Rapid scan spectrophotometer

A spectrometer capable of providing a predetermined wavelength of output light in accordance with a control voltage signal applied to a scanning element is described. The scanning element located at the grating image of the spectrometer is a small mirror attached to the rotor of a galvanometer whose angular position is accurately controlled by a closed-loop electronic control. The spectrum reflected from the mirror is passed through a slit to provide the output light of a predetermined wavelength. Selection of the waveform of the control signal allows the spectrometer to be operated as a dual wavelength spectrometer, to use a linear wavelength scan, or other wavelength scan patterns for absorbance analyses of a sample. The rapid scan capability of this instrument has been utilized to extend the measurement of absorbance changes at one wavelength, corrected for light scattering changes (dual-wavelength spectroscopy), to the measurement of the complete differential absorbance spectrum, similarly corrected for light scattering changes (corrected-differential spectroscopy).

BACKGROUND OF THE INVENTION 
This invention relates to an improved type of rapid scan spectrophotometer 
and method of using it in several modes to obtain the absorption 
characteristics of clear low molecular weight solutions. More 
particularly, the spectrophotometer of this invention utilizes a technique 
for accurately controlling the spectrum being observed at a known time and 
subsequently processing the transmitted spectrum to obtain the absorption 
characteristic of the material being observed. 
Although electronic absorption (uv-visible) spectrophotometry is an 
attractive investigative tool for clear, low molecular weight solutions, 
the application of this technique to the study of biological preparations 
is not straightforward, primarily because these complex and heterogenous 
systems show broad overlapping absorption bands which scatter light 
strongly. Where the parameter of interest is not the total absorbance, but 
the change in absorbance, several prior art spectrometers have been found 
useful. In the prior art split-beam spectrometer light from a 
monochromator is alternately passed through two identical samples which 
are subjected to different conditions. The difference in absorption 
between the samples is then recorded as a function of wavelength. This 
approach allows the measurement of small absorbance changes on a large 
background. However, if the amount of scattered light is time dependent, 
or if it changes as a consequence of the change in experimental 
conditions, false absorbance changes will be recorded. 
The prior art dual wavelength spectrometer provides a means of correcting 
for these scattering changes. As the names implies, light of two different 
wavelengths is used, but both beams pass through the same sample. 
Variations in light intensity at some reference wavelength at which there 
is no absorbance change will reflect only scattering effects. To the 
extent that the relative changes in scattered light are the same at two 
neighboring wavelengths, the intensity at the reference wavelength 
provides a continuous correction for scattering occurring at neighboring 
wavelengths. Also, because the dual wavelength spectrometer rapidly 
monitors light intensities at a specified pair of wavelengths, it can be 
used for kinetic studies. 
It is therefore a primary object of this invention to provide a rapid-scan 
spectrophotometer which can operate in a variety of spectroscopic modes, 
two of which are analogous to those just discussed. The controlled-scan 
spectrophotometer (CSS) of this invention has been operated as a 
dual-wavelength spectrophotometer, but one which uses only a single 
monochromator. 
The CSS has also been used to obtain the complete absorbance spectrum, 
corrected for light scattering changes in the same way that the 
dual-wavelength approach provides correction at a single wavelength. This 
latter technique has been called "corrected-differential" 
spectrophotometry.

SUMMARY OF THE INVENTION 
The difference between the spectrometer of this invention and conventional 
spectrometers is that here an image of the grating is generated optically, 
and a scanning element is located at this grating image. Consequently, the 
wavelength of the light passing through the exit slits can be changed by 
rotating the scanning mirror rather than the massive grating mount, 
enabling rapid wavelength changes. The scanning element is a small mirror 
mounted on the rotor of a galvanometer under closed-loop electronic 
control, which forces the mirror angular position to follow the input 
drive signal rapidly and accurately. This type of scan control allows 
aperiodic and and discontinuous wavelength scans as well as the more 
customary sinusoidal scans. 
With a square wave mirror drive, this instrument can be operated as a 
dual-wavelength spectrometer, even though it has only one monochromator. 
This instrument can also be used to generate rapid, linear wavelength 
scans, by using a periodic ramp mirror drive signal. This last mode of 
operation generates complete transmission spectra, which are stored in 
digital memory. These spectra, together with a priori knowledge of the 
true absorbance at some reference wavelength, enables an absorbance 
spectrum corrected for light scattering changes to be generated. 
