- Email: [email protected]
Retro-diffracted light in fluorescence spectrometers
Volume
125. number 1
CHEMICAL
PHYSICS
LETTERS
21 March
1986
RETRO-DIFFRACTED LIGHT IN FLUORESCENCE SPECTROMETERS A. DUTCH, D.J.S. BIRCH and R.E. IMHOF Department
Received
of Apphed Physrcs, Unrversity of Strathclyde,
26 November
1985; in final form 11 January
Glasgow G4 ONG, UK
1986
Optical monochromators which incorporate a diffraction grating are shown to select not only a transmitted wavelength emerging from the exit slit but also a retro wavelength which is diffracted back through the entrance slit. The relationship between these diffracted wavelengths in Czemy-Turner grating monochromators is described. Sources of error due to the presence of retro light in fluorescence measurements are discussed and methods for their elimination suggested.
1. Introduction The vast majority of monochromators used in luminescence spectroscopy incorporate a diffraction grating rather than a prism as the dispersive element. However, the theory of diffraction not only allows for forward diffracted wavelengths to be transmitted through a monochromator exit slit but also predicts the existence of retro wavelengths which can be diffracted back through the entrance slit. This “bichromatic” behaviour can be most easily observed using a T-geometry configuration since the orthogonal detection channels are optically coupled in a manner which permits efficient feedback of retro light from one monochromator to the other. Although most fluorimeters and fluorometers presently in use are of an L-geometry configuration there are a large number of applications where a T-geometry has advantages and several commercial versions are available. In particular, a T-geometry is useful in the study of dual wavelength emissions and polarisations. It is quicker, more convenient to use and corrects for source or sample instabilities to give more accurate results than using consecutive measurements with a single-channel L-geometry instrument. Perhaps of most importance are the applications of a T-geometry instrument to the study of biophysical problems. For instance, mobility in membranes can be measured using the dual wavelength emissions associated with the monomer-excimer kinetics [l] of pyrene and its de0 009-26 14/86/$03.50 0 Elsevier Science Publishers B.V. (North-Holland Physics Publishing Division)
rivatives [2]. Similarly, the recording of two emission polarisations enables the determination of the rotational anisotropy of fluorescence probes from which protein dynamics [3], membrane lipid order and membrane viscosity [4] can be studied. A T-geometry configuration is a well established approach to these problems but nearly all the early work used filters rather than monochromators to analyse the fluorescence [5-81. We have recently reported a T-geometry pulse fluorometer which uses emission monochromators and time-correlated single-photon counting to record fluorescence and excitation simultaneously [9]. This instrument has provided us with an opportunity to study retro light which seems to have gone largely unrecognised in fluorescence spectroscopy. It is a potential source of error in both T- and Lgeometry instruments which use emission monochromators, irrespective of whether they are steady-state, modulated or pulsed. In this paper we describe the theory of retro light in Czerny-Turner monochromators, illustrate its effect on fluorescence measurements and show how this can be eliminated by either careful choice of transmission wavelength, or by optimisation of the monochromator design.
