- Email: [email protected]
Stray light and noise in confocal microscopy
Moror~ rod Mo
prro Aura Vol. 22. No 3, pp 239—243. 1991 Pnnted n Great Brrtain.
(
0739—626(19! 53.00+0.0(1 99] Pergamon Prens pie
STRAY LIGHT AND NOISE IN CONFOCAL MICROSCOPY C. J. R.
SHEPPARD
Physical Optics Department, School of Physics, University of Sydney. NSW 2006, Australia )Receired 27 March 1991)
Abstract--- The stray light and signal levels in a confocal microscope have been measured. These quantities determine the minimum reflectivity which can be observed with the system, as well as characterizing its noise performance and assisting with choice ofappropriate pinhole size.
INTRODUCTION Confocal microscopy allows imaging of very thin optical sections either in the fluorescence or in the reflection (non-fluorescence) modes. From these optical sections a three-dimensional image of a thick object can be built up. However, the strength of either the fluorescent signal or the reflection signal from light scattered by inhomogeneities within a biological object (Koester et a!., 1989) can be very weak. In practice the signal can be increased by using a larger confocal pinhole, but this has the undesirable effects of decreasing axial and also lateral resolution. It is thus important from a practical point of view to know how these various properties of the confocal system vary with pinhole size. The image signal must be observed in the presence of stray light and noise in the microscope system. The strength of stray light has been considered theoretically by Cox and Sheppard (1986), and experimentally for a fluorescence system by Wells eta!. (1990). However, these papers did not consider the absolute strength of the stray light. Similarly, the strength of the signal was considered by Cox and Sheppard (1986), and measurements reported by Sheppard eta!. (1991) for a reflection system and by Wells et a!. (1990) for fluorescence. Theoretical treatments of signal level for systems in which the confocal aperture is a slit, or in a fluorescence microscope, have also been presented (Sheppard and Mao, 1988; Sheppard eta!., 1991; Gu and Sheppard, 199la). However, to the author’s knowledge no absolute measurements on the strength of stray light have yet been reported. Such measurements help the microscope user to choose an appropriate pinhole size, as well as understanding the noise behaviour of the system. In addition, information on the source of stray light can help with the reduction of its strength. EXPERIMENTAL Experiments were conducted on the confocal microscope in the Department of Engineering Science at the University of Oxford. This microscope, which employs mechanical scanning of the specimen stage under a stationary light spot, is constructed of individual optical components arranged on an optical table. The design allows the flexibility for investigating various optical arrangements. The optical system and scanning stage were developed by the author together with D. K. Hamilton. The latter 239
240
C. J. R. Sheppard
also developed the analog electronic system of the microscope. The optical system uses collimated beams and infinity corrected objectives. If used with objectives which are not corrected for infinite tube-length a correction lens is used. No polarization elements were used in the system to reduce stray light levels. A series of measurements were made of both the signal, from a good reflector as object, and the stray light strength for a range of pinhole sizes. A HeNe laser (633 nm wavelength) was used as source. The measurements were all made with a constant photomultiplier tube supply voltage, and neutral density filters used to alter the signal so that it was in a measurable range. The stray light was measured by removing the object from the system. As the stray light was rather weak the experiment was conducted in a darkened room and the signal passed through a 2 Hz low-pass filter. Figure I illustrates results from a Leitz x 32 lens of numerical aperture 0.5. The points are experimental measurements and the curves theoretical predictions. Here the coordinate t’4 is the normalized pinhole radius, which is related to the true pinhole radius r by
where p is the radius of the optical beam and d the distance from the collimating lens to the pinhole. The values for flare are the measured strengths of the stray light divided by the total signal recorded for a large pinhole. As previously (Sheppard et a!., 1991), very good agreement was found between the variation in the strength of the signal with pinhole size measured and predicted by theory. These measurements were used to calibrate the optical coordinate i’d. It was found that p had a value 3.65 mm and a’ was equal to 200 mm. Measurements were made with pinholes of 5, 10 and 50 ~im in diameter. Ifthe stray light is uniformly distributed over the pinhole plane, its strength is proportional to the pinhole area, as shown by the dotted line in Fig. 1, which gives a good fit to the experimental points. The flare varied between 10-0 and 10-6 of the total signal for the pinhole sizes used. This decreases with decreasing pinhole size and is wellfitted by the theoretical curve shown. For small pinhole sizes the ratio signal/stray light becomes constant at about 2.5 x i0~.This means that the reflectivity of the object can be as low as 4 x l0 8 before the signal is equal in strength to the stray light. To put this in perspective, it has been reported that reflections from features in animal cells arc in the range l0~ to l0~ (Boyde and Petráñ, 1990). Broadly similar results were
0
—‘a a
8 io
2’ >.
