the microscope turret. A procedure type is then chosen  408 . If the excitation characterization  410  is chosen, the excitation measurement is started  412 , the measured output is recorded  414 , and it is determined  416  if more procedures are required. 
     If the detection device characterization  418  is chosen, a detection device trigger type is then chosen  420 , one of a hardware trigger  422 , a synchronous start  424 , or a manual trigger  426 . If the hardware trigger  422  is chosen, a trigger cable is connected  428  between a triggering source and the detection device. The detection device trigger settings are adjusted  430  suitable to the characterization procedure and the triggering source, and extrema of the measured intensity range are determined  432  for the detection device. This determination  432  is common to the three trigger types described above. In a cloud-based implementation use case, triggering may be done through communication (e.g., wireless Internet-based communication) with software running on the microscope computer that gathers and analyzes the images from the microscope detector, all the while controlling the apparatus  102 ,  202  to produce and measure the appropriate calibration signal. 
     If the synchronous start  424  trigger type is chosen, timing settings for the synchronization are matched  434 , and extrema of the measured intensity range are determined  432  for the detection device. 
     If the manual trigger  426  is chosen, camera and detection device settings are established  436 , and extrema of the measured intensity range are determined  432  for the detection device. For all three trigger types  422 ,  424 ,  426 , once extrema of the measured intensity range are determined  432 , the ramp acquisition procedure is started  438 , the acquired detection device data is saved  440 , a model of the detection device is extracted  442 , and it is determined  416  if more procedures are required. Saving the acquired detection device data and extracting a model of the detection device from the saved data may be performed by an external computer executing detector-specific code. The acquired data may be conveyed to the external computer by a wireless link from the apparatus  102 ,  202  to the external computer. In some embodiments, no image data (from the detection device) needs to be saved, because the data may be analyzed ‘on the fly’ by software running on the microscope computer, and the calibration results may be wirelessly uploaded to cloud-based resources. This removes the need to save and transmit large amounts of image data and makes the calibration procedure less time-consuming for two reasons. First, the analysis of the calibration data may be performed automatically and during the image acquisition. Second, as the calibration data is analyzed on the fly, the software may determine when enough data was gathered to calibrate the device, removing the need to collect an over-abundance of data. 
     If more procedures are required, a procedure type is once again chosen  408 . If no more procedures are required, the apparatus  102 ,  202  is removed  444  from the objective position in the turret, and the objective is replaced  446 . 
     For the ramp acquisition procedure, the intensity ramps of the light signal may start below the detection limit of the detection device and may then increase until reaching the saturation point of the detection device. Further, by measuring the intensity-dependent variance, the apparatus  102 ,  202  may be used to analyze the noise characteristics of the detection device over the range of intensities used in the experimental data. Thus, tracking the detection device calibration over time may permit a user to monitor quantitatively the detection device performance and, for example, to compare images from different microscopes or the same microscope at different times. Alternatively, the intensity ramps may be pulsed, varied in pulse length, and the intensity of the ramp may increase or decrease over time to capture light intensity at smaller timescales (such as those present during characterization of fluorescence lifetime imaging field) than may be possible using the previously described intensity ramps. 
     Alternatively, calibration may also be achieved without knowledge of the absolute intensity of the light generated by the calibration apparatus  102 ,  202 . In such scenarios, the power of the light may be lowered to the point where the detector has zero signal and then increased linearly from that bottom point to saturation of the detector. Next, using a noise model and advanced image analysis, it may be possible to measure the number of photons inside a diffraction limited spot. This measurement may be achieved also through the use of the electron to photon conversion factor of the camera, but a noise model may be more exact over a wider range of light intensities. Measuring the actual saturation level of the detector/camera allows also a simpler form of image analysis in which the absolute signals of different objects in an image are compared—without an absolute value for the power—as a reliable ratio of intensities (e.g. x is 10 times brighter then y). 