57
CHEMICAL PHYSICS LETTERS
Volume 125, number f
Hence generally,
2. Retro-light theory Fig. I shows a simple T-geometry configuration for describing the generation and detection of retro light using Czerny-Turner mono~~omators. If 6 is the angle which the incident and reflected rays make with the normal to the grating in zero order (the Ebert angle), then if the grating is rotated through an angle fl towards the monochromator’s entrance slit, the grating equation leads to the following expressions: sin(S f 6) - sin@ - 8) = ~~~~~ for the tran~itted
f
wavelength A, and
2 sin@ - 8) = j&NAB
(2)
for the emergent retro wavelength AR, where N is the number of grating grooves per metre and MT& the order of diffraction for transmitted and retro wavelengths respectively. Solving (1) and (2) leads to the following relationship between hR and h,:
where the + or - refer to the incident and diffracted rays being on the opposite or same side of the normal to the grating respectively. When the grating faces towards the exit slit the following expression holds in both cases: h,
21 March 1986
2 l/2 tans = h!& { [4(cos26)/N2 - M,2 A,]
f ~~~&
f
0)
where the + or - refer to the grating facing towards the exit or entrance slit respectively. These expressions show that for all transmitted wavelengths there exists a corresponding retro wavelength which satisfies the grating equation. Hence, if white light or a broad-band fluorescence spectrum is incident on a mono~hmmator, then selection can occur of not only a transmitted wavelength but aIso a wavelength which is propagated back towards the source, There also exist wavelengths at which both the retro and transmitted wavelengths are equal. From eq. (5) these occur at h, = 2(sin r;)@v[(Mn M$
‘M+ ta&f~~~ f
(6)
where the + or - now refer to the grating facing towards the entrance or exit slit respectively. Fig. 2 describes the retro behaviour as a function of 6. Fig. 2A shows how first-order A, depends on 6 at different retro orders for a 1200 lines per mm grating facing towards the entrance slit. In the case where the gratm ing faces towards the entrance slit, all incoming wavelengths are refIected off the grating and back out of the entrance sht at f? = S . This zero-order retro condition accurs at transmission wavelengths given by A; = (sin 26)/N&+.
(7)
Fig. 2B shows how h$ depends on 6 for first-order transmitted light with gratings of different values of N. The emission mono~~omators used in our T-geometry fluorometer 191 are Spex aviate model 1650 fitted with I200 lines per mm holo~ap~~~y ruled gratings which face towards the entrance slit. Fig. 3 describes the theoretical relationship between transmitted and retro wavelengths for these monochro2nd EMISSION mators at different retro orders and for 6 measured to MONOCHAOMATOR be 25.6”. With the grating facing towards the entrance slit fig” 3A shows that the transmission wavelength for zero order retro occurs in these mono~hromato~ at 650 nm with the retro and transmitted waveleng~s being equal at ~350 nm. Fig, 3B shows what the retro relationship would be for the same monochromator if the grating faced Fig. 1. Generation and detection of retro light in a “lY_geametrv towards the exit slit. It is worthwhile noting that in spectrometer.
21 March1986
CHEMICALPHYSICSLETTERS
Volume125, number 1
S /(DEGREES)
0
10
20 S/t DEGREES)
30
40
Fig.2.Retro dependenceon the Ebert angle6. (A) Equalretro and transmittedwavelengthhR for B 1200 lines/mmgratingfacing towardsthe entrance slit at different retro order MR. (R)TransmissionwavelengthAi for zero-orderretro andgratingsof differena
linesper mmN. this case first-order hR is always greater than XT throughout the near UV and IR up to a common wavelength at ~1500 nm. This has some advantages with regard to eliminating the possibility of re-excitation or shifting the retro light to a wavelength at which it is not detected by a photom~tiplier. In addition, there is no tr~~s~on wavelength at which zero-order retro light exists when the grating faces towards the exit slit. This arrangement is not usually chosen for monochromators because of its increased stray light but it does have some advantages with regard to retro effects.
Similarly, holographic gratings, which have a better stray-light performance than ruled gratings, are increasingly used in fluorescence spectroscopy. However, retro effects can be expected to be less with ruled grat” ings as they occur against the blaze of the grating. Other factors such as the focusing and geometry also influence the relative intensities of the tr~~itted and retro light which are detected, Nevertheless, given such considerations the grating equation itself predicts that retro intensities can indeed be comparable to transmitted intensities.
Volume 125, number 1
CHEMICALPHYSICS LETTERS
21 March 1986
X,/(nm)
-I JUU
420
&/(nm)
540
660
Fig. 3. Relationship between retro wavelength hR and transmitted wavelength XTfor a Spex Minimate model 1650 and 1200 lines per mm grating. (A) Grating facing towards the entrance slit. (B) Grating facing towards the exit slit.
