Patent Description:
<CIT> refers to a multi-mode microimaging method based on programmable LED array illumination. An LED array used as an illumination light source of a microimaging system is directly installed below a sample carrying bench of the microimaging system and the center of the LED array is located at an optical axis of the microimaging system, thereby realizing phase contrast, optical field imaging, and optical dyeing imaging modes.

<CIT> refers to an imaging system for automation of sample monitoring includes an image capture device that cooperates with a lens assembly for imaging the samples.

<CIT> refers to a brightfield transmitted light illumination device for microscopes.

<CIT> refers to a compact, inexpensive fluorescence microscope capable of high-resolution imaging with high light throughput suitable for use in both laboratory and field environments, and methods of use.

In some instances it may also be desirable to translate a microscope objective relative to a specimen location or vice versa. What has been needed are imaging systems and methods that may be used to implement such enhanced contrast techniques as well as other microscopy techniques while allowing for transverse movement of the objective lens relative to a specimen location.

In accordance with claim <NUM> of the present invention a microscope imaging system is provided, including a specimen fixture comprising an illumination side, an imaging side and at least one specimen receptacle which is disposed in a specimen plane of the specimen fixture;.

Some embodiments of a method of microscopic imaging may include aligning an image input axis of an objective at a first position in a specimen plane of a specimen fixture, transmitting a first illumination signal to an illumination screen having a flat illumination surface that faces an illumination side of the specimen fixture, and emitting illumination light from a light pattern on the flat illumination surface of the illumination screen which has a light pattern axis that is aligned with the image input axis of an objective. The method may further include imaging the specimen plane at the first position with the objective. Thereafter, the objective may be translated in an x-y plane which is parallel with the flat illumination surface without moving the illumination screen relative to the specimen fixture. The image input axis of the objective is thereby translated from alignment with the first position in the specimen plane to alignment with a second position in the specimen plane. A second illumination signal may then be transmitted to the illumination screen and illumination light thereby emitted from the light pattern on the flat illumination surface of the illumination screen which has the light pattern axis thereof aligned with the image input axis of the objective at the second position. The method may also include imaging the specimen plane and any specimen or portion thereof at the second position with the objective.

Some embodiments of a microscope imaging system may include a specimen fixture comprising an illumination side, an imaging side and specimen receptacle which is disposed in a specimen plane of the specimen fixture. An illumination screen may be disposed in fixed relation to the specimen fixture facing the illumination side of the specimen fixture, and include a flat illumination surface that is configured to emit an annular light pattern having a light pattern axis. The microscope imaging system may also include a phase contrast objective which faces the imaging side of the specimen fixture, which includes an image input axis that is aligned with the light pattern axis, and which is configured to form an image of the annular light pattern of the illumination screen onto a phase ring of the phase contrast objective without the use of a condenser lens disposed between the illumination screen and the specimen plane and with the annular light pattern of the illumination screen not being effectively disposed at infinity with respect to the phase contrast objective.

Certain embodiments are described further in the following description, examples, claims and drawings. These features of embodiments will become more apparent from the following detailed description when taken in conjunction with the accompanying exemplary drawings.

The drawings are intended to illustrate certain exemplary embodiments and are not limiting. For clarity and ease of illustration, the drawings may not be made to scale, and in some instances, various aspects may be shown exaggerated or enlarged to facilitate an understanding of particular embodiments.

In the case of phase contrast illumination, a condenser lens may not be necessary for a phase contrast illumination device if the light source is an annulus of sufficient brightness and of the correct angular shape. Embodiments discussed herein may be used, in some cases, to replace a phase contrast illumination device or components thereof with an illumination display screen that may be configured to automatically align a light pattern axis of an output light pattern with an image input axis of an objective lens. A large format of such an illumination screen display may allow multiple specimen positions to be illuminated by multiple light patterns on the illumination screen without physical movement or replication of an overhead light. With moving imaging heads or multiple imaging heads, the illumination screen may be configured to adjust a graphical output of light patterns to align the position of emitted illumination corresponding to the graphical output with the position of the moveable imaging head or the active head(s) in an array of imaging heads such as objectives, including microscope objectives. The use of such enhanced techniques without the need of a condenser lens or the like may be desirable in some cases because this may be useful for eliminating moving parts (such as the condenser lens) disposed between the light source such as the illumination screen and the specimen being imaged. Such an arrangement may allow for more robust and reliable movement of the objective relative to the specimen as well as allowing for a simpler imaging system generally.

The ability to control and optimize dynamically the shape, color, position, brightness, time delay, trajectory, frequency and duration of the illumination pattern (or patterns) emitted from an illumination screen or the like may allow for enhanced image contrast during microscopy in many cases. In some cases, portions of such an illumination screen that are not emitting a significant amount of illumination, i.e., those portions of the display that are disposed adjacent to a graphical output or between a plurality of graphical outputs of the display, may be left dark or otherwise in a state that does not emit a significant amount of illumination for purposes of microscopy.

<FIG> show an exemplary embodiment of a microscope imaging system <NUM> that may include a specimen fixture <NUM> having an illumination side <NUM>, an imaging side <NUM> and at least one specimen receptacle <NUM> as shown in <FIG>. The specimen receptacle <NUM> may be disposed in a specimen plane <NUM> of the specimen fixture <NUM>. Referring to <FIG>, the microscope imaging system <NUM> may also include a translation stage <NUM> which is disposed in fixed relation to the specimen fixture <NUM> and which faces the imaging side <NUM> of the specimen fixture <NUM>. An objective <NUM> may be operatively coupled to the translation stage <NUM>, laterally translatable in an x-y plane <NUM> that is substantially parallel to the specimen plane <NUM>, and include an image input axis <NUM> disposed towards the imaging side <NUM> of the specimen fixture <NUM>. The objective <NUM> may also include a object plane <NUM> which is substantially perpendicular to the image input axis <NUM> and adjustable to be coplanar with the specimen plane <NUM>. In some cases, embodiments of the objective <NUM> may include a microscope objective lens with a magnification power of about 10x to about 120x and a working distance of about <NUM> to about <NUM>. The objective <NUM> may also include specialized contrast objectives such as a phase contrast microscope objective lens.

