Patent Application
20120105949
Fluorescence microscopy is an essential tool in microbiology and medicine. In fluorescence, light of one wavelength is absorbed by molecules and re-emitted at a different wavelength. The absorption and emission wavelengths depend on the specific molecules. The separation in wavelengths between absorption and emission allows the background of non-fluorescent light to be filtered from the fluorescence signal, enhancing the sensitivity and providing for quantitative image analysis. In epifluorescence microscopy, the excitation light passes to the sample through the microscope objective that captures the fluorescent light, requiring access to one side of a sample only and allowing fluorescence microscopy on non-transparent objects. An assembly of precision filters and beam-splitters is typically used in epifluorescence. These elements are often conventionally mounted in an interchangeable filter cube that is inserted into a suitably designed microscope by the microscope operator.
Unfortunately, the filter cubes and microscopes are expensive objects. Operators may introduce dust, which can affect image quality, while changing out filter sets. Moreover, the conventional optical arrangement in epifluorescence microscopes passes the fluorescence through an inclined beamsplitter, with adverse effects on the microscope image.
The object of the present invention is to create an epifluorescence microscope that does not suffer from these conventional drawbacks. Embodiments of the present invention achieve a compact form factor without sacrificing optical sensitivity by the novel use of combined optic mounts and light baffles constructed using additive manufacturing processes. The use of additive manufacturing enables stray-light-capturing structures that are not practical to make by other techniques. The compact form of the microscope reduces cost, weight, and improves stiffness with no reduction in optical performance over larger conventional microscopes. Some embodiments of the present invention do not require installation of filters by an operator, reducing the likelihood of dust and contamination on optical surfaces. Some embodiments of the present invention employ a novel light path that avoids passing the fluorescent light through off-axis elements. This optical arrangement provides for the use of a microscope objective having a finite corrected-image distance, such as a DIN objective, rather than infinity-corrected objective that require additional optical elements to form an image. The reduction in complexity can both reduce system cost and improve optical performance by reducing Fresnel losses and imaging artifacts from Fresnel reflections.
As used herein an “additive manufacturing process” comprises any processes in which solid components are produced by a process of adhering, bonding, welding, soldering, brazing, sintering, polymerizing, chemically reacting, photolitically forming or otherwise linking precursor materials such as chemicals, polymers, metals, alloys, powders, beads, grains, micelles, liposomes, emulsions, epoxies, thermosets, thermoplastics, mixtures, aggregates, etc.
Examples of additive manufacturing processes include but are not limited to:
Stereolithography (SLA or SL), which generally may employ photopolymer materials;
Fused Deposition Modeling (FDM), which generally may employ thermoplastics, eutectic metals, etc.;
Selective Laser Sintering (SLS), which generally may employ thermoplastics, metal powders, etc.;
Laminated Object Manufacturing (LOM), which generally uses paper and like materials;
3D Printing (3DP), which uses a range of materials; and
Polyjet Technology, a combination of SLA and FDM, which generally employs photopolymer materials.
In the present invention, at least one element of microscope 102 is manufactured using an additive process. Some embodiments employ a black rigid polyjet-produced material. In some embodiments, the material name is “VeroBlack,” having a hardness of 82 Shore D.
In some embodiments cover 201 supports other elements, such as a microscope objective lens 202, one or more alignment and centering features, such as a post 204, and an electronic interface 206. In some embodiments, the electronic interface 206 carries circuits including power and signaling.
Power circuits may include power for logic, power for the illuminator, power for the camera, etc. In some embodiments, power is converted internally from one voltage to another within the microscope to support the requirements of different electronic devices.
In some embodiments signaling circuits may include digital communications lines, triggering or control lines, video signaling lines, etc. Digital communications may employ differential signaling, e.g., RS485 and the like, I2C, SPI standard communications, USB-1, USB-2, USB-3, Ethernet, IEEE1394, or other standards or custom signaling schemes known in the art.
In some embodiments, triggering or control lines may be used to control strobing of the illuminator and to transmit real-time triggering information to or from the microscope camera.
