Mid-infrared spectrometer attachment to light microscopes

A mid-IR spectrometer attachment performs reflection spectroscopy measurements using commercially available infinity corrected light microscopes without degrading the microscope's performance. The mid-IR spectrometer attachment, which is mounted to and supported by the visible light microscope, introduces infrared radiation into the optical path of the microscope. Radiation from the mid-IR spectrometer source is directed by a trichroic radiation director to a mid-IR objective lens affixed to the microscope nosepiece. The objective lens focuses the radiation on to a subject sample surface in order to acquire either internally or externally reflected infrared spectra by subsequently directing the sample encoded reflected mid-infrared radiation to the radiation director and then to a mid-infrared radiation detection system. The trichroic radiation director can reflect mid-IR, act as a beam splitter for near-IR and transmit visible light to allow the area of mid-IR spectroscopic analysis to be viewed in either visible light or near-IR.

FIELD OF THE INVENTION

The present invention relates generally to the field of mid-infrared (mid-IR) spectrometry, and more specifically to an attachment to infinity-corrected, commercially available light microscopes to provide the techniques of internal and external reflection infrared microspectrometry.

BACKGROUND OF THE INVENTION

Spectroscopic analysis using radiant energy in the infrared region of the electro-magnetic radiation spectrum is a primary technique for chemical analysis of molecular compounds. The infrared spectral region extends from 0.7 to 250-micrometers, however the mid-IR region is generally considered to cover the region from about 2.5 to about 25-micrometers (or parts thereof), which is commonly used for molecular vibrational spectroscopy. While the primary distinction between near-IR and mid-IR regions is based upon whether the underlying molecular frequencies are fundamental or overtone frequencies, instrument components tend to differ and also be specific by region. There is some overlap however, and specifically mid-IR Fourier transform infrared spectrometers typically cover that part of the near-IR region from 1 to 2.5 micrometers.

This invention defines an attachment apparatus and method for infrared spectroscopic or radiometric analysis of microscopic samples of solids or liquids, including biological materials, combining external or internal reflection spectroscopy with visible light and near-IR radiant energy viewing of microscopic samples by using an attachment to standard commercially available visible light microscopes and commercially available video cameras. The magnification optics for infrared spectral analysis are infrared transmitting objective lenses that are used to focus a beam of radiant energy onto a sample, or sample surface, collect the reflected radiant energy, and present that energy to a detector system for spectral analysis.

Since the introduction of commercial infrared microspectrometers, the advantage of combining the capabilities of a visible-light microscope with an infrared spectrometer has been of great importance. Infrared microscopes, such as those disclosed in U.S. Pat. No. 4,878,747 (the '747 patent) issued to Donald W. Sting and Robert G. Messerschmidt, have been used for an ever-expanding range of applications. These specialized microscopes were attached to commercial Fourier transform infrared (FT-IR) spectrometers. Such microscope/FT-IR systems have been used to detect and identify trace contaminants, to analyze multilayered composites, micro-electronic devices, phase distributions in polymeric materials, inclusions in minerals, abnormal cellular materials, DNA, and numerous other materials.

Heretofore, all known combinations of mid-IR spectrometers and visible light microscopes were composed of (1) a combination of a general purpose laboratory spectrometer and an attachment to the spectrometer having visible light illumination and viewing, or (2) a specially designed integrated instrument combining infrared spectroscopy and visible imaging features. In all cases, the resulting products emphasized the infrared spectroscopy capability, utilizing visible microscopy capabilities as a means to support the infrared spectroscopy capability.

Known special infrared microscope systems and attachments to mid-IR spectrometers have become pervasive even though such systems and attachments are costly and complex. The microscope attachments to laboratory FT-IR spectrometers, described in the '747 patent to Sting and Messerschmidt, among others, have become the standard configurations for infrared microspectroscopy systems. These complex microscope attachments typically provide both transmission and reflection capabilities and use variable remote-image-plane masks to define sample areas for infrared analysis. All of this known art, however, consists of special purpose FT-IR microscopes with specialized optical systems that are appended to large bench-top spectrometers, or fully integrated FT-IR microscope systems using some visible light microscope components. No such systems known use an attachment to visible light microscopes, as is contemplated by our invention.

