Patent Application
20060022114
The present invention generally relates to the field of microscopy. In particular, the present invention is directed to a system and method for referencing a position of an objective lens.
In microscopy referencing the image plane of an objective lens to a desired plane with respect to the sample can be difficult. This difficulty may be due to variations over time in the distance between the objective lens and the sample holder. Causes of such variations include changes to the sample stage, variations in sample slides, changes in position of the objective lens, thermo mechanical fluctuations, and any combinations thereof. Knowing the position of the objective lens with respect to a sample holder is valuable for focusing the objective lens.
Typical focusing methods are derived from image analysis through human or software interpretation. In traditional light microscopy, the user manually adjusts the mechanical focus mechanism while simultaneously looking into an eyepiece for an image to appear. As this image comes into focus, the user fine tunes the focus to obtain the clearest image. This is the most rudimentary form of feedback. As camera sensitivity has improved, imaging lower light levels has become possible, for example in fluorescence microscopy single fluorophore imaging is now attainable.
With these high sensitivity imaging systems, there are two basic methods of focusing. A first method requires a user to adjust the focus manually while viewing the image on a video monitor. Although this method works, it requires skill and experience, particularly when initially finding the focus point. In high magnification systems the depth of field is small so that maintaining the focus is difficult given that any “X-Y” movement of the stage will usually require refocusing given that there will be slight movement of the sample in the “Z” axis. In addition, stage drift due to thermal fluctuations is another common source of mechanical drift.
Another method requires a user to employ digital image based software to drive an auto focusing mechanism. Image analysis is used to determine focus by measuring contrast of an acquired digital image. This requires that the system have a motorized focus driven from a computer that simultaneously analyzes the image. To do this, an image from the camera is captured by the computer and analyzed. This provides a control signal that drives the motorized focusing system in a repeating cycle until the proper focus is obtained. This works well to initially get a sample focused. At this point any refocusing must be initiated by a command. While it is refocusing, the camera is dedicated to the focusing process, itself. Unfortunately, this process is slow and precludes image acquisition during the automated focusing.
A reliable way to reference an objective lens image plane to a sample without using an image of the sample in the image plane is needed.
In one embodiment, the present invention provides a system for referencing an objective lens to a desired plane of focus. The system includes an objective lens having a center axis. The system also includes an energy source for directing a first energy of a first wavelength so as to intercept the objective lens at a first location that is radially spaced at a second distance from the center axis. The first energy is directed by the objective lens to a first surface of a dichroic element positioned a predetermined first distance from the desired plane of focus. The first energy then reflects off the first surface and transmits through the objective lens at a second location that is different from the first location at a third distance from the center axis. The system also includes a detector operatively configured to detect the first energy after being transmitted through the objective lens at the second location, the detector providing information relating to the second distance relative to the first distance.
In another embodiment, the present invention provides a system for referencing an objective lens to a desired plane of focus. The system includes an objective lens having a center axis. The system also includes a dichroic element positioned a predetermined first distance from the desired plane of focus. Further, the system includes an energy source for directing a first energy of a first wavelength so as to intercept the objective lens at a first location that is radially spaced at a second distance from the center axis, the first energy being directed by the objective lens to a first surface of the dichroic element, the first energy then reflecting off the first surface and transmitting through the objective lens at a second location that is different from the first location at a third distance from the center axis. Still further, the system includes a detector operatively configured to detect the first energy after being transmitted through the objective lens at the second location, the detector providing information relating to the second distance relative to the first distance.
In yet another embodiment, the present invention provides a method of focusing an imaging device. The method includes (a) providing a dichroic element having a first side and a second side; (b) providing an objective lens having a center axis, a first side, and a second side spaced from the first side, wherein the first side of the objective lens confronts the first side of the dichroic element; (c) passing a first energy through the objective lens at a first distance from the center axis such that the first energy reflects off of the dichroic element and back through the objective lens at a second distance from the center axis; (d) detecting a relationship between the first distance and the second distance; and (e) using the relationship to focus the objective lens.