     The apparatus  102 ,  202  may be used as a stand-alone calibration module or may be used in cooperation with another computer. For use as a stand-alone module, the apparatus  102 ,  202  may include wireless connection capability (e.g., Bluetooth, Zigbee, WiFi, et al.) and may be locally powered by a battery (e.g., Lithium-ion, Lithium polymer, et al.). Additionally, the apparatus  102 ,  202  may be used with an external screen such as a computer monitor. The external screen may be connected to the apparatus  102  by a wired connection or a wireless connection. Alternatively, the apparatus  102 ,  202  may be controlled through a mobile phone (or other mobile device) application. Certain embodiments of the apparatus  102  may cause the microscope to execute certain functions, such as, for example, capturing an image, changing settings or imaging parameters, or activating the microscope light source. Some embodiments of the apparatus  102 ,  202  may be integrated into the microscope itself. For example, the apparatus  102 ,  202  may be situated in the light path of the microscope. In some embodiments, connectivity with a smartphone, a computer, microscope computer (for detector calibration) etc., can take place wirelessly through a cloud-based intermediary. In the case of example apparatus  202 , the microcontroller assembly (e.g., Particle Photon device) is connected to the Particle IoT cloud, which serves as an intermediate between the microcontroller assembly, and other software/hardware that may interact with it. 
     It is further contemplated that the microprocessor(s)  116  within the apparatus  102 ,  202  and/or other external devices (e.g., another computer, monitor or hand-held device) may be configured to execute software to analyze the measurements captured by the external devices and/or apparatus  102 ,  202  during the calibration process. The apparatus  102 ,  202  may be configured to execute an imaging protocol on the microscope and automatically extract key values (e.g., excitation light power output, emission light output, et al.) from the collected measurements. The apparatus  102 ,  202  may use the extracted key values to adjust its own calibration protocol to conduct a full calibration cycle (or parts of a full calibration cycle) autonomously, that is, without user input. 
       FIG. 5  is a diagram of an example internal structure of a processing system  500  that may be used to implement one or more of the embodiments herein. Each processing system  500  contains a system bus  502 , where a bus is a set of hardware lines used for data transfer among the components of a computer or processing system. The system bus  502  is essentially a shared conduit that connects different components of a processing system (e.g., processor, disk storage, memory, input/output ports, network ports, etc.) that enables the transfer of information between the components. 
     Attached to the system bus  502  is a user I/O device interface  504  for connecting various input and output devices (e.g., keyboard, mouse, displays, printers, speakers, etc.) to the processing system  500 . A network interface  506  allows the computer to connect to various other devices attached to a network  508 . Memory  510  provides volatile and non-volatile storage for information such as computer software instructions used to implement one or more of the embodiments of the present invention described herein, for data generated internally and for data received from sources external to the processing system  500 . 
     A central processor unit  512  is also attached to the system bus  502  and provides for the execution of computer instructions stored in memory  510 . The system may also include support electronics/logic  514 , and a communications interface  516 . The communications interface may accept acquired data from the apparatus  102 ,  202  during a calibration procedure, as described with reference to  FIG. 4 . 
     In one embodiment, the information stored in memory  510  may comprise a computer program product, such that the memory  510  may comprise a non-transitory computer-readable medium (e.g., a removable storage medium such as one or more DVD-ROM&#39;s, CD-ROM&#39;s, diskettes, tapes, etc.) that provides at least a portion of the software instructions for the invention system. The computer program product can be installed by any suitable software installation procedure, as is well known in the art. In another embodiment, at least a portion of the software instructions may also be downloaded over a cable communication and/or wireless connection. 
     It will be apparent that one or more embodiments described herein may be implemented in many different forms of software and hardware. Software code and/or specialized hardware used to implement embodiments described herein is not limiting of the embodiments of the invention described herein. Thus, the operation and behavior of embodiments are described without reference to specific software code and/or specialized hardware—it being understood that one would be able to design software and/or hardware to implement the embodiments based on the description herein. 
     Further, certain embodiments of the example embodiments described herein may be implemented as logic that performs one or more functions. This logic may be hardware-based, software-based, or a combination of hardware-based and software-based. Some or all of the logic may be stored on one or more tangible, non-transitory, computer-readable storage media and may include computer-executable instructions that may be executed by a controller or processor. The computer-executable instructions may include instructions that implement one or more embodiments of the invention. The tangible, non-transitory, computer-readable storage media may be volatile or non-volatile and may include, for example, flash memories, dynamic memories, removable disks, and non-removable disks. 
     Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims. 
     While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims.