Inexpensive, High-Quality Optical Relay for Use in Confocal Scanning Beam Imaging

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1 SCANNING VOL. 22, (2000) Received: October 18, 1999 FAMS, Inc. Accepted: February 21, 2000 Inepensive, High-Quality Optical Relay for Use in Confocal Scanning Beam Imaging A.C. RIBES, S. DAMASKINOS, A.E. DIXON Scanning Laser Microscopy Lab, Guelph-Waterloo Program for Graduate Work in Physics, University of Waterloo, Waterloo, Ontario, Canada Summary: An inepensive, high optical-quality relay lens made up of two eyepieces arranged in an afocal assembly for use in confocal scanning laser imaging is described. In the past we have used relays, within our confocal microscopes, made up of achromats with long focal lengths ( 10 cm), which take up large optical tracks and suffer from significant amounts of astigmatism and curvature of field. We quantify aberrations associated with achromat and eyepiece relays using CODE V optical design and analysis software. The eyepiece relay is found to be more compact, better corrected, and not significantly more epensive than its achromat counterpart. In addition to being used to interconnect two scanning mirrors optically as well as scanning mirrors with microscope objectives, it can form part of the optics in a confocal scanning laser MACROscope -Microscope system (Biomedical Photometrics, Inc., Waterloo, Ontario, Canada). Due to design constraints, the MACROscope -Microscope system cannot incorporate a conventional wide-field microscope into its structure such as is done in most commercial confocal microscopes. The eyepiece relay is used as a stand-alone, compact optical link between the scanning mirrors and the microscope objective. This consequently makes the MACROscope -Microscope system more compact and easier to commercialize. Key words: optical relay, eyepiece relay, confocal microscopy/macroscopy, MACROscope, optical analysis PACS: T, F, 42 Introduction A relay lens (DeJager 1980, Smith 1990, Tomkinson et al. 1996) is commonly used in confocal microscopy for optical interconnection of the last scanning mirror with an infinity-corrected microscope objective, as seen in Figure 1. Most commercial confocal microscopes are built around conventional wide-field microscopes that contain a relay lens consisting of an eyepiece followed by a tube lens. Figure 1 shows a schematic of the MACROscope -Microscope (Biomedical Photometrics, Inc., Waterloo, Ontario, Canada) (Dion and Damaskinos 1998; Dion et al. 1995; Ribes et al. 1995, 1996) developed by the confocal microscopy lab at the University of Waterloo s department of physics. It can image (10 s frame rate) specimens from µm up to 7.5 cm 7.5 cm in size by utilizing a sliding turret in order to switch between MACROscope and microscope modes. A full wide-field microscope cannot be easily incorporated within the MACROscope -Microscope, and therefore a stand-alone relay lens must be used. Traditionally we have used a pair of achromats forming a unitary telescope as a relay lens. We report the aberrations associated with using achromats in relay lenses and suggest an alternative relay lens consisting of telescope eyepieces. This eyepiece relay provides less aberrations than achromat relays in a more compact design without raising the price of the relay ecessively. Materials and Methods The Microscope and Relay Address for reprints: Dr. Alfonso Ribes Physics Department University of Waterloo 200 University Ave. Waterloo, Ontario, Canada, N2L 3G1 aribes@uwaterloo.ca The relay lens configurations shown in this article are intended to be used primarily within the contet of a MACROscope -Microscope instrument, as seen in Figure 1. The relay lens optics are therefore evaluated with scan parameters used on our homemade confocal scanning laser microscope. The microscope starts with a 5 mm collimated laser beam typically from a He-Ne (633 nm) or an Ar-Ion (488 nm) laser source. The collimated beam strikes a dichroic beam splitter which preferentially reflects the laser wavelength and transmits all wavelengths 10 to 15 nm longer than the laser wavelength. The collimated

2 A.C. Ribes et al.: Optical relay for use in confocal imaging 283 beam then strikes two G120DT General Scanning galvanometer optical scanners (General Scanning, Inc., Watertown, Mass., USA) which can accommodate 5 mm beams. These scanners are positioned orthogonal to each other such that one scanner moves the beam in the - direction while the other does so in the y-direction. The pivoting motion generated by the scanners is relayed by the unitary telescope or relay lens to the entrance pupil of an infinity-corrected Mitutoyo microscope objective (Mitutoyo, Aurora, Ill. USA). Figure 2 shows a schematic diagram of a relay lens in operation consisting of two identical simple lenses assembled in an afocal (infinite focus) unitary assembly. A collimated laser beam enters lens L1 at an angle θ with respect to the optic ais. The beam pivots about point f 11, the front focal point of lens L1. The beam is focused telecentrically toward the common focal plane of L1 and L2 at f. The beam is referred to as telecentric because the focusing cone is always at right angles to the focal plane independent of the scan angle θ. By symmetry, the behavior of the beam after the common focal plane is the same as before the focal plane, hence a collimated beam emerges from lens L2 pivoting with angle θ about point f 22, the back focal point of Laser Beam epander y Beam splitter Detector Confocal pinhole Detector lens lens L2. The pivoting motion at point f 11 has been relayed to point f 22. The afocal assembly of Figure 2 is also known as a unitary telescope because the input and output beams have the same and scan angle. The relay lens need not be unitary; however, if it is, and identical lens elements are used in L1 and L2, then aberrations such as coma, distortion, and lateral chromatic aberrations are eliminated due to the symmetry principle (Smith 1990). In one particular configuration we use a 2 telescope to epand the 5 mm beams to a 10 mm beam to overfill the entrance pupil of the microscope objective fully. For a 2 telescope, the eit beam (2D) is twice the size of the entrance beam (D), and the eit scan angle is half the size of the entrance angle as shown in Figure 2b. The ratio of entrance and eit beam s and the ratio of entrance and eit scan always follows this inverse relationship. Consequently, if the relay epands the beam to overfill the entrance pupil of a microscope objective, then the entrance scan angle must also be increased to maintain the same field-of-view coverage. After passing through the relay in Figure 1, the collimated laser beam now pivots about the entrance pupil of the microscope objective. For small (<10 ) scan angles, the focus spot displacement from the optic ais is proportional to the scan angle. Light reflected or emitted from the focus spot will return through the microscope objective and the relay lens system, and will be descanned by the scanners. The emitted light will be transmitted by the beamsplitter and focused by a 10 cm focal length achromat onto a confocal pinhole ranging from 10 to 100 µm in size. The fact that both the ecitation and emission beams pass through the relay lens requires high-quality optics to be used if confocality-destroying aberrations are to be avoided. z Scanning mirrors y MACROscope laser scan lens Unitary telescope or relay lens (a) f 11 θ L1 Focal plane f L2 Right-angle cone (telecentric) y f 22 θ z L1 L2 Specimen stage Microscope objective f 11 D 2D f 22 2θ f 2f θ FIG. 1 The confocal scanning laser MACROscope -Microscope. A unitary telescope or relay lens is used to connect optically the last scanning mirror with the entrance pupil of the microscope objective. (b) FIG. 2 (a) A unitary telescope or relay consisting of two identical lenses arranged in an afocal (infinite focus) assembly. (b) A nonunitary optical relay consisting of a 2 epanding telescope.