Although the microscope imaging system embodiment <NUM> is shown as an "inverted" type system with the objective <NUM> facing an upward direction, the same or similar arrangement of components of the microscope imaging system <NUM> could also be used in a non-inverted configuration. In addition, although the microscope imaging system <NUM> is configured to have the specimens <NUM>, specimen receptacles <NUM> and specimen fixture <NUM> disposed in fixed relation to the frame <NUM> with the objective <NUM> configured to translate relative thereto, the inverse arrangement could also be used whereby the objective <NUM> is fixed relative to the frame <NUM>. However, in many cases, it may be preferred to keep the specimens <NUM> stationary to prevent sloshing or otherwise physically agitating the specimens <NUM>. The fixed specimen configuration shown also tends to be more compact.

An illumination screen <NUM> may be disposed in fixed relation to the specimen fixture <NUM>. The illumination screen <NUM> may further include an array of light emitting pixels <NUM> and a flat illumination surface <NUM> which is substantially parallel to the specimen plane <NUM>. The flat illumination surface <NUM> may be disposed facing the illumination side <NUM> of the specimen fixture <NUM>. A controller <NUM> may be operatively coupled to the illumination screen <NUM> and the translation stage <NUM> with conduits <NUM> and be configured to coordinate the transmission of an illumination signal to the illumination screen <NUM> which may be configured to produce emission of a light pattern <NUM> from the flat illumination surface <NUM>. In some cases the controller <NUM> may be configured to coordinate the transmission of a dynamic illumination signal to the illumination screen <NUM> which produces emission of a light pattern <NUM> which has a light pattern axis <NUM> that tracks across the flat illumination surface <NUM> and remains aligned with the image input axis <NUM> of the objective <NUM> as the objective <NUM> is translated in the x-y plane <NUM> from a first position to a second position in the x-y plane <NUM>.

In some cases, the light pattern axis <NUM> may be configured as an axis of symmetry of the light pattern <NUM>. For example, for a light pattern <NUM> configured as an annular light pattern <NUM> as shown in <FIG>, the light pattern axis <NUM> may be disposed in the center of the light emitting annular pattern. The illumination light <NUM> that is emitted from the light pattern <NUM> of the flat illumination surface <NUM> of the illumination screen <NUM> propagates at a variety of angles towards the illumination side <NUM> of the specimen fixture <NUM>. The illumination light <NUM> illuminates the specimen <NUM> and the specimen plane <NUM> disposed adjacent the light pattern axis <NUM> and specimen <NUM>. The illumination light, or scattered derivatives thereof, then propagates to the objective <NUM> and then into the image sensor <NUM> which may be configured to generate an image signal for display on the display screen <NUM>.

The conduits <NUM> that interconnect the controller <NUM> with the illumination screen <NUM>, translation stage <NUM>, image sensor <NUM> and display screen <NUM> discussed below may include any suitable type of conduit configured to transmit energy, information or the like. As such, the conduits <NUM> may include conductive wires, including coaxial cables, fiber optic cables, wireless links or the like. Embodiments of the controller <NUM> may include one or more processors, including microprocessors <NUM>, memory including digital memory <NUM> and any circuitry or other components such as video cards or the like which may be desirable in order to interconnect and use these or other elements of the controller <NUM> and microscope imaging system embodiments <NUM> generally.

In some cases, the specimen fixture <NUM> may include a plurality of specimen receptacles <NUM>. In some instances, the specimen receptacles <NUM> may be disposed on specimen tray embodiments <NUM> which are, in turn, disposed on the specimen fixture <NUM>. The controller <NUM> may be configured to coordinate the transmission of an illumination signal to the illumination screen <NUM> which produces emission of the light pattern <NUM> from the flat illumination surface <NUM> which has a light pattern axis <NUM> that remains aligned with the image input axis <NUM> of the objective <NUM> as the objective <NUM> is translated in the x-y plane <NUM>. In some cases, such tracking of the light pattern axis <NUM> and image input axis <NUM> may occur as the objective <NUM> is being translated from a first position with the image input axis <NUM> aligned with a first specimen receptacle <NUM>' to a second position with the image input axis <NUM> aligned with a second specimen receptacle <NUM>" as shown in <FIG>. For some embodiments, the emission of illuminating light <NUM> may be reduced or eliminated during translation of the objective <NUM> from the first position <NUM>' to the second position <NUM>" and only activated when the objective <NUM> is disposed at either the first position <NUM>' or the second position <NUM>". For some embodiments, the controller <NUM> may be configured to align the image input axis <NUM> of the objective <NUM> with the light pattern axis <NUM> of the light pattern <NUM> by mapping positions of the image input axis <NUM> to corresponding positions on the flat illumination surface <NUM> of the illumination screen <NUM>. For some embodiments, the controller <NUM> may be configured to generate the illumination signal and coordinate the transmission of the illumination signal to the illumination screen.

In some instances, the microscope imaging system <NUM> may further include one or more position sensors <NUM> operatively coupled to the translation stage <NUM> and controller <NUM>. The position sensors <NUM> may include optical encoder strips and corresponding readers as shown or any other suitable type of position sensor <NUM>. The controller <NUM> may be configured to receive position information from the position sensor <NUM> regarding the position of the image input axis <NUM> of the objective <NUM> prior to coordinating transmission of the illumination signal to the illumination screen <NUM>. In some cases, the translation stage <NUM> may be translated by a plurality of servo motors (not shown), stepper motors (not shown) or any other suitable type of motor and the controller <NUM> may be configured to determine the position of the objective <NUM> and image input axis <NUM> by determining the position of each of the motors and optionally accessing a lookup table.

