Edmund Optics(cid:15)(cid:3)(cid:44)(cid:81)(cid:70)(cid:17)(cid:3)
`(cid:40)(cid:91)(cid:75)(cid:76)(cid:69)(cid:76)(cid:87)(cid:3)(cid:20)(cid:19)(cid:19)4(cid:3)
`
`(cid:3)
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`Advances in Filter Technology for Multiphoton Microscopy
`
`Traci R. Jensen*, Gregg W. Jarvis, and Robert L. Johnson, Jr.
`
`Omega Optical, Inc., Brattleboro, VT 05301
`
`ABSTRACT
`
`A new optical interference filter deposition technology is demonstrated that provides the deep blocking and extended
`transmission regions required for multiphoton fluorescence applications. This technology allows for the deposition of high
`phase thickness coatings with many more layers than was previously possible. Theoretical blocking at a level greater than
`optical density 9 is achieved. We present examples of shortpass edge filters and bandpass filters with high transmission and
`deep blocking. A dichroic mirror with high reflection in the near infrared and an extended region of high transmission
`throughout the visible is also presented.
`
`Keywords: Optical filter, interference filter, dielectric thin film, multiphoton fluorescence, shortpass filter, bandpass filter
`
`1. INTRODUCTION
`
`Multiphoton excited fluorescence microscopy is a relatively new technique requiring an optical filter solution unique from
`that of traditional single photon excitation15. Typically it requires a dichroic filter to separate the incoming laser light from
`the fluorescence signal, and a second filter to further attenuate the scattered laser light. A high near-infrared rejection ratio is
`typically required, as most applications utilize femtosecond pulsed Ti:Sapphire lasers. Since there may be as much as 400 nm
`separation between the excitation and emission wavelengths, transmission edge steepness is not as important as it is with
`single photon excitation. The most important filter properties are deep blocking in the near infrared, and high transmission in
`the visible region, with minimal autofluorescence from the filter assembly.
`
`In recent years there have been improvements in optical interference filter deposition technology allowing for the deposition
`of high phase thickness coatings with high spectral contrast. Omega has developed a new deposition technology, hereafter
`referred to as Alpha technology, that allows for the construction of high phase thickness shortpass and longpass edge filters
`with deep blocking and extended regions of high transmission. Traditionally, optical filters are based on a quarter wave stack
`design, in which alternating layers of high and low refractive index materials are deposited on a glass substrate in quarter-
`wave optical thicknesses6. As more layers are deposited, higher blocking and steeper edge slopes are achieved. The number
`of layers that can be deposited using optical monitoring is limited since the growth of the filter is typically monitored in the
`reflection band. Alpha technology avoids monitoring in the reflection band, so the number of layers that can be deposited is
`limited in practice only by the stress characteristics of the film. Zinc sulfide and cryolite (Na5A13F14) are an ideal pair of
`materials for coatings in the visible region, with a good net balance between tensile and compressive stress7. With Alpha
`technology employed in the deposition of ZnS/ cryolite films, coatings in excess of 100 layers have been achieved. Another
`ideal combination of materials for multilayer coatings is TiO2 and Si02, which have the added advantage of durability and
`abrasion resistance.
`
`In this paper we demonstrate the application of Alpha technology to shortpass edge and bandpass filters for multiphoton
`fluorescence applications. These filters are deeply blocking in the near infrared region of the spectrum, and highly
`transmitting in the visible. We present theoretical results showing that blocking as high as optical density 9 is achieved. A
`dichroic mirror with high reflection in the near infrared and high transmission throughout the visible is presented.
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`48
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`Multiphoton Microscopy in the Biomedical Sciences, Ammasi Periasamy, Peter T. C. So, Editors,
`Proceedings of SPIE Vol. 4262 (2001) © 2001 SPIE · 1605-7422/01/$15.00
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`0001
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`2. SHORTPASS EDGE FILTERS
`
`Alpha technology was used to fabricate shortpass edge filters composed of alternating layers of ZnS and cryolite. These
`materials are evaporated onto a glass substrate in an evacuated bell jar using filament heated crucible sources. These are soft
`and hygroscopic materials, so they must be protected from the ambient environment with an epoxy-cemented cover slip. The
`shortpass edge filters are constructed on float glass with no absorption glass components. Figure 1 illustrates an example of a
`shortpass edge filter that is blocking (reflecting) from 750-975 nm and transmitting down to 475 nm. This filter is composed
`of 41 layers, and the maximum blocking is greater than optical density 5. Measurements of blocking are limited to
`approximately optical density 5 (0.001% transmission) due to spectrophotometer signal-to-noise limitations.