In still yet another embodiment, the present invention provides a system for referencing an objective lens to a desired plane of focus. The system includes an objective lens having a center axis. The system also includes a dichroic element positioned a predetermined first distance from the desired plane of focus. Further, the system includes an energy source for directing a first energy of a first wavelength so as to intercept the objective lens at a first location that is radially spaced at a second distance from the center axis, the first energy being directed by the objective lens to a first surface of the dichroic element. Still further, the system includes a detector operatively configured to detect a relative position of the first energy that is reflected from the first surface as a second distance between the objective lens and the dichroic element is varied and provide information related to the relative position for referencing the objective lens to the desired plane of focus.
For the purpose of illustrating the invention, the drawings show a form of the invention that is presently preferred. However, it should be understood that the present invention is not limited to the precise arrangements and instrumentalities shown in the drawings, wherein:
The present invention provides a system and method for referencing an objective lens to a desired plane of focus. In one embodiment, the system includes a source of energy for providing an energy to an objective lens. The energy passes through the objective lens and is reflected off of a dichroic element that is positioned at a known distance from a desired plane of focus. The reflected energy is detected by a detector element. When the objective lens is at a distance from the dichroic element that would focus the objective lens at the plane of the dichroic element (that is, the image plane of the objective lens is positioned at the plane of the dichroic element), the reflected energy will be incident at the detector element at a first location. Using measured deviations from this first location (or another known location representing another condition of the objective lens) and the known distance between the dichroic element and the desired plane of focus, the objective lens can be referenced to the desired plane of focus. This reference can allow the objective lens to be focused on the desired plane of focus either manually or automatically.
Knowing where on detector 170 that energy 125 will be incident at focus to first surface 135 (or some other condition other than focus) can be combined with known distance 145 to reference objective lens 110 to desired plane of focus 150 (e.g. for focusing objective lens 110 at desired plane of focus 150). When objective lens 110 is in focus to first surface 135, first distance 130 and second distance 160 are the same. The relationship of first distance 130 and second distance 160 can be indicated by the position of incidence of energy 125 at detector 170. The location of position 165 on detector 170 is related to the relative position of objective lens 110 with respect to dichroic element 140 and, thus, is related to second distance 160 relative to first distance 130.
A variety of objective lenses are known and are used in a number of imaging applications, any of which may be used as objective lens 110. Example imaging applications include, but are not limited to, simple brightfield microscopy, Epi—fluorescence microscopy, total internal reflection (TIR) microscopy, confocal microscopy, a water immersion objective application, and darkfield microscopy. Numerical aperture (NA) of an objective lens can vary and may depend on the particular imaging application. For example, in TIR microscopy, high NA (e.g., 1.35 and above) objective lenses provide an ability to deliver an excitation beam to a glass-sample interface at the required critical angle. In one example, an objective lens, such as objective lens 110, may include a single objective lens. In another example, an objective lens, such as objective lens 110, may include a plurality of objective lenses configured to work together. The image plane of an objective lens is a fixed distance from the objective lens. The fixed distance depends on the characteristics of the particular objective lens. A desired plane of focus is where the image plane must be to image entities within the desired plane of interest.
A dichroic element, such as dichroic element 140, may include any material that reflects an energy of a first wavelength and allows to be transmitted therethrough an energy of a second wavelength. Example materials for a dichroic element include, but are not limited to, beryllium oxide, magnesium oxide, magnesium fluoride, and any combinations thereof. It is possible to use the energy of the first wavelength to reference the position of the objective lens according to the present invention and to use the energy of the second wavelength as an imaging energy for imaging a sample. In another example, a sample slide may be made up of (in whole or in part) a dichroic material. In yet another example, a sample slide may include a dichroic element on a side of the slide proximate a sample to be imaged. In still yet another example, a sample slide may include a dichroic element on a side of the slide opposite a sample side and closest to an objective lens. A dichroic element that is on a side of a sample slide may be a layered material. In another example, the dichroic element may be a thin film on a side of a sample slide. In a further example, a dichroic element may be any surface mechanically reference to a sample. In still a further example, a dichroic element may be a sample surface of an imaging device. In an additional example, a vacuum coated mineral on glass creates a mirror like lens which reflects certain wavelengths of light while transmitting other wavelengths of light.
