CHAPTER 15 POLYCHROMATOR FOR RECORDING OPTICAL ABSORBANCE CHANGES FROM SINGLE CELLS

STEPHEN J. SMITH Section of Molecular Neurobiolo:§J Yale University School of Medicine New Haven, Connecticut

1. INTRODUCTION 2. OPTICAL ARRANGEMENT 3. ELECTRONIC SIGNAL AMPLIFICATION 4. INSTRUMENTAL RESOLUTION LIMITS

REFERENCES

1. INTRODUCTION

255 256 258 259 260

An instrument fo r recording optical absorbance of s ingle living cells was demonstrated . The apparatus is based on a grating polychromator and measures absorbance simultaneously at several different wavelengths. It was designed for high-resolution recording while leaving cells accessible to multiple microelectrode impalement. The instrument has been used on nerve cells containing the indicator dye arsenazo III to detect cytosolic calc ium concentration transients. The apparatus will be described here, and some of the considerations influencing the design will be discussed.

255

 

256 POLYCHROMATOR FOR RECORDING OPTICAL ABSORBANCE CHANGES

An instrument with multiple spectral channels, such as that described here, may be preferred to a simpler, fixed-wavelength or manually tuned photometer for several reasons. Spectral data may be inherently necessary for a particular analysis, such as discrimination between effects of calcium and protons on the absorbance spectrum of arsenazo III (Brown, et al., 1977; Thomas, 1982) . Spectral information may also help to recognize and ameliorate sources of interference to the desired optical measurement. For example, it is often difficult to completely prevent small movements in living cell preparations, and residual movements may cause changes in light transmittance of a magnitude comparable to indicator dye signals. Differential recording at a properly chosen pair of wavelengths can significantly reduce artifacts due to object movement. The improved speed, accuracy, and convenience of spectral measurement with a multichannel instrument , compared to a manually tuned one , can be decisive in work with living specime ns of limited experimental lifetime.

2. OPTICAL ARRANGEMENT

Optical components of the recording system are shown in schematic form in Fig. I . The arrangement consists of an illuminator to project an intense beam of broad-band-filtered light onto the specimen under study (here labeled ganglion) , a single optical fiber to collect light transmitted through some part of the specimen, and a grating-based polychromator with silicon photodiodes for light measurement. All components are mounted on a tabletop vibration isolating bench within a closable acoustic and electrical shielding enclosure.

Specimens are Kohler-illuminated with a maximum numerical aperture of 0 . 7. The field and aperture stops are omitted for clarity in Fig. I but are useful to reduce stray light and facilitate adjustment. Direct current is delivered to a 12-V, 100-W

FIBER TIP (45fLM diameter)~

GANGLION

CONDENSER LENS

EDGE { LONG-PASS

FILTERS SHORT-PASS

COLLECTOR LENS

LAMP

SINGLE OPTICAL FIBER (200 fLM diameter)

COLLIMATING LENS

DIFFRACTIOt. GRATING

~:JI__---1] WAVELENGTHS' --;-//~ ///

__!\... - / / LONG :::0:---v- - 7" -~-:.-

/ /--/ \"(:: - -// / FOCUSING LENS

MEDIUM -:5S'""\J /// At

SHORT /)1 COLLECTING LENSES

SILICON PHOTODIODES

Figure 1. Schematic diagram of polychromator-based apparatus for spectral measurement of optical absorbance in single living cells.

 

2. OPTICAL ARRANGEMENT 257

tungsten-halogen lamp by an electronic power supply. The lamp housing is water cooled with minimum air volume between the quartz bulb envelope and an aluminum cooling jacket. This arrangement minimizes fluctuations in filament temperature and light output due to air convection currents. Water cooling has additional advantages in minimizing heating of mechanical components and reducing air drafts within the experimental enclosure. both of which contribute to stable recordings and shorten warm-up stabilization periods.

The edge filters shown in Fig. I are used to restrict illuminating light to the broad wavelength band of interest. Possible unwanted effects of illumination at other wavelengths , such as the heat input from longer wavelengths or photochemical actions from shorter wavelengths, are thus minimized. For measurement of calcium transients with arsenazo III, a colored glass long-pass filter with a 530-nm cutoff is used; a combination of heat-absorbing glass and interference short-pass filters are used to eliminate wavelengths longer than 725 nm.

Light transmitted through a restricted specimen area is collected by positioning the tip of a single optical fiber over the desired area using a micromanipulator. The tip is lowered as close as practical to the surface of the specimen. The fiber used is of the graded index type produced for communication purposes and has an outer diameter of 200 j.Lm. In the short lengths employed here (1-2m), such a fiber has an effective light-accepting area equal to this outer fiber diameter. Smaller recording apertures were produced by drawing fiber tips into a taper with heat. Silver was deposited onto the sides of the tapered tip to eliminate stray light coupling. The silver ~as overcoated with varnish to prevent toxicity and deterioration. A fiber tip used in many studies had a tip 45 1-Lm in diameter and accepted light in a cone of 20° semiangle, corresponding to a numerical aperture of 0.35.