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`480
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`520
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`640 680 720
`560 600
`Wavelength (nm)
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`760
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`500
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`600
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`700
`Wavelength (nm)
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`1000
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`1100
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`800:900
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`Figure 1 : Transmission and blocking curves of shortpass edge filter designed for laser in 750-975 nm region.
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`We can calculate the level of blocking with commercially available thin film software. TFCa1c3.4 (Software Spectra, Inc.)
`was used to model the transmission and blocking curves of the filter shown in Figure 1 . The resulting theoretical curves are
`shown in Figure 2. We find that the maximum optical density achieved is 9.3, between the wavelengths of approximately 820
`nm and 860 nm. Blocking is greater than optical density 5 from 736 nm to 970 nm. The difference in edge slope between the
`measured and theoretical curves is attributed to the experimental measuring conditions. All spectra presented in this work
`were measured by a Cary 5 UV-Vis spectrophotometer (Varian) using an f/8 beam. The theoretical curves in Figure 2 were
`calculated using a perfectly collimated f/c' beam. The effect of an increase in the incident angle on a dielectric interference
`filter coating is a shift in the transmitted spectrum to shorter wavelengths. When a slightly divergent beam is used to measure
`a transmission spectrum, the resulting spectrum is an average of the angles of incidence contained within that incident beam.
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`10
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`26)-
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`4.
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`0
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`80 520 560 600 640 680 720 760
`Wavelength (nm)
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`0
`500
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`600
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`700
`900
`800
`Wavelength (nm)
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`1000 1100
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`1 : 2
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`0 4
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`I
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`Figure 2: Theoretical transmission and blocking curves for the shortpass filter shown in Figure 1.
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`Using the same materials it is possible to fabricate a filter that is blocking at lower wavelengths and transmitting to 400 nm.
`Figure 3 shows the transmission and blocking curves of a shortpass edge filter with 45 layers. Blocking is greater than optical
`density 5 in the 650-750 nm wavelength range. In this case the absorption edge of ZnS determines the position of the cut-off
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`Proc. SPIE Vol. 4262
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`20r 7
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`450
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`60
`550
`Wavelength (nm)
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`750
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`Figure 3 : Transmission and blocking curves of shortpass edge filter designed for laser in 650-750 nm region.
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`3. BANDPASS FILTERS
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`Bandpass filters with high transmission, steep slopes, and deep out-of-band blocking are made by combining Alpha shortpass
`and longpass edge filters. The center wavelength position of the filter is determined by the placement of the edges. The
`steepness of each edge may be independently controlled by adjusting the number of layers in the longpass and shortpass
`component filters. The transmission and blocking curves of such a filter are shown in Figure 4. The center wavelength of the
`pass band is 468 nm, and the bandwidth (full width at half maximum) is 40 nm. Peak transmission is approximately 80%, and
`out-of-band blocking is better than optical density 6. This filter is an immersed coating consisting of approximately 100
`alternating layers of ZnS and cryolite. Extra blocking in the 700-1 100 nm region is provided by a 2 mm thickness of BGG22
`absorption glass. A bandpass filter will transmit the fluorescence signal of interest and reject the scattered laser light along
`with all other interfering wavelengths.
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`I I ft
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`7Ô0
`Wavelength (nm)
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`9Ô0
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`iibo
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`20on00 21t\i
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`400
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`500
`450
`Wavelength (nm)
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`550
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`300
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`5Ô0
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`100
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`80
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`60
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`40
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`..cE
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`Figure 4: Transmission and blocking curves of bandpass filter centered at 468 nm.
