Patent Grant
7773302
The present disclosure is directed toward optical filters. In particular, the present disclosure relates to optical filters which may be incorporated into fluorescence imaging and/or quantification systems.
Fluorescence systems are often employed to analyze or image biological samples. In such systems, the sample is typically exposed to light from a broadband or laser source at a wavelength at which a material of interest in the sample, such as a fluorophore or a naturally occurring substance in the material, absorbs light causing it to fluoresce or emit light at a different (typically longer) wavelength. Light emitted from the sample is then detected so that the location, amount, and other properties associated with the material of interest, as well as the sample, can be determined. In addition, an image of the sample can be constructed based on the detected fluorophore, for example.
In many fluorescence systems, light at a given wavelength excites an atom in the material of interest. The atom then relaxes to a lower energy state, and, in doing so, emits light at a different wavelength. Fluorescence systems typically include an optical source, such as a bright arc lamp or a laser, to generate the excitation light, and a photodetector for sensing light emitted by the sample. The photodetector may include a digital camera or the eyes of an observer. In order to reduce the amount of other light reaching detector, such as light from the source, filters are typically employed which are transmissive at wavelengths of light emitted by the sample, but reflective and/or absorbing at other wavelengths. If light at such other wavelengths is adequately suppressed, a so called “spectral darkfield” situation can be achieved in which an image is black or dark when no features of interest are present. Image quality can thus be improved. Without this spectral darkfield property, in most samples no fluorescence could be observed.
Optical filters are also used to direct the excitation light to the sample, and if highly reflective or absorbing at wavelengths associated with the emitted light, can efficiently direct the excitation light at the desired wavelengths to the sample while blocking light from the source at the emitted wavelengths.
Some optical filters include coatings of metal oxides and are physically hard (“hard coatings”), while others include coatings of softer materials, such as sodium aluminum fluoride (“cryolite”) and/or zinc sulfide (“soft coatings”). Filters including soft coatings are commercially available from Omega Optical, Inc.
With improved optical filters, more photons of emitted light and fewer photons of undesired light (e.g., the excitation light) are fed to the photodetector. Thus, weaker signals can be detected, or less excitation light is required to generate a given emitted optical signal, thereby minimizing damage to the sample by intense light from the source. Or, an image can be detected in less time leading to faster measurements. In addition, a higher signal-to-noise ratio (and therefore better resolution) can be achieved in the image, since, for example, the filter can block more excitation light from reaching the photodetector, while transmitting a given intensity of emitted light.
For an optical filter to be useful as a fluorescence filter, it preferably should be able to transmit light with high efficiency over a well-defined band of wavelengths (passband). The spectrum associated with an optical passband filter typically has reduced transmission over a limited range of wavelengths above the high wavelength edge of the passband, as well as a limited range of wavelengths below the lowest wavelength edge. For fluorescence spectroscopy applications, however, the filter spectrum should have substantial blocking of light over a broad range of wavelengths extending well beyond the limited ranges associated with the passband. Generally these two requirements (high transmission in the passband and extended blocking) are at least somewhat mutually exclusive. That is, providing more blocking generally occurs at the expense of reduced transmission in the desired passband. As explained below, wide-band blocking or extended blocking can be enhanced by colored (or absorbing) filter glass. Even with such enhancements, however, typically the most effective means to provide high blocking is with dielectric thin-film reflecting layers—generally the more layers, the more blocking is achievable. Because there tend to be limitations on the number of layers that can be successfully deposited in a single coating run, this requirement means that conventional fluorescence filters to-date have typically required multiple thin-film coatings per filter. For example, filters fabricated by ion-beam sputtering, which deposit many hard coating layers have to-date been made with at least two coatings per filter. Such filters include BrightLine® fluorescence filters commercially available from Semrock, Inc. Filters are disclosed in U.S. Pat. Nos. 6,809,859, 7,068,430, 7,119,960, and 7,123,416, as well as application Ser. No. 10/953,483, all of which are incorporated herein by reference.