DESCRIPTION OF PREFERRED EMBODIMENT 
A block diagram of the controlled scan spectrophotometer of this invention 
is shown in FIG. 1. The light source 1, typically a 250 watt 
tungsten-halide lamp powered by a DC power supply, is focused by a lens 2 
to provide a broad band light source to the modified monochromator 3. A 
plane mirror 5 intercepts the exiting beam 4 before it reaches the exit 
slit of the monochromator, directing the beam to a spherical mirror 6 
(typically 100 mm diameter with a 100 mm focal length). A small mirror 7 
(typically 25 mm by 25 mm) is placed at the image of the grating 8 
generated by the spherical mirror 6. The mirror 7 is mounted on a 
galvanometer 16 and a closed loop galvanometer drive system 9 (for example 
a drive system such as that General Scanning Corporation, Watertown, MA 
Model G-306PD) accurately controls the angular position of the mirror 7 in 
accordance with the voltage output of waveform source 12. Typically, a 
square wave 10 or a ramp waveform 11 is produced on the input line 19 to 
the drive system 9 by the waveform source 12 and one may be selected by 
switch 18. Immediately behind the slit 12, the sample chamber 14 and 
photomultiplier tube 15 are mounted on an optical rail (not shown) which 
allows close, reproducible positioning and provides maximum flexibility in 
the design and use of sample chambers. 
Rotation of the galvanometer 16 (and mirror 7) by the galvanometer drive 
system 9 in response to the voltage from source 12 causes the spacially 
dispersed image of the grating 8 to move across the exit slit 13. The 
wavelength of the light passing through the slit 13 is thus determined by 
the angular orientation of the mirror 7, and hence by the voltage applied 
to the galvanometer drive system 9 from the waveform source 12. Since the 
galvanometer postion is directly related to the current flowing through 
the galvanometer, the current in the galvanometer is sensed and used in a 
feedback loop in the glavanometer drive system 9, as in conventional 
closed loop positioning systems causing the mirror 7 orientation to 
reflect the input voltage from waveform source 12 accurately and rapidly. 
(The mirror stabilizes in its new position with two milliseconds after 
impositon of a large voltage step.) The spectrum can be scanned in any 
desired pattern by electronically synthesizing the appropriate pattern of 
voltages produced by the waveform source 12. Typical waveforms are the 
square waveform 10 and the ramp waveform 11. The scan rate of the mirror 7 
is limited by the mass of the mirror 7 but presents no problem for the 
rates used in this invention. 
In the methods employed in this invention two mirror waveforms 10 and 11 
have been used. For dual-wavelength spectrophotometry, a square wave drive 
signal 10 of suitable amplitude and voltage levels is used to select light 
at the two desired wavelengths at the exit slit 13. For the method of 
corrected-differential spectrophotometry, a periodic ramp 11, which causes 
the linear scan of the wavelength band, is used. These waveforms may be 
generated by conventional well-known circuitry. The amplitude of the 
voltage waveforms determines the scan range. Scan ranges of from 50 to 300 
nm have been used. In principle, the scan range can extend to the full 
range of 600 nm available at the output of the spectrometer grating. 
However the wider scan range results in reduced wavelength accuracy 
because of limitation of the galvanometer-drive system. Scan frequencies 
of 15 and 30 hertz have been used for convenience since they are easily 
obtained from a 60 cycle power source and are rapid enough that the 
conditions of the sample being observed does not perceptibly change. Other 
scan frequencies could easily be provided by conventional circuitry. 
DUAL-WAVELENGTH SPECTROPHOTOMETRY 
In this mode of operation the waveform source 12 provides a square wave 
mirror signal 10 on line 19 which also serves as an input to a pair of 
monostable multivibrators 21, 22 used for timing purposes shown in FIG. 2. 
The output pulse 23 from the multivibrator 21, which occurs during the 
interval when the mirror 7 orientation is changing, resets to zero the 
output from the integrating sample and hold circuit 24. The output pulse 
25 from the second multivibrator 22 occurs during the period when the 
mirror orientation is stable (i.e., light of one wavelength is passing 
through the slit). The signal 26 from the photomultiplier 15 is integrated 
in integrator 27 during the time of occurrence of pulse 25. The final 
value of the integrated signal output 28 of integrator 27 is held by the 
sample hold circuit 24 until the reset pulse 23 starts the cycle over 
again. A subtraction circuit 33 subtracts the output of the sample and 
hold circuit 24 from the photomultiplier tube signal 26. The complement 8 
of the square wave 8 is the input to an identical set of timing and sample 
hold circuits 29' which integrate and then hold the output of this 
subtract circuit when the photomultiplier tube is responding to light at 
the second wavelength. The outputs of the circuits 29, 29' are combined in 
divider 32 to provide the output signal 30 from the processor 20' to the 
recorder 31 of FIG. 1. 