3. Results and discussion There are a number of experiments in fluorescence spectroscopy where the presence of retro light can have a measurable effect on the quality of the results if not eliminated by either working at carefully chosen wavelengths or using filters. Some of the experiments in which it can be a problem include: (1) Differentialfluorometry. In pulse fluorometry this method corrects for source instability by detecting simultaneously excitation light scattered from the 60
sample and fluorescence using dual detection channels with a matched impulse response in a T-geometry configuration [9]. If the excitation corresponds to the retro wavelength of the emission.monochromator the retro light will corrupt the measurement of the excitation pulse. Fig. 4A shows one such measurement when recording the excitation pulse in one detection channel at 3 10 mn for a typical transmitted fluorescence wavelength in the other detection channel of 390 nm. The usual rapid decay of the excitation pulse from the hydrogen flashlamp [9,10] (Edinburgh Instruments
CHEMICAL PHYSICS LETTERS
Volume 125, number 1
21 March 1986
RE-EXCITATION
.
.
ot;x.x ,’ 0
. ..I
, 2
,
,
,
,
,
,
4 6 TIME/ (ns)
a
=.
,
I~.*...,
,
,
10
Fig. 4. Retro distortion in pulse fluorometry. (A) Typical measurement of the ercitation pulse in a T-geometry fluorometer when recording both transmitted and retro light. The delayed peak is caused by retro light. (B) Fluorescence decay of trans-stilbene in cyclohexane showing re-excitation by retro light.
model 199F) when measured using time-correlated single photon counting is seen to be distorted by a second peak at ~4 ns later. This is consistent with the measured round-trip time of the retro light which is detected at this wavelength combination and clearly invalidates reconvolution analysis of a fluorescence decay. An analogous problem would exist with a phase fluorometer operating in this manner but with sinusoidally modulated rather than pulsed excitation. Here the retro effect would appear as an increased phase shift when measuring the excitation which would give an erroneously shorter fluorescence decay time. A configuration which eliminates the common focus for the two detection channels (e.g. by monitoring the source directly [9]) overcomes this problem. (2) Differential polarised jluoromeby. Here two planes of polarisation of the fluorescence are detected with a T-geometry in order to measure time-resolved anisotropy [3,4]. The technique has proved particularly popular in phase fluorometry [6,8] and involves measuring the phase difference between the two detec-
tion channels when the polarisers are parallel and perpendicular. Should both emission monochromators be set to either the zero-order retro condition at h$ or the common retro wavelength, h,, both detection channels will see retro light when the polarisers are parallel but not when they are perpendicular. The detection of retro light will cause an increase in the phase shift associated with each channel. If both detection channels are perfectly symmetric with respect to the positions of the gratings and photomultipliers, the effect will fortunately cancel when the phase difference is considered. However, if this is not the case an error will be introduced. When a T-geometry pulse fluorometer is used to measure two planes of polarisation simultaneously the crossed nature of the polarisers should again reduce the detection of retro light but if a small amount is detected, reconvolution analysis will again be invalidated. The common retro wavelength, h,, can always be easily found for Czerny-Turner monochromators in a T-geometry pulse fluorometer by tuning the excitation monochromator to zero order 61
Volume 125, number 1
CHEMICAL PHYSICS LETTERS
and synchronously scanning equal transmission wavelengths for the detection monochromators until a retro peak appears in the excitation pulse profile. f3) hai w~ve~e~gt~~~o~rne~y and ~uo~met~. T-geometries are particularly useful in fluorimetry when measuring fluorescence at different wavelengths simultaneously [ 111. If one monochromator is tuned to the retro wavelength of the other (or alternatively to a Raman wavelength when the retro light is scattered from the sample) and the retro wavelength occurs within the fluorescence spectrum, the intensity of the signal meas~d by the detection channel viewing the retro light will be anomalously increased. This would