As noted above, colored filter glass has been implemented in order to obtain greater blocking over a wider spectrum. Typically, colored filter glass is often combined with filters formed of soft-coated layers (discussed in U.S. Pat. No. 6,809,859) for such purposes. For example, the long-wave pass emission filters of very low-cost fluorescence filter sets are comprised of a single piece of colored filter glass.
In most soft-coated filters, however, extended-blocking multiple optical coatings are typically provided, each of which blocks light over a band of wavelengths determined by the “stopband width” of a characteristic quarter-wave stack of thin-film layers. Thus, wider blocking ranges require more quarter-wave stack coatings and are thus more difficult to fabricate.
Hard-coated filters are more robust than soft-coated filters and usually achieve blocking via dielectric reflection. Some hard-coated filters are based on a long-wave-pass coating on one side of a single substrate and a short-wave-pass coating on the opposite side, thus producing a bandpass filter, where one or both of the coatings also has built-in extended blocking reflection layers. Other conventional hard-coated filters have been made that have a bandpass filter on one side of a substrate based on a multi-cavity Fabry-Perot type filter coating (quarter-wave-based structure), and then one or more additional coatings with extended blocking layers on the opposite side of the substrate and any additional needed substrates (when there is more than one additional coating). Such filters are described in U.S. Pat. No. 7,119,960 and typically have a narrow passband, which, when measured at the optical density 5 points on the spectral curve, is less than 2% of the center wavelength of the passband. It would be desirable, however, to provide a filter with a wider passband.
Conventional filters typically have limited performance due to the high losses and poor edge steepness associated with colored filter glass or require multiple coating runs leading to higher filter cost. Furthermore, conventional filters that are able to be made at reasonable costs (targeted at more cost-conscious markets like clinical microscopy) typically suffer from poor brightness, poor contrast, and poor reliability and durability. The lower brightness results from the use of colored filter glass in some instances, or from thinner and fewer coatings to reduce coating time, which lead to less steep filter edges (and thus wider exciter-emitter passband separation). Poorer contrast also results from the inability to position the edges optimally (due to poor steepness) as well as lower overall blocking when the coating thickness and the number of coatings are limited. Poor reliability and durability results from the use of soft coatings, which until now have been the only means by which low-cost fluorescence filters could be produced. These filters tend to “burn-out” when exposed to intense radiation for extended periods of time, and because the coatings are porous and absorb water vapor, they can degrade over time, especially in hot, humid, and corrosive environments. In addition, coatings that are not protected from physical contact by an extra glass substrate (such as those found on dichroic beamsplitters) are susceptible to damage when handled or when normal optics cleaning procedures are used.
Accordingly, there is a need for optical fluorescence filters having reduced cost for clinical microscopy applications, for example. There is also a need for such low cost filters to provide more brightness, a lower background light level and/or better contrast. In addition, there is a need for filters that have extremely high reliability and durability, especially in clinical applications, in which doctors and medical technicians must make repeated diagnoses of identical tissue samples, for example, even years after the samples are taken.
Consistent with an aspect of the present disclosure, an optical device is provided which comprises a substrate having a surface and a plurality of hard-coating layers provided on the surface of the substrate. The plurality of hard-coating layers includes alternating first and second layers. The first layers have a first refractive index, nL, and the second layers having a second refractive index, nH, greater than the first refractive index. In addition, the plurality of hard-coating layers has a spectral characteristic, which has a passband. The passband is defined by a first passband wavelength λ1passband and a second passband wavelength λ2passband. The passband has a center wavelength and the minimum spectral distance between the optical density 4 points on the spectral curve is greater than 2% of the center wavelength. The spectral characteristic also has an average transmissivity at least equal to 80% over the passband. Further, the spectral characteristic has an average optical density greater than 4 over a first blocking band of wavelengths extending from a first blocking wavelength, λ1block, to a second blocking wavelength, λ2block, whereby the second blocking wavelength satisfies:
λ2block<0.9*((1−x)/(1+x))*λ1block,
Alternatively, the spectral characteristic has an average optical density greater than 4 over a second blocking band of wavelengths extending from a third blocking wavelength λ3block to a fourth blocking wavelength, λ4block, the fourth blocking wavelength satisfies:
λ4block>1.1*((1+x)/(1−x))*λ3block,
where
A first edge band of wavelengths is associated with a first edge portion of the spectral characteristic adjacent the passband. The first edge band of wavelengths extends from λ1passband to λ1block, such that, at a first transmission wavelength, λ1-50%, within the first edge band of wavelengths, the coating has a transmissivity of 50%.