Fig. I. The measured (points) and theoretical lines) flare (the stray light divided by the signal recorded for a perfect reflector and large pinhole) as a function of normalized pinhole size The measured (points) and theoretical (lines) signal/stray light ratio is also shown. The normalized pinhole radius i’~,was calibrated from measurements of signal strength. and the theoretical plots are fitted to the experimental data.
Stray Light and Noise in Confocal Microscopy
241
obtained using other objectives such as a Zeiss Neofluar IOOX, numerical aperture 1.3, and a Zeiss Epiplan 40 x numerical aperture 0.85. ,
DISCUSSION Koester (1989) has suggested that half-plane apertures can be used to reduce the strength of stray light. It may be true that they can indeed improve optical sectioning, especially when combined with a central obstruction (Koester et al., 1989; Sheppard and Gu, 1990, 1991; Gu and Sheppard, 1991 b). However, our experiments showed that half-plane apertures produced little effect on the stray light, except if they obstructed the central part of the beam. Further experiments confirmed that the major contribution (more than 90%) was reflection from the optical elements of the microscope objective. It should be noted that for the results presented here the beam was stopped down so that it only just filled the objective pupil. As these elements are curved surfaces, only reflection from the region near the optic axis can get through the system to the pinhole. This suggests that a small central obstruction could be used to reduce the stray light level even further. Another way of reducing stray light is to insert an iris diaphragm at the collimating lens which focuses the light on to the pinhole. The further the distance from the objective to the collimating lens, the lower the level of stray light. This method of improving optical sectioning has been discussed by Davidovitz and Egger (1971). Because the light reflected from the objective is brought to a focus by the collimating lens at some particular position, the axial position of the objective alters the strength of the stray light. We have found that if scanning in the axial direction is achieved by scanning of the objective lens, the strength of the stray light changes appreciably through the scan. This may prove an advantage for using axial scanning of the object itself instead. No attempt has been made in these experiments to optimize the stray light by alteration of the axial position of the objective. As the stray light is constant in strength throughout the image, it can be subtracted from the signal electronically to improve contrast at very low signal levels. The noise performance has been considered by Sheppard eta!. (1991). It is found that if the stray light is constant throughout the image the normalized signal to noise ratio is given by S N
/
—
~
—J~(vd)---J~(vd)
(2
where a is a parameter describing the effect ofstray light, which from our measurements has a value 4 x l0 8 for our system with a specimen which is a perfect reflector. Here we have assumed detector noise to be negligible. The finite quantum efficiency of the detector affects both signal and stray light and hence does not appear in the norma!ized signal to noise ratio. The signal to noise ratio (Fig. 2) rises from zero for small values of v4~and 9O% of its theoretical set by shot noise on the total that signal of Ed eventually dropslimit, for large values of Vd. This means thebeam, signalfor tovalues noise ratio is between about 3 and 5000. Interestingly, as the specimen reflectance decreases, the effective value of a increases in proportion, and there is less latitude in the choice of pinhole size. The optimum pinhole size for maximizing signal to noise ratio also decreases. The optimum value of Vd for a given effective value of a is shown in Fig. 3. The solid line is the exact theoretical variation, which it will be noticed, exhibits discontinuities. For a specimen reflectivity of 4 x 10- 8 for example, the optimum pinhole size has reduced to vd=2.3. For large values of a the optimum pinhole size levels off to about Ed = 2.1. The dashed line is an approximate curve, valid for larger values of i’d. This is calculated using the asymptotic values for the Bessel functions, and
242
C. .1. R. Sheppard a
1 0
S/N~
02~
1:0. 2. Signal to noise ratio to the presence ol flare. with sttength ch,iracteriied lii the paranletet’ it alter Sheppard at ii.. 1091
bc 80 Vd
40
20
6
io~2
‘
io.