The output signal 30 of the spectrometer is 
EQU S=.kappa.(V.sub.m -.alpha.V.sub.r)/.alpha.V.sub.r, 
where V.sub.m and V.sub.r are the voltages corresponding to the 
photomultiplier output for incident light of wavelength .lambda..sub.m 
(measurement) and .lambda..sub.r (reference), respectively, and .kappa. 
and .alpha. are adjustable gains. This format is equivalent to the ratio 
detection method in conventional dual-wavelength spectrometers. The gain 
.alpha. of circuits 29' is adjusted to set S to zero initially. Then, if 
.lambda..sub.r is an isobestic point, and if the relative scattering 
changes are the same at two wavelengths, .DELTA.S, the signal at line 30, 
is related to the absorbance change at .lambda..sub.m, .DELTA.A.sub.m, by 
EQU .DELTA.S=.kappa.(10.sup.-.DELTA.Am -1).perspectiveto.-.kappa.(ln 
10).DELTA.A.sub.m, 
where the approximation is valid for small .DELTA.A.sub.m. 
CORRECTED-DIFFERENTIAL SPECTROPHOTOMETRY 
The periodic ramp mirror-drive waveform 11 of waveform source 12 results in 
a linear scan across slot 13 of the dispersed spectrum generated by the 
monochromator 3 with a selectable range of 50-300 nm. The intensity 
spectrum transmitted through the sample 14 is converted to an electronic 
signal by the photomultiplier tube 15 and stored for use in later 
calculations of absorbance changes. For small absorbance changes, several 
sweeps must be summed to obtain a suitable signal-to-noise ratio. FIG. 3 
details the signal processing which occurs prior to storage of the 
intensity spectrum. The swept signal output of photomultiplier tube 15 is 
applied to the first amplifier circuit 50 which provides a voltage offset 
at its output. (V.sub.B is supplied by a precision voltage source 51, such 
as Electronic Development Corp., Model MV 100N.) The signal amplification 
is selected by the setting of gain control 55 of the second amplifier 52. 
Amplification is followed by clipping circuit 53 which prevents 
overloading of the signal averager 54 input circuits. 
Since the ramp mirror drive signal 11 causes a linear wavelength scan, the 
signal averager 54 output (after 1 to N sweeps) is the transmitted light 
intensity as a function of wavelength. The intensity spectrum is recorded 
at gain=1, and then again at high gain, G, with an offset voltage from 
source 51 sufficient to buck out the bulk of the background voltage. The 
sample 14 is perturbed and the new intensity spectrum is recorded at the 
same high gain and offset. The apparent absorbance change .DELTA.A*, is 
then calculated from 
##EQU1## 
where V(n) and v(n) are voltages at the nth data point in the signal 
averager 54 and are related to the transmitted light intensity at the 
wavelength, .lambda..sub.n, which corresponds to the nth data point. The 
voltage v(n) is the result of the application of the bucking voltage and 
gain G to the voltage V at the output of tube 15, which itself is directly 
proportional to the light intensity incident on the photomultiplier tube. 
The voltage v'(n) indicates data taken after perturbation of the sampling 
conditions. 
The contribution of light scattering changes to the apparent absorbance 
change is evaluated at the reference wavelength .lambda..sub.r, 
EQU .DELTA.T=.DELTA.A*(.lambda..sub.r)-.DELTA.A(.lambda..sub.r), 
where .DELTA.A(.lambda..sub.r) is the true absorbance change at 
.lambda..sub.r (as determined by an independent study). It is assumed that 
.DELTA.T is independent of wavelength in the neighborhood of 
.lambda..sub.r, so the true absorbance changes at neighboring wavelengths 
are given by 
EQU .DELTA.A(.lambda..sub.n)=.DELTA.A*(.lambda..sub.n)-.DELTA.T. 