distort both spectral and quantum yield measurements. If these conditions occur in T-geometry phase or pulsed fluorometers the measured lifetimes would also be in error. (4) L-geometry jluorimetry and fluarometry. These measurements are by far the most commonly encountered. Althou~ having a single detection channel, reexcitation of the sample can occur when either the excitation wavelength corresponds to the retro wavelength of the emission monochromator or the transmission wavelength of the emission monochromator is that for zero-order retro light. The effect is likely to be more noticeable the faster the lifetime and when strongly scattering or reflective samples are being studied. We searched for the effect using pulse fluorometry and a range of samples including fluorescent plastic solar collectors, crystals and solutions. The effect is generally difficult to detect but tie have observed it using a solution of trans-stilbene in cyclohexane excited at 285 run with fluorescence at 327.5 nm being detected in second order for a transmission wavelength setting of 655 nm, i.e. at zero-order retro. Even then we could only detect it using front surface excitation and observation of fluorescence at 45” to the cuvette to increase scatter. A 305 nm cut-off filter was placed at the exit slit of the emission monochromator to reduce the detection of scattered light. Fig. 4B shows the excitation pulse, and least-squares reconvolution best fit to the trans-stilbene fluorescence decay which is barely resolved. The systematic deviation in the residuals %4 ns after the main peak ties in with the round-trip time of the retro light and is reflected in the high &i-squared value of 8.2. Fitting over all the decay prior to the effect of reexcitation gave a normalised &i-squared value of 1.25 and decay 62
21 March 1986
time of 101 + 13 ps, which, because of the much longer excitation pulse, agrees as well as can be expected with previous measurements in other solvents [ 12,131. The re-excitation peak was ~dependent of excitation wavelength and disappeared at all other fluorescence wavelengths. Although we have clearly sought suitable conditions for demonstrating that re-excitation due to retro light can distort decay data it might be more easily observed (and hence a greater potential source of error) when using mode-locked laser excitation. This is because of the greater pulse intensity and shorter instrumental pulse duration as compared to flashlamps. The phenomenon of retrodiffracted light is not unique to Czerny-Turner or single grating monochromators. For instance, we have also observed the effect with a Schoeffel GM200 double monochromator for which 6 = 9’ and zeroarder retro occurs at h, = 275 nm. Analogous relationships to those described here can be derived for other monochromator designs. Finally we should add that in addition to the problems associated with retro light it also has uses. For example, it provides a simple means of calibrating a monochromator by using a white light source and a glass plate to act as a beam splitter for separating the incident and retro light. With the grating facing towards the entrance slit the transmission waveleng~ can be varied until zero-order white light is retro reflected, The wavelength calibration is then given by eq. (7).
4. Conclusion Although retro effects in the fluorescence measurements described here are usually small, the increasing precision and sensitivity of instruments means that they might be by no means negligible as a source of error. Moreover, their wavelength-specific nature could easily be misinterpreted as anomalous sample properties. Indeed, retro wavelength effects are also potential sources of error in other branches of spectroscopy in addition to fluorescence. We advise deter~ation of the retro-light ch~acte~stics when using monochromators and in particular when using a T-geometry configuration for fluorescence spectroscopy.
Volume 125, number 1
CHEMICAL PHYSICS LETTERS
Acknowledgement
We would like to thank the SERC for financial support and the tenure of a research studentship by AD. We would also like to thank the University of Strathclyde and Edinburgh Instruments Ltd. for equipment grants.
References [l] J.B. Birks, Photophysics of aromatic molecules (Wiley, New York, 1970). [ 21 H.-J. Galla and W. Hartmann, Chem. Phys. Lipids 27 (1980) 199. [3] I. Munro, I. Pecht and L. Stryer, Proc. Natl. Acad. Sci. US 76 (1979) 56.