$
1’
a ~. lie tiptimitni pinhole si/c I or inaxinti/itto sional ti ttttse ratIo is a litttctton ti lie ellectise utitie nI a 1 ite solid line is the exact sariatioti. I lie dashed line is the .tpprtt\itttItte \,tri,tttttii based tin .ts\iliptttttc s,tlues or the lbessel lttttctiotts
I’ to
b\ differentiation il can he sIio~~ ii that the optiniuni value ol equation 4t~ t4
er
4
~+
liii
S
~o.
i~ is
given b~a root ttl~thc
(~I
ttJr
Il should he noted that br small salues of ii the masimum is broad, so that choice ofait a ppropriate pinhole sue is not en t cal. The measurements described in this pai~errel’er to a particular microscope s slem. In 1/uct we expect that the stray light performance may prove better than in commercial microscopes for the following reasons. F irstly the microscope has an open design: there us no microscope body which itself can produce reflections and add to the stray light level. It might he argued that a microscope body can stop unwanted light travelling from one part oft he system to another, hut in practice we lou can stop such unwanted light paths by placement of suitable light hailles. Secondly. our system has nianv fewer opt cal coin ponen ts than a com mercial in icroscope based on a con ~cntuonal
Stray Light and Noise in Confocal Microscopy
243
microscope. In our system the only component between the beamsplitter and the object is the objective lens (and the correction lens if using non-infinity corrected objectives). Beam-scanning systems would also be expected to exhibit some variation in the stray light during scanning: we would expect the stray light perhaps to be reduced for off-axis image points. CONCLUSION The ratio of signal to stray light has been measured for a confocal microscope system and shown to have a value, for small pinhole sizes, of 2.5 x l0~. This places a lower limit on the specimen reflectivity which can be detected. REFERENCES Boyde, A. and Petráñ, M., 1990. Light budgets, light and heavy losses: one- or two-sided tandem scanning (real-time, direct-view, confocal) microscopy. J. Micro.sc., 160: 335—342. Cox, I. J. and Sheppard, C. J. R., 1986. Information capacity and resolution in anoptical system. J. Opt. Soc. Amer., A3: 1152 1158. Davidovitz, P. and Egger, M.D., 1971. Scanning laser microscope for biological investigations. App!. Opt., 10: 1615 1619. Gu, M. and Sheppard, C. J. R., 1991 a. Confocal fluorescent microscopy with a finite sized circular detector. J. Opu. Soc. Amer., submitted. Gu, M. and Sheppard, C. J. R., l991b. Three-dimensional imaging in confocal fluorescent microscopy with annular lenses. J. Mod. Opt., in press. Koester, C. J., Khanna, S. M., Rosskothen, H. and Tackaberry, R. B. 1989. Incident light sectioning microscope for visualization ofcellular structures in the inner ear. Acta Oto!aryngol. (Stockh.) Suppl., 467: 27 33. Sheppard, C. J. R. and Gu, M., 1990. Optical sectioning in confocal microscopes with annular pupil. Optik. Sheppard, C. J. R. and Gu, M., 1991. Improvement of axial resolution in confocal microscopy using an annular pupil. Opt. Commun., 84: 7 13. Sheppard, C. J. R. and Mao, M.. 1988. Confocal microscopy with slit apertures. J. Mod. Opt., 35: 11691185. Sheppard, C. J. R., Cogswell, C. J. and Gu, M., 1991. Signal strength and noise in confocal microscopy: factors influencing selection of an optimum detector aperture. Scanning, 13: 233-240. Wells. K. S., Sandison, D. R., Strickler, J. and Webb, W. W.., 1990. Quantitative fluorescence imaging with laser scanning confocal microscopy. In Handbook of Biological Confocal Microscopy, J. B. Pawley (ed). Plenum Press, New York, pp. 23 35.
prro Aura Vol. 22. No 3, pp 239—243. 1991 Pnnted n Great Brrtain.