For some embodiments, the translation stage <NUM> may further include a carrier <NUM> which is configured to translate in the x-y plane <NUM> of the translation stage <NUM> with the objective <NUM> being secured in fixed relation to the carrier <NUM> of the translation stage <NUM>. In addition, a focus mechanism may be configured to adjust the position of the object plane <NUM> of the objective <NUM> along the image input axis <NUM> relative to the specimen plane <NUM>. In some cases, such a focus mechanism may include a z-axis actuator <NUM> which is operatively coupled between the carrier <NUM> and the objective <NUM> and may be configured to adjust the position of the object plane <NUM> of the objective <NUM> along the image input axis <NUM> relative to the specimen plane <NUM>. Such z-axis adjustment may also be useful in some instances for keeping illumination light <NUM> emitted from a light pattern <NUM> imaged onto a phase contrast ring <NUM> of a phase contrast objective <NUM> as discussed in more detail below. In particular, z-axis translation of the objective <NUM> relative to the specimen fixture <NUM> or specimen <NUM> disposed thereon may be useful in order to focus the objective onto the specimen <NUM> or portion of interest of the specimen <NUM>. However, such z-axis translation of the objective <NUM> relative to the specimen fixture <NUM> may also result in relative z-axis translation between the objective <NUM> and the illumination screen <NUM>. In some cases, such relative translation between the objective <NUM> and a light pattern <NUM> disposed on the flat illumination surface <NUM> of the illumination screen <NUM> may hinder imaging of the light pattern <NUM> onto the phase contrast ring <NUM> of a phase contrast embodiment of the objective <NUM>. In order to counter such hindering of the imaging of the light pattern <NUM>, the phase contrast ring <NUM> of the phase contrast embodiment of the objective <NUM> may be made wider in order to accommodate for movement or widening of the image of the light pattern <NUM> being imaged thereon. This approach, while useful, may also hinder the transmission of illumination light <NUM> through the objective <NUM> generally and reduce the brightness of the image. Another useful approach to accommodate what would otherwise be relative displacement between the objective <NUM> and the illumination screen <NUM> during focusing or any other z-axis translation of the objective <NUM>, may include translating the illumination screen <NUM> in the z-axis in concert with the z-axis translation of the objective <NUM>. For such an embodiment, one or more motors (not shown) could be operatively coupled between the frame <NUM> and the illumination screen <NUM> and configured to translate the illumination screen <NUM> by a z-axis displacement that matches z-axis displacement of the objective <NUM> by virtue of the z-axis actuator <NUM>. In addition, for some embodiments <NUM>, the illumination screen <NUM> and objective <NUM> may be configured to remain stationary relative to each other, and the specimen fixture <NUM> may be operatively coupled to the frame <NUM> with one or more motors (not shown) which may serve to translate the specimen fixture <NUM> in the z-axis along the image input axis <NUM> of the objective <NUM>. Such a configuration would allow focusing of the objective <NUM> on the specimen <NUM> disposed on the specimen fixture <NUM> without changing the relative separation between the flat illumination surface <NUM> of the illumination screen <NUM> and the objective <NUM>. In some cases, the z-axis actuator <NUM> may include a translation range of up to about <NUM> or more.

Some embodiments of the microscope imaging system <NUM> may also include an image sensor <NUM> operatively coupled to the objective <NUM> and with the controller <NUM> operatively coupled to the image sensor <NUM> by conduits <NUM>. In some cases, the image sensor <NUM> may include a camera, such as a complementary metal-oxide semiconductor (CMOS) camera or a charge-coupled device (CCD) camera. For some embodiments, an optional display screen <NUM> may be operatively coupled to the controller <NUM> and be configured to display an image captured by the image sensor <NUM>. The display screen <NUM> may be coupled to the controller <NUM> by one or more conduits <NUM>. In some cases, the image sensor <NUM> may be operatively coupled directly to the display screen <NUM> by one or more conduits <NUM> (not shown).

Some embodiments of the microscope imaging system <NUM> may include a rigid frame <NUM> which is rigidly coupled to a specimen fixture support <NUM>, the illumination screen <NUM> and translation stage <NUM>. The rigid frame <NUM> may also include a rigid base <NUM> which has a flat bottom surface and which is disposed at the bottom of the rigid frame <NUM>. The rigid frame <NUM> may be made from any suitable high strength material such as steel, aluminum, composite materials such as carbon fiber or the like. The rigid frame <NUM> may serve as a rigid scaffold in order to prevent relative movement between the specimen fixture <NUM>, the illumination screen <NUM>, and the translation stage <NUM> as the objective <NUM> is translated in the x-y plane <NUM> on a carrier <NUM> of the translation stage <NUM>. Although the rigid frame <NUM> is shown having a fairly open configuration, the rigid frame <NUM> could also be enclosed with adjoining removable flat thin panels covering the front, back and top of the outer perimeter of the rigid frame <NUM>. Although the illumination screen <NUM> is nominally secured in fixed relation to the specimen fixture <NUM>, in some cases it may be desirable for the illumination screen <NUM> to be releasably secured to the frame <NUM> in order to provide access to the specimen fixture in order to place or exchange specimens <NUM>, specimen trays <NUM> or the like during use. As such, in some cases it may be useful for the illumination screen <NUM> to be hinged relative to the frame <NUM> such that one or more sides of the illumination screen <NUM> can be pivoted clear of the specimen fixture <NUM> and then easily be returned to its original position parallel to the specimen plane <NUM> by setting it back down.