3 284 Scanning Vol. 22, 5 (2000) Lens Analysis Geometric raytracing and diffraction analyses of the achromat relays and the eyepiece relay were performed with CODE V versions 8.20 and 8.30, which is a comprehensive program for design and analysis of optical systems created by Optical Research Associates (Pasadena, Calif., USA). CODE V was run on various Pentium II-based systems and, for this article, was primarily used to generate point spread functions (PSFs). The PSF gives the intensity as a function of position of a focused laser spot. More specifically, CODE V can find the of the circle needed to enclose a particular fraction of the total focused energy. By averaging the 80 and 90% encircled energy s, an estimate for the 85% encircled energy value is obtained which approimately corresponds to the of an Airy disk. sizes are given as a function of scan angle for various relay configurations. CODE V can only analyze focal assemblies, and therefore an ideal lens is introduced after the relay lens to focus the collimated beam down to a focused spot. Results In the past, the Confocal Microscopy Lab has used a pair of identical achromatic doublets to form a relay lens. Achromats are inepensive and were thought to provide good performance with small scan angles and beam s. The question of relay quality first comes up when looking at highly confocal reflected-light images of flat specular reflectors such as mirrors. The images appeared nonuniform which suggested several possibilities such as instrument misalignment, pupil or beam wobble because of the nontelecentric configuration of the scan mirrors, aberrations in the objective lens, or aberrations in the relay optics which are dealt with in this article. Contributions from the relay optics come about due to aberrations associated with achromats. Achromats are well corrected for spherical aberration, coma, and aial chromatic aberration. A unitary relay will furthermore eliminate lateral chromatic and distortion (Smith 1990). What is left is astigmatism and curvature of field which both depend linearly on the beam and quadratically on the scan angle or field size. Figure 3 shows PSFs for the center and the corner positions on the field of view. A 25 mm focal length ideal lens was used to focus a 5 mm collimated beam emerging from the relay. The relay consists of a unitary telescope made up of two f = 100 mm, 50 mm Edmund Scientific model number D45,353 achromats (Edmund Scientific, Barrington, N.J., USA). The most curved surfaces were placed facing outward so as to minimize spherical aberration. The maimum scan angle or field angle used was 2.8 which gives maimum field coverage when using the Mitutoyo objectives. The following wavelengths were used (for which the achromats and eyepieces were optimized): the blue hydrogen F line (486.1 nm), the yellow helium d line (587.6 nm) and the red hydrogen C line (656.3 nm). Figure 3a shows a PSF for a 0 and 0 y field angle, that is, at the center of the field of view along the optic ais. The dotted white circle denotes the area in which 85% of the spot s energy are contained. The of this circle roughly corresponds to the Airy (Born and Wolf 1991) and is equal to 9.9 µm, which is higher than what is obtained when only the ideal lens is used (8.5 µm). This is to be epected because some residual spherical and aial chromatic aberration still remains. Figure 3b shows a PSF for the corner position in the field corresponding to a field angle of 2.8 in both the and y directions. In this case, the 85% encircled energy is 23.2 µm. The spot is significantly elongated along the radial direction which is typical of astigmatism. Eyepiece Telescope As demonstrated in Figure 3, even a relatively long focal length achromat eperiences aberrations at small scan angles and beam s. Early in the development of the MACROscope -Microscope, the Confocal Microscopy Lab was interested in finding an alternative to the achromat relay which could meet three criteria: Less aberrations. A reduction in off-ais aberrations such as astigmatism and curvature of field was clearly required. A more compact design. Achromat relays occupy four times the focal length of the achromat. The 10 cm focal length achromat relay takes up 40 cm, which is too large to be used in a commercial MACROscope -Microscope system. No radical increases in price. If one is willing to spend more than $10,000, then it is relatively easy to develop a fully corrected relay. To keep costs down below $1,000, the choice of lenses was restricted to stock items. One solution that came to mind almost immediately was to utilize what is being used in commercial confocal microscopes. Figure 4 shows part of a confocal microscope consisting of a scanning mirror coupled to a conventional wide-field microscope using an infinity-corrected objective. The combination of the eyepiece and tube lens (Yonekubo 1982) acts as a nonunitary relay between the scanning mirror and the objective, which at first seems to be an adequate solution. While eyepieces are easily purchased as individual components, this is not the case for tube lenses which are part of the microscope body and in many cases consist of a series of epensive air-spaced components. Instead of a tube lens one can use an eyepiece which is not as well corrected but provides advantages associated with unitary systems. Instead of using eyepieces from microscopes, telescope eyepieces were used. Telescope eyepieces are commonly designed to be used at

A.C. Ribes et al.: Optical relay for use in confocal imaging 285 Intensity 100% 85% Encircled energy 50% 10 µm 0% 10 µm 0 Degree field angle Scan position 2.8 2.8 Degree field angle (a) (b) FIG.

4 A.C. Ribes et al.: Optical relay for use in confocal imaging 285 Intensity 100% 85% Encircled energy 50% 10 µm 0% 10 µm 0 Degree field angle Scan position Degree field angle (a) (b) FIG. 3 Point spread functions or intensity distributions for laser spots located at (a) the center and (b) the corner on the field of view of a simulated microscope objective. The 85% encircled energy roughly corresponds to the Airy disk. Scanning mirror Eyepiece Tube lens Infinity-corrected objective FIG. 4 A typical commercial confocal microscope consisting of a scan assembly coupled to a conventional wide-field microscope. The combination of eyepiece and tube lens within the wide-field microscope makes up a nonunitary relay lens. night when the eye s pupil can reach up to 7 mm in compared with 2 mm in the daytime. Telescope eyepieces offer superior corrections and larger fields of view than microscope eyepieces. The final design chosen for comparison with the achromat relays is shown in Figure 5. The eyepiece relay consists of two Edmund Scientific Erfle (Erfle 1923) eyepieces (f = 32 mm, model number P41,347) arranged back-to-back in an afocal assembly. These lenses are representative of typical, average quality eyepieces and serve as a good baseline for comparison with achromats. For a fair comparison of the eyepiece relay with various achromat relays, a common evaluation method was implemented using CODE V. The entrance pupil, that is, the collimated beam was set to 5 mm. The wavelengths used were 486.1, 587.6, and nm. An ideal lens with a 25 mm focal length was used after the relay to generate a PSF. Point spread functions were evaluated at four positions on the field of view: center (y-field angle = 0 ), halfway (y-field = 1.4 ), edge (y-field = 2.8 ), and corner (field = 2.8, y-field = 2.8 ). The average of the 80 and 90% encircled energy was taken to be the spot. These field angles and beam s closely simulate what is commonly used on our confocal microscope. Table I shows spot s as a function of position for achromat relays with 30, 60, and 100 mm focal lengths, and the Erfle eyepiece relay. In all cases a best-focus routine was used in CODE V to minimize the spot for all field angles simultaneously. Consequently, the half and edge positions tend to give the smallest spot s. The