An energy source, such as energy source 120, may include any energy source that generates an energy (electromagnetic radiation) that can reflect from a surface of a dichroic element according to the present invention. Example energy sources include, but are not limited to, a laser, an arc lamp, a tungsten lamp, a halogen lamp, a light emitting diode, a fluorescent source, other light sources, and any combinations thereof. Example energy used for referencing an objective lens, such as objective lens 110, to a desired plane of focus, such as desired plane of focus 150, includes, but is not limited to, visible, infrared, ultraviolet, near infared and any combinations thereof. In one example, the energy is collimated upon entering the objective lens. In another example, the energy is light (e.g., laser light). In another example, an energy source may provide energy that includes energy of a first wavelength that will reflect from a chosen dichroic element and energy of a second wavelength that will pass through the chosen dichroic element. One example application in which this may be useful is TIR microscopy, where the energy of the second wavelength may be used as an excitation energy for TIR. Energy of the second wavelength may also be used as an imaging energy.
A detector, such as detector 170, may be any detector that detects the reflected energy 125 and can determine a relative position of the reflected energy with respect to a known position of an objective lens, such as objective lens 110, in relation to a dichroic element, such as dichroic element 140. In one example, a detector element detects a relative position of the reflected energy as it exits the objective lens with respect to the position of the incoming energy as it enters the objective lens. Examples of suitable detectors include, but are not limited to, linear detectors, quadrant detectors, photo multipliers, and imaging arrays. The information gained by the detector regarding the relative position of the reflected energy beam can be used to reposition the objective lens with respect to a desired plane of focus. It is possible that this repositioning can be done manually or automatically. A repositioning element can be employed to reposition the objective lens, or to reposition the sample in relation to the lens. An optional control element may be used to interpret the information from the detector and deliver repositioning information to the repositioning element. Many configurations of transferring the information obtained from the detector element to the actual repositioning of the objective lens will be apparent to one of ordinary skill in the art. In one example, a user may visually interpret the information from a linear detector and manually reposition the objective lens until the linear detector indicates a condition of focus of the objective lens on a desired plane of focus. In another example, the detector can deliver position information to a control processor that would drive a servo-mechanical element for repositioning the objective lens and/or the desired plane of focus (e.g. repositioning the sample stage with respect to the objective lens).
A controller may be utilized to process information from a detector, such as detector 470. In one example, information processed by a controller may be used to automatically change the position of an objective lens relative to a desired plane of focus. Changing the relative position may occur in a variety of ways. Examples of ways to change the relative position of an objective lens with respect to a desired plane of focus include, but are not limited to, moving the objective lens; moving the desired plane of focus, such as by moving a sample stage; moving the sample holder; and any combinations thereof. In another example, information processed by a controller may be used to display information related to the relative distance of incoming energy and reflected energy from the center of the objective lens. A controller may include or be in communication with a memory for storing information obtained from a detector, directions for processing information, known or programmed distance between a dichroic element and a desired plane of focus, system calibration information, and/or other information.
As discussed above, in one example, a sample slide may include a dichroic material or, in another example, may have one or more dichroic layers on a surface of the sample slide.
An imaging source 1160 provides a second energy 1162 of a second wavelength that can pass through dichroic element 1127 to a focusing lens 1165. Second energy 1162 then is incident at sample 1140 and passes through dichroic element 1127. Second energy 1162 then passes through objective lens 1105 and to optical element 1122. Optical element 1122 is dichroic in nature reflecting energy 1112 and allowing to second energy 1162 to pass to an imaging element 1170.
Various optical elements may be used to manipulate and/or direct energy prior to incidence at an objective lens and after reflecting back through an objective lens. These optical elements are optional and will depend on the application of the present invention. For example, different optical elements would be required for TIR microscopy as compared with non-TIR microscopy applications. Example optical elements include, but are not limited to, a mirror, a dichroic optical element, a focusing lens, a prism, and any combinations thereof.
A focusing lens may be used to modify an energy before or after passing through an objective lens. Typical laser output can have a particular diameter as it leaves its source (e.g., diameter of about 0.5 millimeter (mm) to about 1 mm). This diameter may increase the further the beam travels from the energy source. Some applications, such as TIR, may benefit from a beam of energy entering an objective lens at a smaller and/or known diameter (e.g., about 100 micrometers (μm) to about 200 μm). Additionally, in certain applications passing the reflected energy beam through a focusing lens prior to being incident at a detector allows for altering a divergent beam to a collimating beam.