Embodiments of the specimen fixture <NUM> may include one or more central apertures <NUM> configured to permit unobstructed imaging of one or more specimen trays <NUM> disposed thereon while still providing stable support for the specimen trays <NUM>. Although the apertures <NUM> may be described as central apertures <NUM>, such apertures may be disposed at any suitable location on embodiments of the specimen fixture <NUM> in order to facilitate imaging of specimens <NUM> disposed thereon. The specimen tray embodiments <NUM> may each include one or more specimen receptacles <NUM> which may be configured to hold specimen <NUM> in a position suitable for microscopic imaging thereof. For example, embodiments of the specimen fixture <NUM> may be configured to position a microscope slide and optional accompanying cover (not shown). In addition, some specimen tray embodiments <NUM> include a plurality of specimen receptacles <NUM> that may be arranged in rows and columns of an array of specimen receptacles <NUM>. In some cases, the specimen tray embodiments <NUM> may include about <NUM> specimen receptacles <NUM> to about <NUM> specimen receptacles <NUM>. Common configurations of specimen trays <NUM> including microtiter trays or plates and the like may include <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM> specimen receptacles <NUM> in the form of specimen wells.

The specimen fixture support <NUM> which is secured in fixed relation to the frame <NUM> and which provides mechanical support for the specimen fixture <NUM> may also include a central aperture <NUM> to allow unobstructed imaging of the specimen trays <NUM> disposed on the specimen fixture <NUM>. Both the specimen fixture support <NUM> and specimen fixture <NUM> may include positioning pins <NUM> that may be secured to respective illumination surfaces of the specimen fixture support <NUM> and specimen fixture <NUM>. The positioning pins may be configured to laterally secure the specimen trays <NUM> to the specimen fixture <NUM> and to laterally secure the specimen fixture <NUM> to the specimen fixture support <NUM>. As such, the positioning pins <NUM> or any other suitable structure may be configured to define a perimeter that closely fits to an outer lateral surface of the specimen trays <NUM> or specimen fixture <NUM>.

For some embodiments, the illumination screen <NUM>, as shown in <FIG>, may include a pixelated display screen such as a liquid crystal display (LCD) screen, a plasma display screen or the like. In some instances, the flat illumination surface <NUM> may be configured to emit illumination light <NUM> from a pixelated structure including about <NUM> to about <NUM> pixels per inch. In some cases, the illumination screen <NUM> may have a brightness of about <NUM> candelas per square meter to about <NUM> candelas per square meter. In addition, the illumination screen <NUM> may be configured to generate a light pattern <NUM> with illumination light <NUM> having an adjustable wavelength of about <NUM> to about <NUM>. Any of the illumination screen embodiments <NUM> discussed herein may also use any suitable array of alternative light sources such as light emitting diodes (LEDs), vertical cavity surface emitting lasers (VCSELs) or the like.

For some illumination screen embodiments <NUM>, such as an illumination screen embodiment <NUM> utilizing LCD display technology, the illumination light <NUM> emitted therefrom may be polarized or partially polarized. For some applications, the emission of polarized or partially polarized illumination light <NUM> may have certain benefits, but in other applications, such polarized illumination light <NUM> may be detrimental to the process. As such, in some cases, it may be desirable to include an optional element such as an optical layer <NUM> disposed over the flat illumination surface of the illumination screen <NUM>, as shown <FIG>, that may be configured to reduce or eliminate the polarization of illumination light emitted from certain illumination screen embodiments <NUM>. Embodiments of such optical layers <NUM> may include a waveplate or retarder type optical layer <NUM> that serves to reduce or eliminate the polarization of illumination light embodiments <NUM> emitted from the flat illumination surface <NUM> of the illumination screen <NUM>. Such a waveplate or retarder layer may be used to modify the polarization state of the illumination light <NUM> without reducing the intensity or direction of the illumination light <NUM>. Some such optical layer embodiments <NUM> may serve to rotate linear polarization or transform linear polarization into circular polarization and may include birefringent, crystalline or polymer materials that create a phase shift between polarization components. Some specific examples of such optical layers may include <NUM>/<NUM>-waveplates, <NUM>/<NUM> waveplates as well as zero order and achromatic waveplates or retarders. In some cases, such an optical layer may be disposed at any position between the sample <NUM> and the illumination screen <NUM>. For the embodiment shown in <FIG>, the optical layer is disposed on the outer surface of the flat illumination surface <NUM> of the illumination screen <NUM>.

Some embodiments of a method of microscopic imaging while using microscope imaging system embodiments such as those discussed herein may include aligning the image input axis <NUM> of the objective <NUM> at a first position in the specimen plane <NUM> of the specimen fixture <NUM> and transmitting a first illumination signal to the illumination screen <NUM>. The illumination screen <NUM> may include the flat illumination surface <NUM> that faces the illumination side <NUM> of the specimen fixture <NUM>. Illumination light <NUM> is then emitted from the light pattern <NUM> of the flat illumination surface <NUM> of the illumination screen <NUM> which corresponds to the illumination signal transmitted. The light pattern <NUM> generated by the first illumination signal may be positioned to have a light pattern axis <NUM> that is aligned with the image input axis <NUM> of an objective <NUM>. The method may further include imaging the specimen plane <NUM> at the first position <NUM>' (see <FIG>) with the objective <NUM>. Thereafter, the objective <NUM> may be translated in the x-y plane <NUM> which is parallel with the flat illumination surface <NUM>. The objective <NUM> may be so translated without moving the illumination screen <NUM> relative to the specimen fixture <NUM> such that the image input axis <NUM> of the objective <NUM> is translated from alignment with the first position <NUM>' in the specimen plane <NUM> to alignment with a second position <NUM>" in the specimen plane <NUM>. A second illumination signal may then be transmitted to the illumination screen <NUM> and corresponding illumination light <NUM> emitted from the light pattern <NUM> of the flat illumination surface <NUM> of the illumination screen <NUM> which has a light pattern axis <NUM> that is aligned with the image input axis <NUM> of the objective <NUM> at the second position <NUM>". The method may also include imaging the specimen plane <NUM> at the second position with the objective <NUM>.