A second edge band of wavelengths is associated with a second edge portion of the spectral characteristic adjacent the passband. The second edge band of wavelengths extends from λ2passband to λ3block, such that, at a second transmission wavelength, λ2-50%, within the second edge band of wavelengths, the coating has a transmissivity of 50%,
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and together with the description, serve to explain the principles of the invention.
a-2c illustrate spectral characteristics associated with examples of the filter shown in
a-7c illustrate filter sets consistent with additional aspects of the present disclosure; and
Consistent with the present disclosure, a filter having high transmission, steep edges, and extended blocking is realized with a single coating provided on one side of a substrate. Instead of providing a plurality of quarter-wavelength-based Fabry-Perot type cavities, the single coating includes a portion that serves as a first edge filter for blocking wavelengths exceeding some predetermined wavelength, and another portion that acts as a second edge filter to block wavelengths below another wavelength. When these coating portions are formed on one another, their corresponding spectra are superimposed, and the resulting spectrum includes a wide passband (greater than 2% the center wavelength, measured as the minimum spectral distance between the optical density 4 points on the spectral curve) with blocking on either side. The coating also includes at least one additional portion for extended blocking. As a result, a filter having a spectrum with high transmissivity in the passband, steep passband edges, and extended blocking can be obtained in a single coating without the need to provide additional coatings on multiple substrates. Accordingly, multiple conventional filters are not necessary to obtain these desirable spectral characteristics. Overall costs are therefore reduced. In addition, a filter set (including an exciter filter, beam splitter and emission filter) having just three filter components can be realized, leading to a simpler system design with improved reliability.
Reference will now be made in detail to various exemplary embodiments of the present disclosure, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts.
An exemplary filter 100 in accordance with the present disclosure is shown in
The first (112), second, (114), third (116), and fourth (118) coating portions are typically formed with high-precision, ion-assisted, ion-beam sputtering thin-film deposition techniques. Such known techniques, which may include optical monitoring, can be used to accurately deposit hundreds of layers. In particular, deposition of the first (112) and second (114) coating portions may be controlled in accordance with known algorithms and may be further controlled with known optical monitoring of the deposited materials. Deposition of the third (116) and fourth (118) coating portions may also be controlled with known algorithms. Optical monitoring of the deposition of the materials that constitute the third (116) and fourth (118) coating portions, however, may not be necessary. Rather, these depositions may be timed for specified periods of time instead of being subject to continuous optical monitoring. Known optimization algorithms may also be applied to further adjust the overall thickness of each of coating portions 112, 114, 116, and 118 and/or the thicknesses of individual high and low refractive index layers that constitute coating portions 112, 114, 116, and 118. In addition, consistent with the present disclosure, first coating portion 112 may be omitted if extended blocking (described in greater detail below) at longer wavelengths is not required. In that case, coating 111 includes coating portions 114, 116, and 118. Alternatively, if extended blocking at shorter wavelengths is not required, fourth coating portion 118 may be omitted, such that coating 111 includes coating portions 112, 114, and 116.
An exemplary spectral characteristic 200-3 of filter 100 consistent with the present disclosure is shown in
λ2block<0.9*((1−x)/(1+x))*λ1block,
where
A value for λ2block as determined by the above equations typically indicates that the blocking on the short-wavelength side of the passband occurs over a wider region than that which would result from a single quarter-wave stack of layers. The equations are adapted from the analysis in Section 5.2 (specifically Equations 5.15) from the text book Thin-Film Optical Filters (Third Edition, H. A. Macleod, Institute of Physics Publishing, Bristol and Philadelphia, 2001), which is incorporated herein by reference. Blocking beyond that which would result from a single quarter-wave stack of layers (which itself is present due to function of the quarter-wave stack in forming the filter edge) is referred to as “extended blocking.” Such extended blocking over a wavelength region results from a more complex layer structure than merely a quarter-wave stack, and includes, for example, multiple quarter-wave stacks optimally combined into a single coating, or a “chirped” quarter-wave stack in which each of the high and low index layer thicknesses are monotonically increasing or decreasing over at least a portion of the coating.