In some embodiments one or more electronic circuits may employ a connector 208 situated elsewhere, of example at an end of the housing 110. In some embodiments, a connector may provide power. In some embodiments a connector may conform to USB, Ethernet, or IEEE1394 physical and signaling standards. In some embodiments, a custom connector may be employed. Some embodiments may power the microscope in part or full by power from a bus, e.g., USB, IEEE1394, power over Ethernet, and the like.
In other embodiments, at least one electronic circuit is made wirelessly, e.g., via radio techniques, inductive coupling, capacitive coupling, etc. Some preferred embodiments expose a reduced number of or no conductors to the exterior of the microscope, potentially providing a maximum protection against damage from spills. In some embodiments, the microscope transmits video information wirelessly. In some embodiments, the microscope control signals are transmitted wirelessly. In some embodiments, the microscope contains a power source, e.g., a battery, ultracapacitor, and the like.
Some embodiments of the present invention contain a heat sink/cooling plate in the base 210.
If the light source 302 has a narrow spectral bandwidth like an LED or laser, the light source 302 may comprise a plurality of individually controllable emitters having different spectral outputs to provide for tuning of the excitation wavelength. The design of such a compound emitter may be complicated by the need not to produce a marked shift in the illumination pattern when switching sources. Spatial interleaving or optical interleaving of emitter elements may be employed to produce a spatially stable illumination pattern.
Light from the light source 302 passes through a first condenser optic 304 and a second condenser optic 306 that are shown as being lenses. Some alternative embodiments of the condenser optics may employ reflective, diffractive, Fresnel, holographic optics and the like to direct illuminator light efficiently along the ray path 301 instead of refractive condenser lenses. An aperture 308 prevents stray rays from the light source 302 from entering the microscope or impinging on an excitation filter 310 at a significant angle, which may be important for maintaining a sharp pass-band cut-off if filter 310 is an interference filter. Excitation filter 310 removes components of the spectrum emitted by the illuminator 302 that overlap the fluorescence signal spectrum substantially. This filter 310 may be a colored glass or molecular filter. However, in preferred embodiments, this filter may be an interference filter or a combination of molecular absorption and interference filter because of the enhanced control over cut-off frequency and reduced autofluorescence provided by an interference filter. Filter autofluorescence may generate a false background signal and limit the sensitivity of the microscope.
In some preferred embodiments, the illumination wavelength may be adjusted by changing elements 302, 310, or a combination. If the illuminator 302 has a broad spectral output, it may be preferable to change the excitation filter 310 characteristics. This may be accomplished by the use of an excitation filter having a spatially varying passband and physically displacing the filter, angle-tuning the excitation filter by tilting it more or less with respect to the ray path 301, arranging a plurality of filters having different passbands in a selectable fixture, the use of an electrically tunable filter such as an acousto-optic module, etc. In some embodiments, particularly when the illuminator has a narrow spectral emission, a plurality of illuminator elements, e.g., 302 and 310, or 302, 304, 306, 308, and 310 may be changed in a group.
In some embodiments, these adjustments or changes may be manual. In some embodiments, these changes may involve removing and replacing elements in the system. In such embodiments, care should be exercised in the design to avoid the introduction of dust to the microscope, at least in locations where it produces a visible defect in the microscope image. In preferred embodiments these adjustments or changes may be mechanized, e.g., via a DC motor, solenoid, brushless DC motor, stepper motor, and the like.
The illuminator rays substantially follow path 301 to a beamsplitter 312. In some preferred embodiments, this beamsplitter has a dichroic characteristic: passing the illumination or excitation wavelength selectively and reflecting the fluorescence or emission wavelength selectively. In other embodiments, this beamsplitter may have a substantially neutral spectral response and an approximately 50% reflectivity. Such an embodiment may be favorable for supporting multiple excitation and emission wavelengths without the need to change the beamsplitter. The advantage of using a dichroic beamsplitter is significantly greater fluorescence signal strength and a reduction in bleed through of the excitation light on the camera image.