In some instances, such a method may further include emitting illumination light <NUM> from the light pattern <NUM> and maintaining the alignment of the light pattern axis <NUM> with the image input axis <NUM> of the objective <NUM> as the objective <NUM> is being translated in the x-y plane <NUM> from the first position <NUM>' to the second position <NUM>" without moving the illumination screen <NUM> relative to the specimen fixture <NUM> such that translation of the light pattern axis <NUM> tracks the translation of the image input axis <NUM> of the objective <NUM> by virtue of a dynamic illumination signal that adjusts a position of the light pattern <NUM> to correspond to an x-y position of the objective <NUM>. In some instances, such a method may further include imaging the specimen <NUM> or specimen receptacle <NUM> at the first position <NUM>' in the specimen plane <NUM> and imaging the specimen <NUM> or specimen receptacle <NUM> at the second position <NUM>" in the specimen plane <NUM>.

In <FIG>, the objective lens <NUM> of a microscope imaging system <NUM> is shown beneath and pointed towards a specimen plane or stage <NUM> where the object to be imaged or specimen <NUM> is located. Illumination screen <NUM> is shown with emitted illumination <NUM> from an annular illumination pattern <NUM> facing the specimen <NUM> and microscope objective lens <NUM>. Specimen <NUM> is an object of interest to be imaged. A light pattern or shape <NUM> of an illumination object or zone is being emitted from a flat illumination surface <NUM> of the illumination screen <NUM> upon the specimen <NUM>. In the side view cutaway view of <FIG>, the inverted microscope objective <NUM> may be translated in the x-y plane <NUM> and in along a z-axis beneath the specimen plane <NUM> which is trans-illuminated from above by the illumination light pattern <NUM>. Embodiments discussed herein may include the use of a flat illumination surface <NUM> of a standard computer monitor or TV display such as the illumination screen <NUM> as a method of displaying illumination patterns <NUM> and emitting corresponding illumination light patterns <NUM> for increased contrast used for microscopy and the like.

Referring to <FIG>, the objective <NUM> is shown moving relative to the specimen plane <NUM> by the arrow <NUM> in <FIG>. The illumination light pattern <NUM> is also shown moving across the flat illumination surface <NUM> of the illumination screen <NUM> in corresponding displacement to remain in alignment with the objective <NUM>. <FIG> shows a specimen <NUM> being tracked by keeping it in the center of the field of view of the objective <NUM> automatically, and the illumination light pattern <NUM> may be maintained in alignment with the corresponding objective <NUM> as indicated by arrows <NUM>. <FIG> illustrates an illumination light pattern <NUM> that may change position over time while the objective <NUM> images the specimen plane <NUM>. In some instances, this technique may yield information on a <NUM>-dimensional shape of the specimen <NUM>. In some cases, the illumination light pattern <NUM> may include a regularly repeating intensity variation <NUM> of a light pattern <NUM> in one dimension both stationary and sweeping across the specimen plane <NUM> as shown in <FIG>. In some cases, the illumination light pattern <NUM> may include a regularly repeating intensity variation in multiple dimensions both stationary and sweeping across the specimen plane <NUM> as shown in <FIG>.

Some of the microscope imaging system embodiments <NUM>, <NUM>' (discussed below) herein may be directed generally to transmitted and oblique-illumination sources for microscope imaging or any other suitable form of imaging. In some cases, for a particular specimen <NUM>, whether using a contrast enhancement technique or not, the quality of images generated from the objective <NUM> and image sensor <NUM> may vary depending upon changes in the direction from which illumination <NUM> arrives, and from changes in the pattern of the illumination light <NUM>. By using a display screen <NUM> it may be possible to emit illuminating light <NUM> from a range of directions and in a variety of patterns. This makes possible manual or automated searches for optimal images by altering the direction and pattern of the illuminating light <NUM> and analyzing the images using software running on a computer such as processor <NUM>.

Some microscope imaging system embodiments <NUM>, <NUM>' discussed herein may be directed to methods and devices for the use of a TV or monitor and its graphical display as a flexible illumination screen <NUM> with positional, multicolored, patterned, and dynamic display as bright field or transmitted illumination for enhanced contrast. The large format of display screen embodiment <NUM> including projector embodiments may allow the positions of multiple specimens <NUM> to be illuminated by emitting illuminating light <NUM> from certain portions of a flat illumination screen <NUM> of a display screen <NUM> without physical movement or replication of an overhead light fixture. The use of a planar illumination screen <NUM> for an illumination source allows a variety of shapes and patterns on such a screen to be optimized for the transmitted light pattern <NUM>. The addition of an optional lens array or shade array (not shown) to manipulate the displayed light patterns <NUM> and illumination source into desired optical properties is contemplated herein as well.

Some microscope imaging system and method embodiments <NUM>, <NUM>' discussed herein may also be directed to methods and devices for the use of a projector and its associated projected graphics and light patterns <NUM> as a flexible illumination source with positional, multicolored, patterned, and dynamic display as bright field or transmitted illumination for enhanced contrast. Embodiments discussed herein are also directed to methods and devices for the automatic optimization of image quality based on feedback from a live image, such as may be generated by the image sensor <NUM>.

By using a sufficiently large display screen <NUM> positioned so its output light <NUM> reaches a specimen <NUM> and then enters the objective lens <NUM> of a microscope, it is possible to cause an image to appear on the flat illumination surface <NUM> of the illumination screen <NUM> such that if the displayed light pattern <NUM> is of appropriate shape, color, and intensity, that image can serve as a light source for the microscope. In the case of phase contrast microscopy, as discussed above, it is possible to cause to appear on the display screen <NUM> an annulus of particular angular shape with reference to the center of the object field or image input axis <NUM> of a phase contrast objective lens, such that a phase contrast image is generated.

A light pattern <NUM> having an annular configuration displayed may be manually or automatically aligned with a corresponding image input axis <NUM> of the objective lens <NUM>. In the case of automatic alignment, software running on a computer that receives images from the image sensor <NUM> in the microscope imaging system <NUM>, <NUM>' may execute an algorithm that calculates the optimum position and shape of the light pattern annulus <NUM> displayed by analyzing the images and altering the annulus configuration.