In addition, spectral characteristic 200-3 has an average optical density greater than 4, and may be more than 5, over second blocking band of wavelengths 225 extending from a third blocking wavelength λ3block to a fourth blocking wavelength, λ4block, the fourth blocking wavelength satisfies:
λ4block>1.1*((1+x)/(1−x))*λ3block,
Theoretically, the factors 0.9 and 1.1 in the above formulas do not define the upper and lower bounds of λ2block and λ4block, respectively. In practice, however, due to uncertainties in the precise values of the refractive indexes of the deposited layers that constitute coating 111 (such as inability to measure the index precisely and slight variations of the index with wavelength and environmental conditions) and other non-idealities (such as measurement uncertainty), the values of λ2block and λ4block that are actually observed can extend slightly below and above, respectively, that which is theoretically predicted. Accordingly, the above formulas take into account such non-idealities by incorporating a factor of 0.9 in the formula for λ2block and a factor of 1.1 in the formula for λ4block.in order to reflect that which may actually be observed.
A value for λ4block as determined by the equation above may ensure that the blocking on the long-wavelength side of the passband is comprised of extended blocking, or blocking over a wider range than would result from a single quarter-wave stack of layers, in analogy to the description of short-wavelength-side extended blocking above.
A first edge band of wavelengths 230 is associated with first edge portion 210 adjacent passband 205. First edge band of wavelengths 230 extends from λ1passband to λ1block, such that, at a first transmission wavelength, λ1-50%, within first edge band of wavelengths 230, coating 111 has a transmissivity of 50%, and λ1passband, λ1block, and λ1-50%, satisfy:
(λ1passband−λ1block)/λ1-50%<2%.
Further, a second edge band of wavelengths 240 is associated with a second edge portion 220 of spectral characteristic 200 adjacent passband 205. Second edge band of wavelengths 240 extends from λ2passband to λ3block, and, as shown in
(λ3block−λ2passband)/λ2-50%<2%.
A first portion 201 of spectral characteristic 200-3 extending from λ2block to λ1EB has reduced transmission and constitutes a range of extended blocking associated with first coating portion 118. A second portion 202 of spectral characteristic 200 extending from λ1EB to a center wavelength λ0 of passband 205 constitutes part of a long-wave-pass edge filter spectrum attributable to third coating portion 116, and a third portion 203 extending from center wavelength λ0 to λ2EB constitutes part of a short-wave pass edge filter spectrum attributable to second coating portion 114. Extended blocking of portion 204 of spectral characteristic 200-3 extends from λ2EB to λ4block, and is attributable to coating portion 112.
In the above exemplary transmission characteristic 200-3, λ2block may be substantially equal to 400 nm and λ4block may be substantially equal to 700 nm. In addition, spectral characteristic 200-3 may have an average OD greater than 2 over a band of wavelengths extending from λ4block (e.g., 700 nm) to 1000 nm or 1100 nm. λ4block may also be substantially equal to 900 nm. Further, consistent with the present disclosure, the passband may have a bandwidth, measured as the minimum spectral distance between λ1block and λ3block,(both of which typically having an associated optical density of 4, and being referred to as “OD 4 points”), which is greater than 2% of the center wavelength λ0. Accordingly, for example, for a center wavelength λ0 of 550 nm, the passband bandwidth (i.e., the minimum spectral distance between λ1block and λ3block) is greater than 11 nm. Exemplary passband bandwidths may be between 10 nm and 80 nm and exemplary center wavelengths may be within 380 nm to 700 nm.
As noted above, first coating portion 112 may be omitted. In that case, the resulting spectral characteristic will lack extended blocking over longer wavelengths beyond λ2EB (see spectral characteristic 200-1 in
Returning to
Filters consistent with the present disclosure may be incorporated into commercially available fluorescence microscopes, such as the BX41 microscope available from Olympus America Inc.