21 March 1986
[4] M. Heyn, FEBS Letters 108 (1979) 359. [5] R. Schuyler and I. Isenberg, Rev. Sci. Instrum. 42 (1971) 813. [6] W.W. Mantullin and G. Weber, J. Chem. Phys. 66 (1977) 4092. [7] D.M. Jameson, G. Weber, R.D. Spencer and G. Mitchell, Rev. Sci. Instrum. 49 (1978) 510. [ 8 J J.R. Lakowicz and F.G. Prendergast, Biophys. J. 24 (1978) 213. [ 91 D.J.S. Birch, R.E. Imhof and A. Dutch, Rev. Sci. Instrum. 55 (1984) 1255. [lo] D.J.S. Birch and R.E. Imhof, Rev. Sci. Instrum. 52 (1981) 1206. [ 111 D.J.S. Birch, R.E. Imhof and A. Dutch, J. Luminescence 31,32 (1984) 703. [ 12 ] M. Sumitani, N. Nakashima; K. Yoshihara and S. Nagakura, Chem. Phys. Letters 51 (1977) 183. [ 131 S.H. Courtney and G.R. Flemming, J. Chem. Phys. 83 (1985) 215.
63
125. number 1
CHEMICAL
PHYSICS
LETTERS
21 March
1986
RETRO-DIFFRACTED LIGHT IN FLUORESCENCE SPECTROMETERS A. DUTCH, D.J.S. BIRCH and R.E. IMHOF Department
Received
of Apphed Physrcs, Unrversity of Strathclyde,
26 November
1985; in final form 11 January
Glasgow G4 ONG, UK
1986
Optical monochromators which incorporate a diffraction grating are shown to select not only a transmitted wavelength emerging from the exit slit but also a retro wavelength which is diffracted back through the entrance slit. The relationship between these diffracted wavelengths in Czemy-Turner grating monochromators is described. Sources of error due to the presence of retro light in fluorescence measurements are discussed and methods for their elimination suggested.
1. Introduction The vast majority of monochromators used in luminescence spectroscopy incorporate a diffraction grating rather than a prism as the dispersive element. However, the theory of diffraction not only allows for forward diffracted wavelengths to be transmitted through a monochromator exit slit but also predicts the existence of retro wavelengths which can be diffracted back through the entrance slit. This “bichromatic” behaviour can be most easily observed using a T-geometry configuration since the orthogonal detection channels are optically coupled in a manner which permits efficient feedback of retro light from one monochromator to the other. Although most fluorimeters and fluorometers presently in use are of an L-geometry configuration there are a large number of applications where a T-geometry has advantages and several commercial versions are available. In particular, a T-geometry is useful in the study of dual wavelength emissions and polarisations. It is quicker, more convenient to use and corrects for source or sample instabilities to give more accurate results than using consecutive measurements with a single-channel L-geometry instrument. Perhaps of most importance are the applications of a T-geometry instrument to the study of biophysical problems. For instance, mobility in membranes can be measured using the dual wavelength emissions associated with the monomer-excimer kinetics [l] of pyrene and its de0 009-26 14/86/$03.50 0 Elsevier Science Publishers B.V. (North-Holland Physics Publishing Division)
rivatives [2]. Similarly, the recording of two emission polarisations enables the determination of the rotational anisotropy of fluorescence probes from which protein dynamics [3], membrane lipid order and membrane viscosity [4] can be studied. A T-geometry configuration is a well established approach to these problems but nearly all the early work used filters rather than monochromators to analyse the fluorescence [5-81. We have recently reported a T-geometry pulse fluorometer which uses emission monochromators and time-correlated single-photon counting to record fluorescence and excitation simultaneously [9]. This instrument has provided us with an opportunity to study retro light which seems to have gone largely unrecognised in fluorescence spectroscopy. It is a potential source of error in both T- and Lgeometry instruments which use emission monochromators, irrespective of whether they are steady-state, modulated or pulsed. In this paper we describe the theory of retro light in Czerny-Turner monochromators, illustrate its effect on fluorescence measurements and show how this can be eliminated by either careful choice of transmission wavelength, or by optimisation of the monochromator design.