Some microscope imaging system embodiments <NUM>, <NUM>' discussed here may greatly simplify a hardware configuration as a single illumination screen <NUM> may be used as the light source hardware. No lenses or masks need to be used in many cases. Embodiments may also include the use of a sufficiently large illumination screen <NUM> such that it may be possible to position the output light <NUM> as needed for imaging a large area. This may make it possible to use a simpler design of an imaging system <NUM>, <NUM>' wherein the specimen(s) <NUM> remain stationary and the imaging optics such as the objective <NUM> move to scan an area. Using software control, only the patterns of illuminating light <NUM> emitted from the illumination screen <NUM> need to be moved or otherwise translated. The illumination screen <NUM> itself may be stationary, so there are no physical parts that need be moved to maintain illumination alignment with the imaging optics <NUM>. The display may include a low profile, i.e., small height, making this highly advantageous in live cell imaging in incubators. Because the illumination screen <NUM> may be homogenous in light produced, variability across labware may be less in some cases. In some instances, for the microscope imaging system embodiments <NUM>, <NUM>' discussed herein, a vertical separation distance between the flat illumination surface <NUM> of the illumination screen <NUM> and the specimen plane <NUM> of the specimen fixture <NUM> may be about <NUM> to about <NUM>.

Some microscope imaging system embodiments <NUM>, <NUM>' discussed herein may include automation of alignment of the light pattern axis <NUM> of the output light pattern(s) with the image input axis <NUM> of the objective lens(es) <NUM>. The output light <NUM> may also be optimized in some cases to provide a means of discovering new light patterns <NUM> which produce contrast. Arbitrary and variable shapes and colors of light may be used in a manual or automated search for the illumination condition that provides valuable images. Light patterns <NUM> may include bullseyes, checkerboards, half-moons, pinpoints, boxes, crosshairs, circles, squares, polygons, freehand lasso, pie chart rays and the like. Some embodiments <NUM>, <NUM>' discussed herein may also include the display of a phase annulus <NUM> and its size, position and thickness may be adjusted and optimized, including dynamically.

Some microscope imaging system embodiments <NUM>, <NUM>' discussed herein may also include single or multiple images acquired with structured light patterns <NUM> that may be used in combination with mathematical image analysis techniques to extract information about specimens <NUM>. This may include the ability to orbit, raster, or spiral a small symmetrical spot of light around the field of view (FOV) while sequential images may be taken for enhanced <NUM>-D imaging, DOF imaging, ptychography imaging and the like. Embodiments <NUM>, <NUM>' discussed herein may also include using the color of the display to optimize an imaging experiment. Using green to maximize detection by image sensor <NUM> and red to reduce the energy of the illumination, for example.

In some cases, embodiments <NUM>, <NUM>' discussed herein may also include the ability to reduce the energy of the illumination light <NUM> by pulsing the illumination light <NUM> at the frame rate or some offset. Sample lensing or general matrix effects may also be compensated for in some cases in order to flatten the image brightness by displaying or projecting a compensatory image light pattern <NUM>. In some instances, illumination conditions may be stored in memory <NUM> and retrieved by the processor <NUM> to reduce the effort needed to acquire new images. Embodiments <NUM>, <NUM>' discussed herein may also include the ability to function over a range of distances from the objective lens <NUM> because the light patterns <NUM> may be scaled to control their angular size. This makes some embodiments useful for both robotic applications that require large operating clearance and incubator applications that require a compact system <NUM>, <NUM>'. The illumination screen <NUM> may also be used in some instances to provide an overhead shroud for fluorescence microscopy; shielding room light, protecting people's eyes from UV, etc. Embodiments discussed herein may also include shade arrays with and without lenses- honeycomb dimensional grid for limiting projection angles used in conjunction with a lens. Embodiments discussed herein may also include a variety of lenses, including ball lenses, concave lenses, convex lenses, Fresnel lenses, polarized lenses, reducing lenses, expanding lenses, directional lenses, and the like.

In some cases, an illumination light pattern <NUM> from an illumination screen may be used to effect biological and chemical mechanisms- photocaged compounds, photolysis, optogenetics, and optical chemical synthesis. Embodiments discussed herein may also include using the illumination light pattern <NUM> as an attractant or deterrent to a biological response.

Some embodiments of the illumination screen <NUM> may include an LCD, CRT, TFT, retinal display, DLP, LED, OLED type illumination screen <NUM> or the like. The illumination screen <NUM> may be of sufficient dimensions such that it emits light of one or more colors positioned so that the emitted light enters the objective lens <NUM>. For such embodiments, the displayed illumination light <NUM> may be adjusted, including adjustment of the color of the illumination light <NUM>, adjustment of the position of the light pattern <NUM> on the flat illumination screen <NUM>, and adjustment of the time dependence of the illumination light <NUM>. For some embodiments <NUM>, <NUM>' a light pattern image <NUM> may be used in feedback to optimize the illumination including automatically. One exemplary light pattern <NUM> may include a single circle of light centered above the objective <NUM> for "aperture illumination" with the center of the circle of light pattern <NUM> or light pattern axis <NUM> being aligned with the image input axis <NUM> of the objective <NUM>. As discussed in more detail below, a plurality of microscope objectives <NUM> may be illuminated sequentially or simultaneously. In addition, many positions of a single microscope objective <NUM> may be illuminated sequentially or simultaneously. In some cases, wherein a plurality of microscope objectives <NUM> which are too close together may have individual illumination sources that include respective overlapping illumination light patterns <NUM> projected sequentially. Embodiments of the electronic image sensor <NUM> may be used for forming a system of sufficient sensitivity that permits transmission of the images to a computer including the controller <NUM> or components of a computer such a the microprocessor <NUM>. Software running on the computer or microprocessor <NUM> of the controller <NUM> may be configured to control the illumination screen <NUM> and access the images from the same image sensor <NUM>. Embodiments of the microscope imaging system <NUM>, <NUM>' may include an objective lens <NUM> of a type designed to generate any of bright field images, dark field images, phase contrast images, or other enhanced images.