(
0739—626(19! 53.00+0.0(1 99] Pergamon Prens pie
STRAY LIGHT AND NOISE IN CONFOCAL MICROSCOPY C. J. R.
SHEPPARD
Physical Optics Department, School of Physics, University of Sydney. NSW 2006, Australia )Receired 27 March 1991)
Abstract--- The stray light and signal levels in a confocal microscope have been measured. These quantities determine the minimum reflectivity which can be observed with the system, as well as characterizing its noise performance and assisting with choice ofappropriate pinhole size.
INTRODUCTION Confocal microscopy allows imaging of very thin optical sections either in the fluorescence or in the reflection (non-fluorescence) modes. From these optical sections a three-dimensional image of a thick object can be built up. However, the strength of either the fluorescent signal or the reflection signal from light scattered by inhomogeneities within a biological object (Koester et a!., 1989) can be very weak. In practice the signal can be increased by using a larger confocal pinhole, but this has the undesirable effects of decreasing axial and also lateral resolution. It is thus important from a practical point of view to know how these various properties of the confocal system vary with pinhole size. The image signal must be observed in the presence of stray light and noise in the microscope system. The strength of stray light has been considered theoretically by Cox and Sheppard (1986), and experimentally for a fluorescence system by Wells eta!. (1990). However, these papers did not consider the absolute strength of the stray light. Similarly, the strength of the signal was considered by Cox and Sheppard (1986), and measurements reported by Sheppard eta!. (1991) for a reflection system and by Wells et a!. (1990) for fluorescence. Theoretical treatments of signal level for systems in which the confocal aperture is a slit, or in a fluorescence microscope, have also been presented (Sheppard and Mao, 1988; Sheppard eta!., 1991; Gu and Sheppard, 199la). However, to the author’s knowledge no absolute measurements on the strength of stray light have yet been reported. Such measurements help the microscope user to choose an appropriate pinhole size, as well as understanding the noise behaviour of the system. In addition, information on the source of stray light can help with the reduction of its strength. EXPERIMENTAL Experiments were conducted on the confocal microscope in the Department of Engineering Science at the University of Oxford. This microscope, which employs mechanical scanning of the specimen stage under a stationary light spot, is constructed of individual optical components arranged on an optical table. The design allows the flexibility for investigating various optical arrangements. The optical system and scanning stage were developed by the author together with D. K. Hamilton. The latter 239
240
C. J. R. Sheppard
also developed the analog electronic system of the microscope. The optical system uses collimated beams and infinity corrected objectives. If used with objectives which are not corrected for infinite tube-length a correction lens is used. No polarization elements were used in the system to reduce stray light levels. A series of measurements were made of both the signal, from a good reflector as object, and the stray light strength for a range of pinhole sizes. A HeNe laser (633 nm wavelength) was used as source. The measurements were all made with a constant photomultiplier tube supply voltage, and neutral density filters used to alter the signal so that it was in a measurable range. The stray light was measured by removing the object from the system. As the stray light was rather weak the experiment was conducted in a darkened room and the signal passed through a 2 Hz low-pass filter. Figure I illustrates results from a Leitz x 32 lens of numerical aperture 0.5. The points are experimental measurements and the curves theoretical predictions. Here the coordinate t’4 is the normalized pinhole radius, which is related to the true pinhole radius r by
where p is the radius of the optical beam and d the distance from the collimating lens to the pinhole. The values for flare are the measured strengths of the stray light divided by the total signal recorded for a large pinhole. As previously (Sheppard et a!., 1991), very good agreement was found between the variation in the strength of the signal with pinhole size measured and predicted by theory. These measurements were used to calibrate the optical coordinate i’d. It was found that p had a value 3.65 mm and a’ was equal to 200 mm. Measurements were made with pinholes of 5, 10 and 50 ~im in diameter. Ifthe stray light is uniformly distributed over the pinhole plane, its strength is proportional to the pinhole area, as shown by the dotted line in Fig. 1, which gives a good fit to the experimental points. The flare varied between 10-0 and 10-6 of the total signal for the pinhole sizes used. This decreases with decreasing pinhole size and is wellfitted by the theoretical curve shown. For small pinhole sizes the ratio signal/stray light becomes constant at about 2.5 x i0~.This means that the reflectivity of the object can be as low as 4 x l0 8 before the signal is equal in strength to the stray light. To put this in perspective, it has been reported that reflections from features in animal cells arc in the range l0~ to l0~ (Boyde and Petráñ, 1990). Broadly similar results were
0
—‘a a
8 io
2’ >.