57
CHEMICAL PHYSICS LETTERS
Volume 125, number f
Hence generally,
2. Retro-light theory Fig. I shows a simple T-geometry configuration for describing the generation and detection of retro light using Czerny-Turner mono~~omators. If 6 is the angle which the incident and reflected rays make with the normal to the grating in zero order (the Ebert angle), then if the grating is rotated through an angle fl towards the monochromator’s entrance slit, the grating equation leads to the following expressions: sin(S f 6) - sin@ - 8) = ~~~~~ for the tran~itted
f
wavelength A, and
2 sin@ - 8) = j&NAB
(2)
for the emergent retro wavelength AR, where N is the number of grating grooves per metre and MT& the order of diffraction for transmitted and retro wavelengths respectively. Solving (1) and (2) leads to the following relationship between hR and h,:
where the + or - refer to the incident and diffracted rays being on the opposite or same side of the normal to the grating respectively. When the grating faces towards the exit slit the following expression holds in both cases: h,
21 March 1986
2 l/2 tans = h!& { [4(cos26)/N2 - M,2 A,]
f ~~~&
f
0)
where the + or - refer to the grating facing towards the exit or entrance slit respectively. These expressions show that for all transmitted wavelengths there exists a corresponding retro wavelength which satisfies the grating equation. Hence, if white light or a broad-band fluorescence spectrum is incident on a mono~hmmator, then selection can occur of not only a transmitted wavelength but aIso a wavelength which is propagated back towards the source, There also exist wavelengths at which both the retro and transmitted wavelengths are equal. From eq. (5) these occur at h, = 2(sin r;)@v[(Mn M$
‘M+ ta&f~~~ f
(6)
where the + or - now refer to the grating facing towards the entrance or exit slit respectively. Fig. 2 describes the retro behaviour as a function of 6. Fig. 2A shows how first-order A, depends on 6 at different retro orders for a 1200 lines per mm grating facing towards the entrance slit. In the case where the gratm ing faces towards the entrance slit, all incoming wavelengths are refIected off the grating and back out of the entrance sht at f? = S . This zero-order retro condition accurs at transmission wavelengths given by A; = (sin 26)/N&+.
(7)
Fig. 2B shows how h$ depends on 6 for first-order transmitted light with gratings of different values of N. The emission mono~~omators used in our T-geometry fluorometer 191 are Spex aviate model 1650 fitted with I200 lines per mm holo~ap~~~y ruled gratings which face towards the entrance slit. Fig. 3 describes the theoretical relationship between transmitted and retro wavelengths for these monochro2nd EMISSION mators at different retro orders and for 6 measured to MONOCHAOMATOR be 25.6”. With the grating facing towards the entrance slit fig” 3A shows that the transmission wavelength for zero order retro occurs in these mono~hromato~ at 650 nm with the retro and transmitted waveleng~s being equal at ~350 nm. Fig, 3B shows what the retro relationship would be for the same monochromator if the grating faced Fig. 1. Generation and detection of retro light in a “lY_geametrv towards the exit slit. It is worthwhile noting that in spectrometer.
21 March1986
CHEMICALPHYSICSLETTERS
Volume125, number 1
S /(DEGREES)
0
10
20 S/t DEGREES)
30
40
Fig.2.Retro dependenceon the Ebert angle6. (A) Equalretro and transmittedwavelengthhR for B 1200 lines/mmgratingfacing towardsthe entrance slit at different retro order MR. (R)TransmissionwavelengthAi for zero-orderretro andgratingsof differena
linesper mmN. this case first-order hR is always greater than XT throughout the near UV and IR up to a common wavelength at ~1500 nm. This has some advantages with regard to eliminating the possibility of re-excitation or shifting the retro light to a wavelength at which it is not detected by a photom~tiplier. In addition, there is no tr~~s~on wavelength at which zero-order retro light exists when the grating faces towards the exit slit. This arrangement is not usually chosen for monochromators because of its increased stray light but it does have some advantages with regard to retro effects.