5 286 Scanning Vol. 22, 5 (2000) ais position (0 ) spot for the f = 100 mm achromat relay is larger than 9.9 µm because it has been defocused in order (primarily) to reduce the corner position spot. The Erfle relay performs similarly at the on-ais, half, and edge positions when compared with the f = 100 mm achromat relay, and it outperforms the achromat relay by 23% at the corner position. This is to be epected since Erfle eyepieces are superior to achromats off-ais while only offering a nominal improvement on-ais. The Erfle relay is twice as compact as the f = 100 mm achromat relay, and it is twice as costly, which is still relatively inepensive given that an achromat costs about $100. The f = 60 mm achromat relay is as compact as the Erfle relay but provides a substantially inferior performance. The corner position spot for the f = 60 mm achromat relay is nearly 2.5 larger than that of the Erfle at the corner position. The f = 30 mm achromat relay has a focal length similar to the Erfle eyepiece. The amount of correction provided by the Erfle eyepiece compared with an achromat is clearly evident. The f = 30 mm achromat relay spot is at best three times the size of the spot produced by the Erfle relay. The relationship between achromat focal length and astigmatism is simple: the shorter the focal length, the larger the astigmatism. Good achromat relays are therefore large relays. The use of eyepieces gives broader range of choices since cost/quality is included as another variable. Lower quality eyepieces include Kellner, Orthoscopic, and Plössl types, while Nagler eyepieces (Tele Vue Optics, Suffern, N.Y., USA), for eample, offer higher quality than Erfle s at an increased cost. the field of view, but occupies a 40 cm optical track which is too large for all but laboratory instruments and therefore is not desirable on a commercial MACROscope -Microscope instrument. The Erfle eyepiece relay has been used to show that a compact, well-corrected relay is possible even when very short focal length lenses are used. This opens the possibility of combining achromats with eyepieces to achieve an optimum cost, quality, and compactness combination. In a recent eperiment, an f = 55 mm Televue Plössl eyepiece was combined with an f = 120 mm achromat to achieve an epanding telescope that could accommodate an objective carousel. This particular combination will allow for all objectives to be properly overfilled due to the epansion ratio, as well as providing optical performance similar to the Erfle relay. In addition to being used as an optical coupler between scan mirrors and a microscope objective, the eyepiece relay can be used between two scan mirrors to produce a beam that pivots at a single point (Stelzer 1995). An all-reflective relay for this purpose has been described by Amos (1991). The relay consists of two concave mirrors arranged in an afocal assembly and has the advantage of being corrected at all wavelengths including ultraviolet and infrared. If parabolic mirrors are used (Rosin et al. 1974, Wetherell 1987, Wetherell et al. 1974), then very high-quality relays can be achieved. The eyepiece telescope described in this paper is as well corrected as a parabolic mirror relay with similar track length. The eyepiece telescope is easier to align and substantially cheaper due to the high cost of optical quality parabolic mirrors (about 4 times more than eyepieces). Discussion The authors have shown that for small scan angles (2.8 ) and beam s (5 mm) associated with confocal microscopy, relay lenses consisting of a pair of achromatic doublets show effects from aberrations such as astigmatism and curvature of field. The longer the focal length, the less aberrations the relays will produce. An f = 100 mm achromatic telescope performs well, ecept near the corners in f 11 Entrance beam Eyepiece relay First Erfle eyepiece FIG. 5 A unitary relay lens made up of two identical Edmund Scientific Erfle eyepieces arranged in an afocal assembly. f 22 Eit beam Second Erfle eyepiece TABLE I size comparison table for f = 30, 60, 100 mm achromat relays and the Erfle (f = 32 mm) eyepiece relay. The laser spot positions (field angles) on the field of view are as follows: ais (0 ), half-way (1.4 ), edge (2.8 ), and corner ( ) f = 100 mm f = 32 mm Erfle Achromat relay eyepiece relay Position (µm) Position (µm) Ais 12.4 Ais 11.8 Half 11.0 Half 12.0 Edge 12.1 Edge 12.9 Corner 18.6 Corner 14.3 f = 60 mm f = 30 mm Achromat relay Achromat relay Position (µm) Position (µm) Ais 18.9 Ais 41.5 Half 15.1 Half 35.4 Edge 19.2 Edge 38.8 Corner 34.9 Corner 64.7