An imaging element, such as imaging element 11170, may be any system that allows a user to perceive the image of interest. The imaging element can be electronic, mechanical, optical, or any combinations thereof. Examples of suitable imaging elements include, but are not limited to, a lens, a CCD, microscope, a camera, a particle detector, amicro-optic biological screening systems, and any combinations thereof. One of ordinary skill in the art would be familiar with various imaging elements for use in microscopy and other applications.
The present invention does not require an image to determine proper focus. Even in the most simple configuration, (i.e., no electronics), the systems and methods of the present disclosure allow a user to reference an objective lens to a desired plane of focus that is a known distance from a dichroic element. Manual and/or automatic focus is then achievable. In more advanced systems, continuous high speed focus feedback is possible. An imaging energy is not required to make the objective lens reference. Thus, there is no need to interrupt digital image acquisition for focus feedback corrections to compensate for any drift in focus. Since no image is required, it is possible to maintain proper focus while simultaneously changing the fluid in a sample. This can allow numerous types of dynamic experiments in biology, chemistry, and physics. Microfluidics in microscopy is another application among a host of possibilities that require close focus control.
Further, the system and method of the present disclosure uses an element that is at a predetermined distance from the desired plane of focus as the reference point for focusing an objective lens. This is important in that whenever a sample is translated in the X-Y planes, there is Z axis error between the sample and the objective lens which affects the focus. Z axis errors also arise due to thermo-mechanical drift of the stage and other microscope components. Drift as little as, for example, 100 nanometers (nm) can be easily distinguished in a high magnification system as focus error. Referencing the dichroic element according to the present invention provides compensation for Z axis errors.
Another benefit that arises from the use of a dichroic element, such as dichroic element 140, includes the ability to reflect one or more wavelengths of energy more efficiently for use in referencing the position of the objective lens to a desired plane of focus, such as desired plane of focus 150. Reflection off of a plane glass surface, or other conventional sample substrate, allows typical energy beams, such as an IR laser beam, to only reflect a partial amount of each wavelength of energy that is incident at the glass surface. Thus, only a portion of the intensity of the energy could be reflected to a detector, while a portion (possibly, a substantial portion) of the energy of that wavelength is allowed to pass through the sample substrate to the sample. Depending on the wavelength of energy used for referencing the objective lens, this passed-through energy could be detrimental to a sample. Reducing energy exposure to a sample has important implications when working with certain fluorescent and biological preparations, particularly in long time lapse applications where focus drift are a long standing problem. Additionally, either the energy incident at the glass surface would need to be of higher intensity or the detector would need to be more sensitive than in a situation where the reflected light were of full intensity. The present invention uses a dichroic element, such as dichroic element 140, that can be selected to allow desired imaging wavelengths to safely pass to a sample, while more efficiently reflecting a greater intensity of an energy of a particular wavelength used for referencing the objective. This greater signal would allow higher resolution focus detection while preserving efficient light transmission for imaging.
Additionally, glass alone will only reflect a certain range of wavelengths incident at particular angles. Today, microscopy and imaging has ever increasing demands for using multiple light paths and various selected wavelengths. A dichroic element according to the present invention allows more flexibility in selecting wavelengths of light that can pass and be reflected at a wider range of angles. A dichroic element can also be more selective of which wavelengths are allowed to pass and reflect than would be available without such an element. A non-transparent surface, such as a surface of a semiconductor, does not allow any energy to pass through, thereby restricting many imaging configurations. TIR reflection requires TIR conditions at an interface between two materials and high numerical aperture objective lenses. This significantly restricts the use of this type of reflection and requires the presence of an evanescent field, which may not be desirable in all applications. A dichroic element can be used in TIR and non-TIR applications to reference an objective to a desired plane of focus.
Although the invention has been described and illustrated with respect to exemplary embodiments thereof, it should be understood by those skilled in the art that the foregoing and various other changes, omissions and additions may be made therein and thereto, without parting from the spirit and scope of the present invention.
This application claims the benefit of U.S. Provisional Patent Application No. 60/591,911, filed Jul. 28, 2004, entitled “Focus Feedback System and Method Using Reflected Laser Light as a Signal Source,” which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60591911 | Jul 2004 | US |