Similarly, holographic gratings, which have a better stray-light performance than ruled gratings, are increasingly used in fluorescence spectroscopy. However, retro effects can be expected to be less with ruled grat” ings as they occur against the blaze of the grating. Other factors such as the focusing and geometry also influence the relative intensities of the tr~~itted and retro light which are detected, Nevertheless, given such considerations the grating equation itself predicts that retro intensities can indeed be comparable to transmitted intensities.
Volume 125, number 1
CHEMICALPHYSICS LETTERS
21 March 1986
X,/(nm)
-I JUU
420
&/(nm)
540
660
Fig. 3. Relationship between retro wavelength hR and transmitted wavelength XTfor a Spex Minimate model 1650 and 1200 lines per mm grating. (A) Grating facing towards the entrance slit. (B) Grating facing towards the exit slit.
3. Results and discussion There are a number of experiments in fluorescence spectroscopy where the presence of retro light can have a measurable effect on the quality of the results if not eliminated by either working at carefully chosen wavelengths or using filters. Some of the experiments in which it can be a problem include: (1) Differentialfluorometry. In pulse fluorometry this method corrects for source instability by detecting simultaneously excitation light scattered from the 60
sample and fluorescence using dual detection channels with a matched impulse response in a T-geometry configuration [9]. If the excitation corresponds to the retro wavelength of the emission.monochromator the retro light will corrupt the measurement of the excitation pulse. Fig. 4A shows one such measurement when recording the excitation pulse in one detection channel at 3 10 mn for a typical transmitted fluorescence wavelength in the other detection channel of 390 nm. The usual rapid decay of the excitation pulse from the hydrogen flashlamp [9,10] (Edinburgh Instruments
CHEMICAL PHYSICS LETTERS
Volume 125, number 1
21 March 1986
RE-EXCITATION
.
.
ot;x.x ,’ 0
. ..I
, 2
,
,
,
,
,
,
4 6 TIME/ (ns)
a
=.
,
I~.*...,
,
,
10
Fig. 4. Retro distortion in pulse fluorometry. (A) Typical measurement of the ercitation pulse in a T-geometry fluorometer when recording both transmitted and retro light. The delayed peak is caused by retro light. (B) Fluorescence decay of trans-stilbene in cyclohexane showing re-excitation by retro light.
model 199F) when measured using time-correlated single photon counting is seen to be distorted by a second peak at ~4 ns later. This is consistent with the measured round-trip time of the retro light which is detected at this wavelength combination and clearly invalidates reconvolution analysis of a fluorescence decay. An analogous problem would exist with a phase fluorometer operating in this manner but with sinusoidally modulated rather than pulsed excitation. Here the retro effect would appear as an increased phase shift when measuring the excitation which would give an erroneously shorter fluorescence decay time. A configuration which eliminates the common focus for the two detection channels (e.g. by monitoring the source directly [9]) overcomes this problem. (2) Differential polarised jluoromeby. Here two planes of polarisation of the fluorescence are detected with a T-geometry in order to measure time-resolved anisotropy [3,4]. The technique has proved particularly popular in phase fluorometry [6,8] and involves measuring the phase difference between the two detec-
tion channels when the polarisers are parallel and perpendicular. Should both emission monochromators be set to either the zero-order retro condition at h$ or the common retro wavelength, h,, both detection channels will see retro light when the polarisers are parallel but not when they are perpendicular. The detection of retro light will cause an increase in the phase shift associated with each channel. If both detection channels are perfectly symmetric with respect to the positions of the gratings and photomultipliers, the effect will fortunately cancel when the phase difference is considered. However, if this is not the case an error will be introduced. When a T-geometry pulse fluorometer is used to measure two planes of polarisation simultaneously the crossed nature of the polarisers should again reduce the detection of retro light but if a small amount is detected, reconvolution analysis will again be invalidated. The common retro wavelength, h,, can always be easily found for Czerny-Turner monochromators in a T-geometry pulse fluorometer by tuning the excitation monochromator to zero order 61
Volume 125, number 1
CHEMICAL PHYSICS LETTERS