After each intensity spectrum is averaged, the digital contents of the 
signal averager, typically a Biomac Model 1000, are recorded at each data 
point and subsequently processed in a conventional calculator or 
mini-computer to provide .DELTA.A*(.lambda..sub.n) and then 
.DELTA.A(.lambda..sub.n) in accordance with the equation therefore. 
CONCLUSION 
The rapid scanning capability and the use of a closed-loop galvanometer 
system of the controlled-scan spectrophotometer provides a versatile 
instrument. Nonlinear and discontinuous scanning patterns can be used with 
greater efficiency than the sinusoidal pattern of a resonant mirror 
system, and the possibility of nonperiodic patterns permits other 
applications which would be impossible with a resonant system. Thus, in 
the dual-wavelength mode, the use of a square-wave pattern rather than a 
sinusoidal pattern means that much more time per scan is spent at the two 
wavelengths of interest, permitting longer integration times for reduction 
of photon noise. Again, the use of a periodic ramp in the 
corrected-differential mode means that much less time is wasted in 
bringing the mirror back to start the scan again. 
The operation of the instrument as a dual-wavelength spectrometer, and its 
response to insertion of neutral density filters is a clear demonstration 
of its ability to correct for changes in light scattering. Insertion of 
these filters into the beam causes changes equivalent to 0.001 A or less, 
i.e., the spectrometer corrected &gt;99% of the induced "scatter." The 
sensitivity is not quite that of commercial units, but this could be 
improved by using better sample-and-hold amplifiers (with less noise) and 
a more accurate divide circuit. As it is, the novel design of this 
invention requires only one monochromator, provides good sensitivity at 
much lower cost, and offers an important advantage over a conventional 
unit: additional wavelength pairs could be simultaneously monitored by 
adding steps of appropriate amplitude to the square-wave mirrordrive 
waveform and adding data processing circuits identical to those already in 
use. In comparison, a 4-wavelength spectrometer recently reported by 
another involved much effort and ingenuity in modifying a standard 
dual-wavelength spectrometer with considerable loss in time resolution. 
The operation of this instrument has been described in a new mode, that of 
corrected-differential spectroscopy. The wavelength band is linearly 
scanned, and the transmitted intensity spectrum is stored in a memory 
unit. If the intensity spectrum is recorded before and after perturbation 
of the same conditions, the resulting differential absorbance spectrum can 
be calculated. This apparent absorbance change, .DELTA.A*(.lambda..sub.n), 
includes the real absorbance change .DELTA.A(.lambda..sub.n), and an 
artificial change due to changes in light scattering, 
.DELTA.T(.lambda..sub.n), 
EQU .DELTA.A*(.lambda..sub.n)=A(.lambda..sub.n)+.DELTA.T(.lambda..sub.n). 
The variations in light scattering have a broad effect on the transmitted 
light intensity; in particular, it is assumed that the relative changes in 
light intensity caused by fluctations in light scattering are essentially 
identical at neighboring wavelengths. If a similar assumption is made with 
regard to the scattering changes produced by the perturbation of the 
sample conditions, then the effects of light scattering enter as an 
additive constant [.DELTA.T]. (This is an inherent assumption of 
dual-wavelength spectroscopy.) Thus, if the true absorbance change at some 
reference wavelength, .lambda..sub.r, is known, .DELTA.T can be evaluated, 
and the true absorbance changes at neighboring wavelengths determined. (In 
practice, an isosbestic point is often chosen as the reference 
wavelength.) Although the precise value of the scattering contribution may 
vary from scan to scan, measurements at the reference wavelength permit 
this value to be evaluated for each absorbance calculation. Consequently, 
repetitive scans of the intensity spectrum can be summed and used in place 
of a single scan. The corrected-differential spectrum is equivalent to the 
point spectrum which would be obtained by recording the absorbance changes 
by dual-wavelength spectroscopy as .lambda..sub.m is successively 
incremented and the experiment repeated. The corrected-differential 
technique permits this information to be collected in a single experiment. 
It is evident that those skilled in the art, once given the benefit of the 
foregoing disclosure, may make numerous other uses and modifications of, 
and departures from the specific embodiments described herein without 
departing from the inventive concepts. Consequently, the invention is to 
be construed as embracing each and every novel combination of features 
present in, or possessed by, the apparatus and techniques herein disclosed 
and limited solely by the scope and spirit of the appended claims.