Our invention provides for the use of both external-reflection and internal-reflection microspectroscopy techniques. Internal-reflection microspectroscopy provides certain advantages over both transmission and external reflection microspectroscopy, particularly in the ability to analyze thick samples. With the introduction of internal-reflection microspectrometry, as shown in U.S. Pat. No. 5,093,580 to Donald W. Sting, and U.S. Pat. No. 5,200,609 to Donald W. Sting and John A. Reffner (also known as attenuated total reflection microspectrometry or micro-ATR) reflection microspectrometry has gained ever-greater importance. Furthermore, our invention extends the capabilities of internal-reflection microspectroscopy by using the unique ATR technology disclosed in U.S. Pat. Nos. 5,703,366 and 5,552,604 issued to Sting and Milosevic to create a novel infinity-corrected ATR objective used for microspectroscopy.

All previous forms of infrared microspectroscopy apparatus were designed from the perspective of the spectroscopist, whereas this invention is designed from the perspective of those using visible light microscopes. Our invention treats the infrared spectroscopy capability as an adjunct to a visible light microscope, and thereby provides extension of the visible microscope's capabilities. It is a primary object of the present invention to provide an FT-IR spectrometer attachment that is easily attached to a commercially available light microscope without compromising any of the available visible light microscope features, options, and capabilities.

SUMMARY OF THE INVENTION

The present invention provides an optical system, apparatus and method to use a mid-IR spectrometer system as an attachment to commercial light microscopes for molecular analysis of materials. In this invention a small spectrometer, in combination with optical, mechanical and electronic components, form an apparatus that can be directly attached to a light microscope for measurement of infrared spectra of microscopic samples or sample domains. Because it can be readily attached directly to existing microscopes, using conventional mechanical connectors that are typically used for microscopes, costs are significantly lower than the current art method of using a dedicated infrared microscope that is attached to a laboratory FT-IR spectrometer. Furthermore, because of the ease of use and accessibility of such low cost infrared spectroscopy capability to material scientists, biologists, and pathologists, as well as others using conventional visible light microscopes, it is expected that significant interdisciplinary benefits will occur.

Using our invention, infrared spectra are acquired using either the external-reflection or the internal-reflection spectroscopy technique. By using reflection spectroscopy techniques, nearly all types of samples can be analyzed. A thin film of material for example, can be mounted on an infrared reflective, but visibly transmissive, substrate such as low-E glass to be analyzed by reflection-absorption, a special case of external-reflection, whereby infrared radiation from the spectrometer is directed onto and through the sample film to the low-E glass substrate, where the radiation is reflected and subsequently passes through the film a second time, whereupon the radiation ultimately is directed to a detector for analysis. An absorption spectrum is thereby acquired, but the measurement was made using the external-reflection technique. For external-reflection spectroscopy, the external-reflection infrared objective lens does not contact the sample, as it must with the ATR objective lens which is used for internal-reflection spectroscopy.

Any thick or thin sample that is placed in contact with the internal-reflection element of an ATR objective lens can result in an ATR spectrum. Because the infrared spectrum of most samples can be measured by using either internally or externally reflected radiation, the infrared spectrometer attachment can provide molecular analyses in a simple and economical manner.