and synchronously scanning equal transmission wavelengths for the detection monochromators until a retro peak appears in the excitation pulse profile. f3) hai w~ve~e~gt~~~o~rne~y and ~uo~met~. T-geometries are particularly useful in fluorimetry when measuring fluorescence at different wavelengths simultaneously [ 111. If one monochromator is tuned to the retro wavelength of the other (or alternatively to a Raman wavelength when the retro light is scattered from the sample) and the retro wavelength occurs within the fluorescence spectrum, the intensity of the signal meas~d by the detection channel viewing the retro light will be anomalously increased. This would distort both spectral and quantum yield measurements. If these conditions occur in T-geometry phase or pulsed fluorometers the measured lifetimes would also be in error. (4) L-geometry jluorimetry and fluarometry. These measurements are by far the most commonly encountered. Althou~ having a single detection channel, reexcitation of the sample can occur when either the excitation wavelength corresponds to the retro wavelength of the emission monochromator or the transmission wavelength of the emission monochromator is that for zero-order retro light. The effect is likely to be more noticeable the faster the lifetime and when strongly scattering or reflective samples are being studied. We searched for the effect using pulse fluorometry and a range of samples including fluorescent plastic solar collectors, crystals and solutions. The effect is generally difficult to detect but tie have observed it using a solution of trans-stilbene in cyclohexane excited at 285 run with fluorescence at 327.5 nm being detected in second order for a transmission wavelength setting of 655 nm, i.e. at zero-order retro. Even then we could only detect it using front surface excitation and observation of fluorescence at 45” to the cuvette to increase scatter. A 305 nm cut-off filter was placed at the exit slit of the emission monochromator to reduce the detection of scattered light. Fig. 4B shows the excitation pulse, and least-squares reconvolution best fit to the trans-stilbene fluorescence decay which is barely resolved. The systematic deviation in the residuals %4 ns after the main peak ties in with the round-trip time of the retro light and is reflected in the high &i-squared value of 8.2. Fitting over all the decay prior to the effect of reexcitation gave a normalised &i-squared value of 1.25 and decay 62
21 March 1986
time of 101 + 13 ps, which, because of the much longer excitation pulse, agrees as well as can be expected with previous measurements in other solvents [ 12,131. The re-excitation peak was ~dependent of excitation wavelength and disappeared at all other fluorescence wavelengths. Although we have clearly sought suitable conditions for demonstrating that re-excitation due to retro light can distort decay data it might be more easily observed (and hence a greater potential source of error) when using mode-locked laser excitation. This is because of the greater pulse intensity and shorter instrumental pulse duration as compared to flashlamps. The phenomenon of retrodiffracted light is not unique to Czerny-Turner or single grating monochromators. For instance, we have also observed the effect with a Schoeffel GM200 double monochromator for which 6 = 9’ and zeroarder retro occurs at h, = 275 nm. Analogous relationships to those described here can be derived for other monochromator designs. Finally we should add that in addition to the problems associated with retro light it also has uses. For example, it provides a simple means of calibrating a monochromator by using a white light source and a glass plate to act as a beam splitter for separating the incident and retro light. With the grating facing towards the entrance slit the transmission waveleng~ can be varied until zero-order white light is retro reflected, The wavelength calibration is then given by eq. (7).
4. Conclusion Although retro effects in the fluorescence measurements described here are usually small, the increasing precision and sensitivity of instruments means that they might be by no means negligible as a source of error. Moreover, their wavelength-specific nature could easily be misinterpreted as anomalous sample properties. Indeed, retro wavelength effects are also potential sources of error in other branches of spectroscopy in addition to fluorescence. We advise deter~ation of the retro-light ch~acte~stics when using monochromators and in particular when using a T-geometry configuration for fluorescence spectroscopy.
Volume 125, number 1
CHEMICAL PHYSICS LETTERS
Acknowledgement
We would like to thank the SERC for financial support and the tenure of a research studentship by AD. We would also like to thank the University of Strathclyde and Edinburgh Instruments Ltd. for equipment grants.
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