Fig. I. The measured (points) and theoretical lines) flare (the stray light divided by the signal recorded for a perfect reflector and large pinhole) as a function of normalized pinhole size The measured (points) and theoretical (lines) signal/stray light ratio is also shown. The normalized pinhole radius i’~,was calibrated from measurements of signal strength. and the theoretical plots are fitted to the experimental data.
Stray Light and Noise in Confocal Microscopy
241
obtained using other objectives such as a Zeiss Neofluar IOOX, numerical aperture 1.3, and a Zeiss Epiplan 40 x numerical aperture 0.85. ,
DISCUSSION Koester (1989) has suggested that half-plane apertures can be used to reduce the strength of stray light. It may be true that they can indeed improve optical sectioning, especially when combined with a central obstruction (Koester et al., 1989; Sheppard and Gu, 1990, 1991; Gu and Sheppard, 1991 b). However, our experiments showed that half-plane apertures produced little effect on the stray light, except if they obstructed the central part of the beam. Further experiments confirmed that the major contribution (more than 90%) was reflection from the optical elements of the microscope objective. It should be noted that for the results presented here the beam was stopped down so that it only just filled the objective pupil. As these elements are curved surfaces, only reflection from the region near the optic axis can get through the system to the pinhole. This suggests that a small central obstruction could be used to reduce the stray light level even further. Another way of reducing stray light is to insert an iris diaphragm at the collimating lens which focuses the light on to the pinhole. The further the distance from the objective to the collimating lens, the lower the level of stray light. This method of improving optical sectioning has been discussed by Davidovitz and Egger (1971). Because the light reflected from the objective is brought to a focus by the collimating lens at some particular position, the axial position of the objective alters the strength of the stray light. We have found that if scanning in the axial direction is achieved by scanning of the objective lens, the strength of the stray light changes appreciably through the scan. This may prove an advantage for using axial scanning of the object itself instead. No attempt has been made in these experiments to optimize the stray light by alteration of the axial position of the objective. As the stray light is constant in strength throughout the image, it can be subtracted from the signal electronically to improve contrast at very low signal levels. The noise performance has been considered by Sheppard eta!. (1991). It is found that if the stray light is constant throughout the image the normalized signal to noise ratio is given by S N
/
—
~
—J~(vd)---J~(vd)
(2
where a is a parameter describing the effect ofstray light, which from our measurements has a value 4 x l0 8 for our system with a specimen which is a perfect reflector. Here we have assumed detector noise to be negligible. The finite quantum efficiency of the detector affects both signal and stray light and hence does not appear in the norma!ized signal to noise ratio. The signal to noise ratio (Fig. 2) rises from zero for small values of v4~and 9O% of its theoretical set by shot noise on the total that signal of Ed eventually dropslimit, for large values of Vd. This means thebeam, signalfor tovalues noise ratio is between about 3 and 5000. Interestingly, as the specimen reflectance decreases, the effective value of a increases in proportion, and there is less latitude in the choice of pinhole size. The optimum pinhole size for maximizing signal to noise ratio also decreases. The optimum value of Vd for a given effective value of a is shown in Fig. 3. The solid line is the exact theoretical variation, which it will be noticed, exhibits discontinuities. For a specimen reflectivity of 4 x 10- 8 for example, the optimum pinhole size has reduced to vd=2.3. For large values of a the optimum pinhole size levels off to about Ed = 2.1. The dashed line is an approximate curve, valid for larger values of i’d. This is calculated using the asymptotic values for the Bessel functions, and
242
C. .1. R. Sheppard a
1 0
S/N~
02~
1:0. 2. Signal to noise ratio to the presence ol flare. with sttength ch,iracteriied lii the paranletet’ it alter Sheppard at ii.. 1091
bc 80 Vd
40
20
6
io~2
‘
io.