Another object is to use infinity-corrected reflecting objectives and complementary optical components both to direct radiant energy onto a microscopic area and to allow visualization of the magnified image of the specimen and of a highly correlated measure of the mid-IR radiation. The near-IR radiation from the infrared source is used to get this magnified image and correlated measure through an integral video system. Visualization of the mid-IR radiation is achieved by bringing together three distinctly separate ideas in a novel way. First, infrared spectrometers, and specifically mid-IR FT-IR spectrometers, provide a source of infrared radiation that includes some near-IR radiation. Second, commercially available video camera arrays are sensitive to near-IR radiation. Finally, commercially available optical elements are readily made that transmit or reflect radiation differently for different wavelength regions. Using these facts in a novel way caused us to define a new term, a “trichroic’ element, meaning an optical element with defined functions in three different wavelength regions. For example, in the preferred embodiment of the mid-IR attachment, the trichroic element largely transmits visible light radiation, it both transmits and reflects near-IR radiation, and it largely reflects mid-IR radiation. The specifics of how the trichroic element is used in conjunction with the preferred embodiment is discussed in detail when describingFIGS. 7 and 8. Using this novel idea and others has allowed us to incorporate the mid-IR spectrometer attachment into infinity-corrected light microscopes to provide unique and significant benefits to the microscopist. In all embodiments, the inclusion of the mid-IR attachment on the microscope maintains a simple optical system without compromising any of the standard features and capabilities of the light microscope.

One embodiment of the invention provides an optical system, which meets the Koehler illumination criterion of focusing the source element of the radiation at the pupil (aperture) of the objective lens. Visible light illumination systems typically meet this criterion, and this embodiment of our invention meets the Koehler illumination criterion for both visible and infrared radiation. To our knowledge, infrared microspectrometer systems have never before been designed to meet the Koehler illumination criterion. This embodiment of our invention, which meets this criterion, we believe, will be of increasing importance to infrared microspectrometry as infrared array detectors become more readily available at affordable prices.

Other objects of this invention will be apparent from the following description, which is provided to enable any person skilled in the art to make and use the invention, and which sets forth the best mode contemplated by the inventors of carrying out their invention. Various modifications to the specific embodiments disclosed herein, within the general principles of the invention as defined herein, will be apparent to those skilled in the art.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Turning now in more detail to the invention, and initially toFIG. 1, a commercially available infinity corrected visible light microscope is schematically shown as1. Said microscope is generally composed of a frame2, a sample support stage3, transmitted visible light source4aand/or a reflected visible light source4b, nosepiece5, infinity corrected objective lens6, and visible light viewing means, such as a binocular or trinocular viewer with eyepieces7. The visible light optics can be used for visual imaging, but mid-IR objective lenses6are needed for infrared spectral analysis at wavelengths greater than about 4 micrometers.

All of the optical elements are aligned to the visible light optical path8of the microscope1. A sample9is typically placed on a sample substrate10, which is supported by the sample support stage3. The sample support stage3typically has adjustments to provide for three dimensional spatial movements to place the sample9or some attribute of the sample9at the focus of the infinity corrected objective lens6so that observation by the visible light viewing means7is readily effected.

For samples that are transparent or translucent, the transmitted visible light source4acan be used to provide sample illumination in conjunction with condenser12. In that case, the visible light beam is transmitted from visible light source4aalong the visible light microscope optical centerline8through the condenser12, the sample substrate10, then through the sample9and the objective lens6. From objective lens6, the visible light continues along optical centerline8and finally to the visible viewing means7.

Samples that are visibly opaque might require that the reflected visible light source4bto be used to illuminate the sample. In that case, visible light from illuminator4bis introduced along the microscope optical path8via a visible beam splitter, which directs light downward to the objective lens6, which focuses the light onto the surface of the visibly opaque sample9. Some light is reflected by the surface of sample9and collected by the objective lens6, which collimates the reflected visible light and directs it back to the beamsplitter of visible illuminator4b, whereby some of the visible light is transmitted through the beamsplitter to the visible light viewing means7.

In addition, certain samples might require special illumination techniques such as polarized light or radiation of specific frequencies to create florescence or other special visible effects. In such cases, visible light sources4aor4bmight be readily replaced with such special illumination means, as is known in the art.

Commercially available microscope systems provide for significant flexibility and capabilities, the present invention adds the capability of mid-IR microspectroscopy without degrading those flexibilities and capabilities. The mid-IR spectrometer attachment is mechanically and optically compatible with a plurality of commercial infinity-corrected visible light microscopes.