In a phase contrast system generally, the image of an annulus of light is projected at optical infinity by passing the light <NUM> through a condenser lens (not shown). This light illuminates the specimen <NUM>. The fraction of this light <NUM> that passes through the specimen <NUM> without deviation enters a phase contrast type objective <NUM>. Such objectives are designed to focus this light <NUM> onto an internal phase ring. This may be useful in order to achieve the phase contrast effect. If an annular light source is used to illuminate a specimen <NUM> without a special phase contrast objective lens then its image may not projected at infinity and the undeviated light will not be focused on the phase ring inside the objective. In some cases, this may degrade the phase contrast effect.

The phase contrast effect may generally be improved or restored by designing a special phase contrast objective lens <NUM> which is configured to focus an image of the annulus of light onto the internal phase ring of a phase contrast objective for the case of an annular light source that is positioned at some known distance from the objective, without an interposed lens as in the standard phase contrast system.

<FIG> is a schematic representation of an objective <NUM> that is configured as a typical phase contrast type objective including a phase ring <NUM> disposed within the objective optical train. The phase ring <NUM> is represented by two schematic cross section segments. The objective is disposed below a specimen <NUM> which is being illuminated by an annular light pattern <NUM> represented by two schematic cross section segments of the annular light pattern <NUM>. Generally speaking, such a phase contrast microscopy configuration would include a condenser lens (not shown) aligned with the optical axis of the system and disposed between the annular light pattern <NUM> and the specimen <NUM>. Such a condenser lens would typically be configured to correct the angle of the illumination light <NUM> emitted from the light pattern <NUM> so as to effectively present the light pattern <NUM> at a distance of infinity from the objective <NUM> and specimen <NUM>. For such an arrangement, the light pattern <NUM> would be imaged by the objective lens <NUM> onto the phase ring <NUM> disposed within the objective so as to yield a high degree of phase contrast for the image generated by the phase contrast objective <NUM>. However, such a condenser lens may be inconvenient to include and position in such a system, particularly such a system wherein it may be desirable to translate the objective <NUM> relative to the specimen <NUM>. Without such a condenser lens, the light pattern <NUM> is not properly imaged onto the phase ring <NUM> as indicated by the displacement of the point of intersection <NUM> of the rays of illumination light <NUM> being positioned below the phase ring <NUM> within the objective <NUM>. Such an arrangement may not be effective to produce a useful amount of phase contrast during use.

In order to correct this effect, as discussed above, a special phase contrast objective <NUM> may be used that includes an objective lens <NUM>' (and any other associated optics required to achieve the desired result) that is configured to image the light pattern <NUM> onto the phase ring <NUM> without the use of a condenser lens and with a light pattern <NUM> that is not effectively positioned at a distance of infinity from the objective <NUM> and specimen <NUM>. <FIG> shows a schematic representation of an objective <NUM> disposed below a specimen <NUM> which is being illuminated by an annular light pattern <NUM> similar to the arrangement of <FIG>. However, the objective lens <NUM>' of the system of <FIG> has been modified so as to image the light pattern <NUM> onto the phase ring <NUM> even without the use of a condenser lens such as is typically used in a phase contrast system light source. As can be seen in <FIG>, the point of intersection <NUM> of the rays of illuminating light <NUM> are coplanar with the phase ring <NUM> of the objective <NUM> and thus an proper amount of phase contrast will be generated by the system even without the use of a condenser lens in the system. Such a specially configured objective <NUM> and corresponding objective lens <NUM>' (including any other associated optics required to achieve the desired result) may be used for any of the microscope imaging systems or methods discussed herein.

As such, some embodiments of a microscope imaging system <NUM> may include the specimen fixture <NUM> having the illumination side <NUM>, the imaging side <NUM> and specimen receptacle <NUM> which is disposed in the specimen plane <NUM> of the specimen fixture <NUM>. The illumination screen <NUM> may be disposed in fixed relation to the specimen fixture <NUM> facing the illumination side <NUM> of the specimen fixture <NUM>, and include a flat illumination surface <NUM> that is configured to emit an annular light pattern <NUM> having a light pattern axis <NUM>. The microscope imaging system <NUM> may also include a phase contrast objective <NUM> which faces the imaging side <NUM> of the specimen fixture <NUM>, which includes the image input axis <NUM> that is aligned with the light pattern axis <NUM>, and which is configured to form an image of an annular light pattern <NUM> of the illumination screen onto a phase ring <NUM> of the phase contrast objective <NUM> without the use of a condenser lens (not shown) disposed between the illumination screen <NUM> and the specimen plane <NUM> and with the annular light pattern <NUM> of the illumination screen <NUM> not being effectively disposed at infinity with respect to the phase contrast objective <NUM>.