The spectral characteristic 600 of emission filter 326 is shown in
λ2block<0.9*((1−x)/(1+x))*λ1block,
In addition, spectral characteristic 600 has an average optical density greater than 4 over an upper blocking band of wavelengths 625 extending from wavelength λ3-2block to wavelength, λ4-2block, λ4-2block satisfying:
λ4block>1.1*((1+x)/(1−x))*λ3block,
where
A lower edge band of wavelengths 630 is associated with a lower edge portion 610 adjacent passband 605. Lower edge band of wavelengths 630 extends from λ1-2passband to λ1-2block, such that, at wavelength λ1-2-50%, within lower edge band of wavelengths 630, coating 111 has a transmissivity of 50%, and λ1-2passband, λ1-2block, and λ1-2-50%, satisfy:
(λ1-2passband−λ1-2block)/λ1-2-50%<2%.
Further, an upper edge band of wavelengths 640 is associated with an upper edge portion 620 of spectral characteristic 600 adjacent passband 605. Upper edge band of wavelengths 640 extends from λ2-2passband to λ3-2block, such that, at wavelength λ2-50%, within upper edge band of wavelengths 640, coating 511 (
(λ3-2block−λ2-2passband)/λ2-2-50%<2%.
A first portion 601 of spectral characteristic 600 extending from λ2-2block to λ1-2EB has reduced transmission and constitutes a range of extended blocking associated with coating portion 518. A second portion 602 of spectral characteristic 600 extending from λ1-2EB to a center wavelength λ2-0 of passband 605 constitutes part of long-wave-pass edge filter spectrum attributable to third coating portion 516, and a third portion 603 extending from center wavelength λ2-0 to λ2-2EB constitutes part of a short-wave pass edge filter spectrum attributable to second coating portion 514. Extended blocking of portion 604 of spectral characteristic 600 extends from λ2-2EB to λ4-2block, and is attributable to coating portion 512. Passband 605, measured as the minimum spectral distance between λ1-2block and λ3-2block, has a bandwidth similar to that of the passband bandwidth of spectral characteristic 200-3 discussed above in connection with
It is noted that if extended blocking is not required at longer wavelengths, coating portion 512 may be omitted, such that extended blocking of portion 604 would not be provided for wavelengths greater than λ2-2EB. In that case, spectral characteristic 600 would resemble spectral characteristic 200-1 shown in
Returning to
Alternatively, the locations of detector 332 and source 305 may be switched, as well as the locations of filters 315 and 326. In this example, dichroic beam splitter 320 passes excitation light, which has a wavelength within passband 205, and reflects light at the emission light wavelength (in passband 605), such that the emission light is reflected toward detector 332.
In
a illustrates a filter set 701 including right-angle prisms constituting substrates 705 and 710. Coating 111 of filter 100 may be provided in contact with side surface 720 of substrate 705, while coating 412 of filter 320 may be provided on hypotenuse surface 721. In addition, coating 511 of filter 326 may be provided on side surface 724 of substrate 710. As further shown in
Filter set 702 shown in
Exemplary spectra associated with filter sets consistent with the present disclosure will next be described with reference to
Similar plots were obtained in connection with a filter set suitable for use with samples including fluorescein isothiocyanate (FITC), as shown in
As discussed above, the present disclosure describes a filter in which a coating, preferably provided on a single substrate, has sharp passband edges as well as extended blocking. Filter sets employing such filters can be realized with three or fewer substrates, thereby simplifying system design and reducing costs.
Tables 1 and 2 below list exemplary individual layer thicknesses associated with the exciter filter, dichroic beamsplitter, and emitter filter spectra discussed above. Tables 1 and 2 correspond to the above described filter sets for use in connection with Calcofluor White and FITC dyes, respectively.
It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.
This application claims the benefit of U.S. Provisional Application No. 60/841,552 filed Sep. 1, 2006, and U.S. Provisional Application No. 60/842,950 filed Sep. 8, 2006, the contents of both of which are incorporated herein by reference.
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| Number | Date | Country | |
|---|---|---|---|
| 20080055716 A1 | Mar 2008 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60841552 | Sep 2006 | US | |
| 60842950 | Sep 2006 | US |