FIG. 2schematically displays a top view of the preferred embodiment of the present invention, shown generally by100. The mid-IR spectrometer attachment100includes the mid-IR spectrometer source101, which provides a source of infrared radiation102directed toward radiation directing means, or radiation director103, which is a trichroic element in the preferred embodiment. Radiation director103provides the means to couple and align the source of infrared radiation102with the visible optical path8of the microscope1, as shown inFIG. 4. Radiation director103reflects infrared radiation102such that it is aligned with the optical centerline8of the microscope1for interaction with a sample via an infinity corrected infrared objective lens6(seeFIG. 4). After interaction with the sample9, that has been manipulated by the stage3(seeFIG. 4) adjustments to be at the focus of the infrared objective6, internally or externally reflected infrared radiation102that is now sample encoded is collected by objective lens6and returned along the microscope optical path8, whereby it is reflected a second time by radiation director103to the infrared detector system104. Infrared detector system104is composed of directing and focusing mirrors104a, detector104b, and detector electronics104cthat are commonly used in the art. Directing and focusing mirrors104acan be a single mirror or multiple mirrors, largely depending upon the physical space constraints imposed by the size and shapes of microscope components. Detector104bis typically a high sensitivity cooled MCT detector as is standard practice for infrared microscopes, however, any high sensitivity detector with sufficient broadband width is acceptable. Increasingly, multi-element infrared detectors are being used for infrared microspectroscopy, and we contemplate using multi-element (or multi-pixel) detectors to provide spatial spectroscopic images of samples. Specifically,FIG. 6discloses a mid-IR. attachment optical configuration intended to have the special benefit of nearly constant infrared radiation density at the sample9when using a multi-element detector104b. Such constant radiation density at the sample9minimizes spectral artifacts resulting from different elements (pixels) of the detector being at significantly different radiation levels. All of the mid-IR spectrometer attachment100components are affixed to a base plate105, which cooperates with a cover106(seeFIG. 3) to provide an enclosure to accommodate a purged environment for the infrared radiation102.

FIG. 3displays the present invention, showing the various elements,101through106as they might schematically appear in a side view relative to the microscope optical path8. Male flange107and female flange108are complementary to each other and mate to complementary mechanical details on the visible illuminator4band the visible viewing means7as assembled inFIG. 4. Adjustment screws109in female flange detail108are used to center the viewing means7onto optical centerline8. Likewise, adjustment screws (not shown) on the visible illuminator4bprovide for alignment of the attachment100to the optical centerline8of the microscope. Such alignment for concentricity is well known and practiced by those in the art, and is thereby not discussed in detail.

FIG. 4schematically displays a side view of the mid-IR attachment100of the present invention, shown in a usable position with a commercially available infinity corrected visible light microscope1. As shown, this attachment comprises a small spectrometer that is sized to be mounted to and supported by the visible light microscope. InFIG. 4, both visible light sources4aand4bare shown, however the presence of4aor4bis dependent only on the visible illumination requirements of the sample being observed and thereby both are not jointly required for the present invention. For many situations, visible illuminator4bis not required, thereby allowing attachment100to be mounted on and connected directly to frame2. Furthermore, an infinity corrected mid-IR objective lens6is shown in the nosepiece5. It is common practice to use nosepieces that accommodate multiple objective lenses; a single objective lens is shown only for simplicity.

The mid-IR spectrometer attachment100is shown in place on a generic commercial light microscope1with both a transmitted light illuminator and a reflected light illuminator4b. Since there are several manufacturers and designs of microscopes, the mechanical fixtures that couple the mid-IR spectrometer attachment100to the microscope1might vary. The only requirements for the microscope1are that it is able to use infinity corrected objectives, and there are no glass elements between the mid-IR spectrometer attachment100and the nosepiece5.

Furthermore, while the reflected light visible source4bis shown to be below the mid-IR spectrometer attachment100of the present invention, with slight modification the reflected visible light source4bcan be placed above the mid-IR spectrometer attachment100without compromising the spirit of the invention.