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`A dichroic mirror for multiphoton fluorescence microscopy applications is illustrated in Figure 5.This filter is designed for
`use at a 45° angle of incidence to reflect the laser line and transmit the fluorescence signal. It is highly reflecting at
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`4. DICHROIC FILTERS
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`50
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`Proc. SPIE Vol. 4262
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`edge of the filter, at approximately 390 nm. In order to achieve transmission below 400 nm, it would be necessary to use a
`different material with an absorption edge further into the UV. Hafnium oxide has been shown to be a good high index
`material for use in the UV8, with an absorption edge at approximately 215 nm.
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`6
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`C
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`—5)43 ,&. '
`aLT350
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`100
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`80
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`60
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`40
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`.! E
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`C
`350
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`450
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`550
`650
`Wavelength (nm)
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`750
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`0003
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`wavelengths longer than 750 nm, and highly transmitting down to approximately 400 nm. This filter is an exposed coating
`composed of Ti02 and Si02, which are hard oxide coatings. Since deep blocking is not required, a standard quarter wave
`stack design is used. A hard oxide antireflection (AR) coating has been applied to the uncoated side of the substrate to boost
`visible transmission. To insure minimal autofluorescence, the filter is deposited on a fused silica substrate. By changing the
`deposition monitor wavelength it is possible to shift the edge position to accommodate other laser lines. However, the
`absorption edge of titania is located at approximately 400 nm. In order to achieve transmission further into the UV, a material
`with an absorption edge lower than that of Ti02 must be used.
`
`IOU
`
`300 400 500 600 700 800 900 1000
`Wavelength (nm)
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`Figure 5: Transmission curve of dichroic mirror measured
`at 45ang1e of incidence.
`
`5. SUMMARY
`
`A new deposition technology has been applied to the production of shortpass edge and bandpass filters for multiphoton
`fluorescence applications. This technology allows for the deposition of a large number of dielectric layers, so deep blocking
`of the scattered laser light is achieved. The shortpass edge filters have wide regions of transmission and approximately 100
`nm wide regions of blocking, providing flexibility with tunable laser sources. A bandpass filter with high transmission in the
`pass band and deep out-of-band blocking has been presented. This would be ideal for applications in which the laser
`excitation wavelength is fixed and there is only one fluorescence signal of interest. An example of a dichroic mirror with high
`reflection throughout the near infrared and high transmission throughout the visible has been presented for microscopy
`applications.
`
`6. REFERENCES
`
`1 .
`
`J. Wei, E. Okerberg, J. Dunlap, C. Ly, and J. B. Shear, "Determination of Biological Toxins Using Capillary
`Electrokinetic Chromatography with Multiphoton-Excited Fluorescence," Anal. Chem. 72, pp. 1360-1363, 2000.
`2. J. B. Shear, B. B. Brown, and W. W. Webb, "Multiphoton-Excited Fluoroscence of Fluorogen-Labeled
`Neurotransmitters," Anal. Chem. 68, pp. 1778-1783, 1996.
`3. A. Van Orden, H. Cai, P. M. Goodwin, and R. A. Keller, "Efficient Detection of Single DNA Fragments in Flowing
`Sample Streams by Two-Photon Fluorescence Excitation," Anal. Chem. 71, pp. 2108-21 16, 1999.
`4. D. N. Fittinghoff, P. W. Wiseman, and J. A. Squier, "Widefield Multiphoton and Temporally Decorrelated Multifocal
`Multiphoton Microscopy," Optics Express 7, pp. 273-279, 2000.
`5. D. C. McCarthy, "A Start on Parts for Multiphoton Microscopy," Photonics Spectra 33, pp. 80-88, 1999.
`6. H. A. Macleod, Thin Film Optical Filters, McGraw-Hill, New York, 1989.
`J. D. Rancourt, Optical Thin Films User's Handbook, McGraw-Hill, New York, 1987.
`7.
`8. T. R. Jensen, R. L. Johnson, Jr., J. Ballou, W. Prohaska, and S. E. Morin, "Environmentally Stable UV Raman Edge
`Filters," Soc. Vac. Coaters 43rd Tech. Con. Proc., 239-243 (2000).
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`Proc. SPIE Vol. 4262
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