$
1’
a ~. lie tiptimitni pinhole si/c I or inaxinti/itto sional ti ttttse ratIo is a litttctton ti lie ellectise utitie nI a 1 ite solid line is the exact sariatioti. I lie dashed line is the .tpprtt\itttItte \,tri,tttttii based tin .ts\iliptttttc s,tlues or the lbessel lttttctiotts
I’ to
b\ differentiation il can he sIio~~ ii that the optiniuni value ol equation 4t~ t4
er
4
~+
liii
S
~o.
i~ is
given b~a root ttl~thc
(~I
ttJr
Il should he noted that br small salues of ii the masimum is broad, so that choice ofait a ppropriate pinhole sue is not en t cal. The measurements described in this pai~errel’er to a particular microscope s slem. In 1/uct we expect that the stray light performance may prove better than in commercial microscopes for the following reasons. F irstly the microscope has an open design: there us no microscope body which itself can produce reflections and add to the stray light level. It might he argued that a microscope body can stop unwanted light travelling from one part oft he system to another, hut in practice we lou can stop such unwanted light paths by placement of suitable light hailles. Secondly. our system has nianv fewer opt cal coin ponen ts than a com mercial in icroscope based on a con ~cntuonal
Stray Light and Noise in Confocal Microscopy
243
microscope. In our system the only component between the beamsplitter and the object is the objective lens (and the correction lens if using non-infinity corrected objectives). Beam-scanning systems would also be expected to exhibit some variation in the stray light during scanning: we would expect the stray light perhaps to be reduced for off-axis image points. CONCLUSION The ratio of signal to stray light has been measured for a confocal microscope system and shown to have a value, for small pinhole sizes, of 2.5 x l0~. This places a lower limit on the specimen reflectivity which can be detected. REFERENCES Boyde, A. and Petráñ, M., 1990. Light budgets, light and heavy losses: one- or two-sided tandem scanning (real-time, direct-view, confocal) microscopy. J. Micro.sc., 160: 335—342. Cox, I. J. and Sheppard, C. J. R., 1986. Information capacity and resolution in anoptical system. J. Opt. Soc. Amer., A3: 1152 1158. Davidovitz, P. and Egger, M.D., 1971. Scanning laser microscope for biological investigations. App!. Opt., 10: 1615 1619. Gu, M. and Sheppard, C. J. R., 1991 a. Confocal fluorescent microscopy with a finite sized circular detector. J. Opu. Soc. Amer., submitted. Gu, M. and Sheppard, C. J. R., l991b. Three-dimensional imaging in confocal fluorescent microscopy with annular lenses. J. Mod. Opt., in press. Koester, C. J., Khanna, S. M., Rosskothen, H. and Tackaberry, R. B. 1989. Incident light sectioning microscope for visualization ofcellular structures in the inner ear. Acta Oto!aryngol. (Stockh.) Suppl., 467: 27 33. Sheppard, C. J. R. and Gu, M., 1990. Optical sectioning in confocal microscopes with annular pupil. Optik. Sheppard, C. J. R. and Gu, M., 1991. Improvement of axial resolution in confocal microscopy using an annular pupil. Opt. Commun., 84: 7 13. Sheppard, C. J. R. and Mao, M.. 1988. Confocal microscopy with slit apertures. J. Mod. Opt., 35: 11691185. Sheppard, C. J. R., Cogswell, C. J. and Gu, M., 1991. Signal strength and noise in confocal microscopy: factors influencing selection of an optimum detector aperture. Scanning, 13: 233-240. Wells. K. S., Sandison, D. R., Strickler, J. and Webb, W. W.., 1990. Quantitative fluorescence imaging with laser scanning confocal microscopy. In Handbook of Biological Confocal Microscopy, J. B. Pawley (ed). Plenum Press, New York, pp. 23 35.