In addition, a video camera11is shown, attached to the visible light viewing means7to provide for electronic viewing of the sample and/or sample matrix. While such video camera11is used in a conventional way to observe a sample, etc., it is furthermore used in a novel, unique way to view the near-IR radiation from the infrared spectrometer source101. Since the near-IR radiation and the mid-IR radiation are co-mingled as infrared radiation102, observing the near-IR radiation is a direct measure of the mid-IR radiation, which is not observable by the video camera11. Commercially available video cameras11are typically solid-state video cameras, and some are Charge Coupled Devices, or CCD cameras, although our invention will work with any video camera that is sensitive to near-IR radiation, and/or mid-IR radiation. When used as a mass consumer market video camera, these cameras typically have filters for blocking near-IR radiation that must be removed for our use. An example of such a commercially available video camera, or CCD camera is CBCAmerica Model No. CMLH512-L12 that is sensitive to near-IR radiation. It is used to provide an electronic signal for visual light representation of the near-IR radiation from the infrared spectrometer source. In the absence of visible radiation, we are able to observe the location and extent of the mid-IR spectrometer source radiation by observing the near-IR radiation, which is commingled with the mid-IR radiation. The net effect is that we are able to observe the extent of the mid-IR spectrometer radiation as it interacts with the sample or specific areas of the sample. The trichroic element allows for simultaneous viewing in the near-IR and sample analysis in the mid-IR utilizing infrared energy initially emanating from the same source. To those skilled in the art, this is an extremely important feature since it provides direct observation as to what is being spectroscopically measured.

Such direct observation significantly simplifies the analysis process and assures that “what you see is what you analyze”. Since the infrared detector104bwill detect all infrared radiation that is in its field of view, it is sometimes necessary to restrict the source of the infrared radiation102to be contained within the boundaries of the sample of interest. Such use of sample defining masks is well known in the art and thereby is not described in detail herein. In our invention, in order to achieve more specificity, one sample defining mask101c, along with optics101band101dare used as shown inFIG. 5as part of the mid-IR spectrometer source101. When used in conjunction with a radiation director103, such as a trichroic element that will be further explained below, and a video camera11, ample confirmation of “what you see is what you analyze” is provided.

Furthermore, infrared alignment and optical system confirmation are made dramatically easier with the video camera11. First, the camera11is aligned to the microscope1with attachment100, for example as shown inFIG. 4, using visible light means4aand/or4b. The radiation directing means103and the other optical components of attachment100are then adjusted using visible light to grossly align the attachment100. Then, by switching the visible light source off and leaving the infrared source on, and observing (with the video camera11) near-IR radiation that is commingled with mid-IR radiation, the attachment100is finely adjusted, again using adjustments (not shown) on radiation directing means103and the other optical components of attachment100.

FIG. 5displays a top elevation schematic of the mid-IR attachment100of the present invention, including a sample defining mask101c, wherein the infrared beam102is focused by mirror lens101bat the sample defining mask101cand re-collimated by mirror lens101dand directed to radiation director103. The mid-IR spectrometer source101is herein defined to include mirror lenses101band101d, and sample defining mask101c.

FIG. 6displays a top elevation schematic of the mid-IR attachment100of the present invention wherein a visible light source101m, is attached to base plate105and is incorporated into the mid-IR attachment100of the present invention in order to eliminate the need for and associated cost of a separate visible illumination means4b. Both the infrared radiation102and the visible light beam101nare subsequently focused, masked twice, and refocused and re-collimated to achieve the criterion of Koehler illumination for both infrared radiation and visible light illumination.

In the figure, infrared radiation102and visible light illumination101nfrom visible light source101mare commingled at a trichroic element101eand made to follow the same optical path102. The trichroic element101eis designed to largely reflect mid-IR radiation and near-IR radiation, while largely transmitting visible light101n.

The commingled visible and infrared radiation, now referred to as102, is focused by mirror lens101fto the aperture defining mask101g,and through sample defining mask101h, and on to mirror lens101jwhich simultaneously creates an image at infinity of all the radiation at sample defining mask101hand an image of the radiation at mask101gat the aperture of the infinity corrected objective lens6, once it has been reflected by flat mirror101kand radiation director103. For this configuration, as shown inFIG. 6, trichroic element103is a different trichroic element than that of the configurations shown inFIGS. 2 and 5, and is designed to largely reflect mid-IR radiation (reflectivity of around 95%) and to act as a beamsplitter for both near-IR radiation and visible light. This trichroic element design permits the visible light source to be incorporated in the attachment while eliminating the need for a separate vertical visible light illuminator.