Beamsplitter 312 has the unfortunate consequence of producing a stray reflection of the illuminator rays along path 311. Surfaces that these stray rays land on are in the field of view of the camera and require careful attention to avoid contamination of the fluorescence image.
A fraction of excitation rays pass through the beamsplitter 312 and follow path 313 through the objective lens set 202 and onto sample 314. A component of the fluorescence produced by those rays passes back through the objective lens set substantially along path 315. When these rays reach the top surface 316 of beamsplitter 312, a significant part of the rays reflect substantially along path 317. Because these rays are reflected by the top surface of the beamsplitter, the beamsplitter produces no image aberrations.
This lack of aberrations is an important improvement over conventional epifluorescence microscopes in which the fluorescence rays pass through the tilted beam splitter on their way to forming an image, which might introduce aberrations, particularly for non-infinity corrected objectives.
The rays 317 reflect off a folding mirror 318 into rays 319, and then reflect off mirror 320 into rays 321. The purpose of the mirrors 318 and 320 is to keep the microscope body size compact. In some alternative embodiments, more, fewer, or no folding mirrors are used. The rays 321 pass through an emission filter 322 that provides a sharp cut off to block excitation wavelengths from passing while efficiently passing emission, or fluorescence, wavelengths.
In some embodiments, filter 322 can be changed or adjusted to provide good sensitivity for different fluorophores. In some embodiments, the filter 322 is adjusted or changed in a manner analogous to 310. However, angle tuning and acousto-optical filtering of the fluorescence may produce image aberrations. In some embodiments, adjustments or changeouts of 310 and 322 are ganged. In some embodiments, adjustments or changeouts of 310, 312, and 322 are ganged. In some embodiments, filter changeouts or adjustments are ganged with changes in 302.
Element 324 is a camera. In some embodiments the camera is monochromatic. In other embodiments, the camera has additional filters for color separation.
In some embodiments, the camera employs a charge-coupled device sensor. In other embodiments, the camera employs a CMOS sensor. In some embodiments, the camera has avalanche signal amplification, e.g., an electron-multiplied CCD. Some embodiments employ multi-channel plates for photon amplification.
In the embodiment in
This body 410 contains a plurality of features 411 and 412 that act as internal baffles to enhance the absorption of stray light rays. It further contains an internal aperture 413 and apertures 414 and 416 for mirrors 318 and 320, respectively. Such baffles and apertures dramatically reduce stray rays, providing for enhanced fluorescence detection sensitivity, however they may be cost prohibitive to produce using conventional machining, casting, or molding. The novel use of additive manufacturing to produce this body provides the design freedom to combine many conventionally challenging features into one or a few bodies economically.
Circuitry for driving the illuminator 302 may be formed on a printed circuit board 419. Having this driver board, the illuminator, and camera, three-heat generating elements of the microscope in intimate contact with the heat sink and exchanger 210 prevents excessive internal temperatures. In some embodiments, adhesive pads that enhance heat transfer are employed to make good thermal contact between heat generators and the heat sink. In some embodiments, thermally conductive greases may be used, provided these greases do not outgas or attack materials in the camera and that care is taken to avoid contamination of optical elements. In other embodiments, thermally conductive epoxies or mechanical pressure may be used to enhance heat transfer efficiency.
In some embodiments, additional cavities can be engineered into the solid-filled regions 432 to enhance trapping of stray light and to reduce the body fabrication times.
In some embodiments of the present invention, the body 410 is manufactured in pieces, e.g., split along the centerline similar to the view in
Some embodiments of the present invention are employed as swappable modules in a system such as shown in
In some embodiments, the epifluorescence microscope modules may be swapped with power on. In some embodiments the epifluorescence microscope contains a device, such as a serial EEPROM or microcontroller, that can be queried and written about information including some of the following items: the hardware version, firmware version, illuminator wavelength, characteristic of filters and beam splitters, microscope objective, indexes that identify the types of filters, beam splitters, objectives, and illuminators contained within the microscope, and the like.