In some instances, the microscope imaging system <NUM> may further include the translation stage <NUM> which is disposed in fixed relation to the specimen fixture <NUM> and which faces the imaging side <NUM> of the specimen fixture <NUM>. In addition, the phase contrast objective <NUM> may be operatively coupled to the translation stage <NUM> and be laterally translatable in the x-y plane <NUM>, the x-y plane <NUM> being substantially parallel to the specimen plane <NUM> in some cases. Such a microscope imaging system embodiment <NUM> may further include the controller <NUM> which is operatively coupled to the illumination screen <NUM> and the translation stage <NUM> and be configured to coordinate the transmission of an illumination signal to the illumination screen <NUM> which produces emission of the annular light pattern <NUM> from the flat illumination surface <NUM>. The annular light pattern <NUM> may include a light pattern axis <NUM> that remains aligned with the specimen receptacle <NUM> and the image input axis <NUM> of the phase contrast objective <NUM> as the phase contrast objective <NUM> is translated in the x-y plane <NUM> from a first position to a second position. For some embodiments, the controller <NUM> may be configured to align the image input axis <NUM> of the phase contrast objective <NUM> with the light pattern axis <NUM> by mapping positions of the image input axis <NUM> to corresponding positions on the flat illumination surface <NUM> of the illumination screen <NUM>. In some cases, the translation stage <NUM> further includes a carrier <NUM> which may be configured to translate in the x-y plane <NUM> and the phase contrast objective <NUM> may be secured in fixed relation to the carrier <NUM> of the translation stage <NUM>. In some instances, a focus mechanism may be configured to adjust the position of an object plane of the phase contrast objective <NUM> along the image input axis <NUM> relative to the specimen plane <NUM>. For such embodiments, the focus mechanism may include the z-axis actuator <NUM> which may be operatively coupled between the carrier <NUM> and the phase contrast objective <NUM> and may be configured to adjust the position of the object plane of the phase contrast objective <NUM> along the image input axis <NUM> relative to the specimen plane <NUM>. In some cases, such a system may include a plurality of phase contrast objectives <NUM>.

To that end, any of the microscope imaging system and method embodiments discussed herein may include a plurality of objectives <NUM> in order to provide alternative contrast enhancement capabilities or for any other suitable purpose. Referring to <FIG>, some microscope imaging system embodiments <NUM>' may include a plurality of objectives <NUM> which are operatively coupled to the translation stage <NUM> on the imaging side <NUM> of the specimen fixture <NUM>, which are laterally translatable in the x-y plane <NUM> that is substantially parallel to the specimen plane <NUM>, which each include an image input axis <NUM> disposed towards the imaging side <NUM> of the specimen fixture <NUM> and which each include an imaging plane or object plane <NUM> which is substantially perpendicular to the image input axis <NUM> and that may be adjusted along a z-axis of the objective <NUM> to be coplanar with the specimen plane <NUM>. An image sensor <NUM> operatively coupled to each of the respective objectives <NUM> with the plurality of objectives <NUM>. In some cases, the plurality of objectives <NUM> may be secured in fixed relation relative to each other in the x-y plane <NUM>. In some instances, the plurality of objectives <NUM> may be each translatable in the x-y plane <NUM> independent of each other.

Referring to <FIG>, in some cases, a plurality of objectives <NUM> may be used to image a plurality of corresponding specimens <NUM> using a plurality of illumination light patterns <NUM> aligned with each respective microscope objective <NUM>. For some embodiments, sequential illumination may be included in order to avoid interference from adjacent patterns of illumination <NUM>. Specifically in the case of overlapping phase annulus <NUM> wherein the spacing of the objectives <NUM> may be less than the diameter of the annulus <NUM>. In some instances, the microscope objectives <NUM> may be of different types, and the illumination light patterns <NUM>, <NUM>', <NUM>" and <NUM>‴ shown in <FIG> may be different and appropriate for each respective objective <NUM>. Illumination light patterns <NUM>, <NUM>', <NUM>" and <NUM>‴ may be any shape from a pinpoint to an infinite plane which may include the entire flat illumination surface <NUM> of the illumination screen <NUM>. In some cases, a plurality of objectives <NUM> may be moved relative to the specimen plane <NUM> and the corresponding illumination light patterns <NUM> remain aligned with the image input axes <NUM> of the respective objectives <NUM> as shown in <FIG>.

Embodiments illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising," "consisting essentially of," and "consisting of' may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation and use of such terms and expressions do not exclude any equivalents of the features shown and described or portions thereof, and various modifications are possible. The term "a" or "an" can refer to one of or a plurality of the elements it modifies (e.g., "a reagent" can mean one or more reagents) unless it is contextually clear either one of the elements or more than one of the elements is described. Thus, it should be understood that although embodiments have been specifically disclosed by representative embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and such modifications and variations are considered within the scope of this disclosure.

Claim 1:
A microscope imaging system (<NUM>), comprising:
a specimen fixture (<NUM>) comprising an illumination side (<NUM>), an imaging side (<NUM>) and at least one specimen receptacle (<NUM>) which is disposed in a specimen plane (<NUM>) of the specimen fixture (<NUM>);
a translation stage (<NUM>) which is disposed in fixed relation to the specimen fixture (<NUM>) and which faces the imaging side (<NUM>) of the specimen fixture (<NUM>);
an objective (<NUM>) which is operatively coupled to the translation stage (<NUM>), which is laterally translatable in an x-y plane (<NUM>) that is substantially parallel to the specimen plane (<NUM>), which includes an image input axis (<NUM>) disposed towards the imaging side (<NUM>) of the specimen fixture (<NUM>) and which includes a object plane (<NUM>) which is substantially perpendicular to the image input axis (<NUM>) and adjustable to be coplanar with the specimen plane (<NUM>);
an illumination screen (<NUM>) disposed in fixed relation to the specimen fixture (<NUM>), the illumination screen (<NUM>) including an array of light emitting pixels (<NUM>) and a flat illumination surface (<NUM>) which is substantially parallel to the specimen plane (<NUM>) and which faces the illumination side (<NUM>) of the specimen fixture (<NUM>); and
a controller (<NUM>) which is operatively coupled to the illumination screen (<NUM>) and the translation stage (<NUM>) and which is configured to coordinate transmission of an illumination signal to the illumination screen (<NUM>) which produces emission of a light pattern (<NUM>) from the flat illumination surface (<NUM>) which has a light pattern axis (<NUM>), wherein the light pattern axis (<NUM>) comprises an axis of symmetry of the light pattern (<NUM>), that remains aligned with the image input axis (<NUM>) of the objective (<NUM>) as the objective (<NUM>) is translated in the x-y plane (<NUM>) from a first position to a second position.