The mid-IR spectrometer source101in this embodiment is herein defined to include spectrometer101a, trichroic element101e, condensing mirror101f, radiation mask101g, sample defining mask101h, lens mirror101j, directing mirror101k, visible light source101m, along with the associated visible light path101nand the infrared light path102commingled with101n.

In addition to the cost benefit associated with eliminating visible light source4b, there are benefits associated with microscope alignment and instrument integrity assurance. Furthermore, with the availability of multi-element mid-IR array detectors, it is significant that the Koehler criterion be met in order to insure evenly distributed infrared illumination of the sample and subsequently the detector, for non-absorbing samples.

Referring now toFIG. 7, please note the trichroic element103is an optical filter element that has three distinct functions. It is substantially transparent to visible light (FIG. 7a) and thereby allows the visible light microscope1to operate in a normal fashion (seeFIGS. 7 and 8). A sample can be illuminated from either above (seeFIG. 4), using visible illuminator4b, or below, using illuminator4a, allowing the sample9to be observed by the visible light viewing means7(seeFIG. 7a). For mid-infrared radiation (FIG. 7b), the trichroic element103is highly reflective and behaves like a mirror. For example, depending on the composition and thickness of the layers making up the trichroic element103, reflectivity in the mid-infrared can be as much as 95% or more (seeFIG. 8). Radiation102from the infrared spectrometer source101is reflected by the trichroic element103toward the microscopic objective6. The radiation reflects off the sample9or a reflective substrate10under the sample9, back through reflecting objective6, and back to trichroic element103. It then makes a second reflection at the trichroic element103and is directed to the mid-IR detector system104, composed of additional optics104a, detector104band detector electronics104c. In the near infrared the trichroic element103behaves like a beamsplitter, as depicted inFIG. 7c. The near infrared radiation from the source101travels along the identical path as the mid-infrared radiation, commingled in the infrared radiation102. When it arrives at the trichroic element103, some radiation is reflected to objective6in the same way as the mid infrared radiation. The rest passes through and is not used. The objective6directs radiation to reflect off the sample9or a reflective substrate10under the sample9, and back through the reflecting objective6, and on to trichroic element103, whereat some of the near-IR radiation is reflected to the infrared detector system104and some passes through the trichroic element103and on to the visible viewing means7, which incorporates video camera11.

The radiation that passes through the trichroic element103is detected by video camera11, which is sensitive to near infrared radiation. The output of the camera can be sent to a monitor for visible viewing. The radiation could be directed to the eyepieces of visible viewing means7, but it is invisible to the human eye. It should be noted although shown in three separate diagrams, all three modes of operation occur simultaneously. It will be appreciated by those of skill in the art that, depending on the desired reflectance (FIG. 8) for infrared and transmittance for visible and near-IR radiation, the coatings and substrate can be chosen appropriately. For example, reflectance of mid-IR from 80% to 95%, and even up to about 99%, can be achieved. U.S. Pat. No. 5,160,826 to Cohen, et al., which is hereby incorporated by reference in its entirety, discloses a coated window that substantially transmits visible radiation while simultaneously reflecting infrared radiation. Trichroic elements need to be specified by functionality by spectral region and can be ordered from optical component manufacturers such as Spectral Systems of Fishkill, N.Y.

These three functions are novel and important. Transparency of visible light allows normal visible microscopy. Reflectivity in the mid-infrared allows spectroscopic analysis. Since the near-infrared radiation travels virtually the same path as the mid-infrared radiation for optical paths with little or no chromatic aberration, it will illuminate an area that coincides with the area of spectroscopic analysis. Therefore, the camera11views the part of the sample9that is being analyzed by the mid-infrared radiation. In addition, simultaneous near-infrared and visible viewing, permit precise positioning of the sample9on the microscopic stage3to select the desired portion of the sample to analyze.