6 A.C. Ribes et al.: Optical relay for use in confocal imaging 287 Conclusions The effect on beam quality in a confocal microscope due to optical relays made up of achromats has been quantified for three different focal lengths. As the focal length increases, aberrations decrease. However, even for relatively large focal lengths (f = 100 mm), small scan angles (2.8 ), and small beam s (5 mm), significant aberrations are present near the corners on the field of view. Although it was clear from the beginning that higher quality lenses (triplets, laser scan lenses, etc.) of similar focal length would perform better than achromats, the degree of improvement was not clear. The authors have shown that an optical relay made up of Erfle telescope eyepieces with focal lengths equal to 32 mm is better corrected than an f = 100 mm achromat relay. In addition, the eyepiece relay is twice as compact while remaining under $500 in cost. It has been shown that utilization of telescope eyepieces is well worth the increase in cost, yielding higher quality, more compact optical systems. This innovative design makes possible the commercial viability of the MACROscope -Microscope as it has improved the optical quality and reduced the optical path length of the system without increasing the cost significantly. Acknowledgments The authors would like to thank the Natural Sciences and Engineering Research Council and Photonics Research Ontario for grants that have supported the development of the MACROscope -Microscope. MACROscope is a registered trademark of Biomedical Photometrics. References Amos WB: Achromatic Scanning System. U.S. Patent 4,997,242 (1991) Born M, Wolf E: Principles of Optics, 6 th Edition. Pergamon Press, Oford (1991) 398 DeJager D: Camera viewfinder using tilted concave mirror erecting elements. SPIE 237, (1980) Dion AE, Damaskinos S: Apparatus and Method for Scanning Laser Imaging of Macroscopic Samples. U.S. Patent 5,760,951 (1998) Dion AE, Damaskinos S, Ribes A, Beesley KM: A new confocal scanning beam laser MACROscope using a telecentric, f-theta laser scan lens. J Microsc 178(3), (1995) Erfle H: Ocular. U.S. Patent 1,478,704 (1923) Ribes AC, Damaskinos S, Dion AE, Carver GE, Peng C, Fauchet PM, Sham TK, Coulthard I: Photoluminescence imaging of porous silicon using a confocal scanning laser MACROscope /Microscope. Appl Phys Lett 66(18), (1995) Ribes AC, Damaskinos S, Tiedje HF, Dion AE, Brodie DE: Reflected-light, photoluminescence and OBIC imaging of solar cells using a confocal scanning laser MACROscope /Microscope. Solar Energy Mat Solar Cells 44(4), (1996) Rosin S, Amon M, Laakmann P: Afocal parabolic reflectors. Appl Opt 13(4), (1974) Smith WJ: Modern Optical Engineering, 2 nd Edition. McGraw-Hill, New York (1990) 239, 372 Stelzer EHK: The intermediate optical system of laser-scanning confocal microscopes. In Handbook of Biological Confocal Microscopy, 2 nd Edition,, Chapter 11 (Ed. Pawley JB). Plenum Press, New York (1995) Tomkinson TH, Bentley JL, Crawford MK, Harkrider CJ, Moore DT, Rouke JL: Rigid endoscopic relay systems: A comparative study. Appl Opt 35(34), (1996). Wetherell WB: All-reflecting afocal telescopes. SPIE 751, (1987) Wetherell WB, Rimmer MP: Confocal paraboloids: Some comments. Appl Opt 13(10), 2192 (1974) Yonekubo K: Afocal Relay Lens System. US Patent 4, 353, 624 (1982)

Point Spread Function. Confocal Laser Scanning Microscopy. Confocal Aperture. Optical aberrations. Alternative Scanning Microscopy

Point Spread Function. Confocal Laser Scanning Microscopy. Confocal Aperture. Optical aberrations. Alternative Scanning Microscopy Bi177 Lecture 5 Adding the Third Dimension Wide-field Imaging Point Spread Function Deconvolution Confocal Laser Scanning Microscopy Confocal Aperture Optical aberrations Alternative Scanning Microscopy