The polychromator consists of the arrangement of components shown at the right of Fig. I. The collimating lens is a camera lens of 50 mm focal length andf = 1.8. The end of the optical fiber is positioned at the back focal plane of this lens so that light exiting the fiber is collimated for projection onto the diffraction grating. The grating is a replica ruled 1200 lines/mm and blazed at 500 nm. The grating is oriented such that the desired wavelengths (530-725 nm for arsenazo III studies) of the first-order diffracted light on the high-efficiency side of the blazed grating pattern fall onto the focusing lens . The focusing lens has a 300-mm focal length and a 30-mm clear aperture. A spectrally dispersed image of the exit tip of the fiber, enlarged 6 x, is thus formed along a curved surface at a radius of 300 mm centered on the focusing lens. An array of up to five detector modules (of which only 3 are shown in Fig. 1, for clarity), are positioned along this surface.

Each detector module consists of a narrow aluminum housing holding an aspheric collecting lens (8-mm focal length, 12-mm diameter) and a silicon photodiode. These elements are positioned such that a reduced image of the 30-mm focusing lens falls entirely on the 1-mm-diameter active area of the photodiode. The use of a small-area photodetector minimizes detector noise power, which is proportional to detector active area. Use of a collecting lens of diameter larger than the mounting package in which the photodiode chip is supplied reduces the spectral " dead" band between adjacent detectors at minimum spacing. With the optics specified, each detector

 

258 POLYCHROMATOR FOR RECORDING OPTICAL ABSORBANCE CHANGES

module samples a spectral band approximately 20 nm in width, while adjacent cbannel-band center spacing can be as little as 30 nm. These parameters can be adjusted by changing the focal length of the focusing lens or by masking the detector module aperture .

Detector channels are tuned to the desired spectral region by adjusting the lateral position of individual detector modules along the polychromator output focusing surface. A narrow band interference filter of known peak wavelength is inserted in the illuminator path to facilitate placement of detectors. Spectral tuning is then calibrated by temporarily replacing the normal collecting fiber with a similar fiber coupled to a calibrated monochromator light source. Tuning curves are generated for each channel by reading photodiode output as a function of a monochromator wavelength setting.

3. ELECTRONIC SIGNAL AMPLIFICATION

Amplification and processing of the photocurrent analog signal from silicon photodiodes are carried out in several stages. Low-level preamplification takes place at a transimpedance amplifier (current-to-voltage converter) located very near the photodiode to reduce electromagnetic interference noise pickup and stray capacitance. Signal switching, scaling, and differential amplification take place at a rack-mounted control amplifier located outside the isolating enclosure.

The transimpedance amplifier is based on an FET operational amplifier with a bias current of less than I pA and low input voltage noise. The feedback network consists of a parallel RC combination. A 500-M.O. feedback resistor (matching the photodiode shunt resistance) is used unless a smaller value i required to avoid exceeding the amplifier voltage output limit when photocurrent is large. Parallel capacitance is adjusted to obtain a critically damped transient response (using an LED to produce a step change of light input).

The control amplifier allows gain adjustment of each photocurrent signal. To produce an optical absorbance change signal of constant calibration, the gain of each photocurrent channel is adjusted to make the " resting" or reference photocurrent signal equal to a fixed reference level of I 0. 00 V. The optical absorbance signal of interest normally takes the form of some small devia tion from this reference level . For instance, Ca-arsenazo Ill signals in an excitable cell may produce peak changes in light transmittance on the order of one part in I 000. Thus, if photocurrent gain is adjusted to provide a 10.00-V signal prior to a stimulus, the signal might change to 9.99 V as the result of that stimulus. To permit efficient recording of such small changes, a differential amplifier is arranged to subtract the I 0.00-V reference voltage from the photocurrent signal and amplify the result by another factor of I 0. The output is then zero for the resting photocurrent level and I V per I% change in reference to that level. For small changes in light transmittance, such signals are readily converted to absorbance change units by a fixed multiplicative constant (e.g., see Smith and Zucker, 1980) .

Finally, the control amplifier contains a unity gain differential amp I ifier to provide an output proportional to the difference between any two switch-selected spectral

 

4. INSTRUMENTAL RESOLUTION LIMITS 259

channe ls . For small changes in light signa ls, the output of this amplifier is proportional to changes in the ratio of transmittance at the two selected wavelengths, or differential absorbance. Such a signal is useful for emphasizing absorbance changes of known spectral character , such as a change in indicator dye absorbance, while tending to reject many interfering signals such as changes in ti ssue light scattering or some instrumental instabilities.

4. INSTRUMENTAL RESOLUfiON LIMITS

The usefulness of optical absorbance measurement on single living cells is often constrained by resolution or s ignal- to-noise ratio limitations. The minimum resolvable signal may be determined by instrumental resolution limits or by factors inherent to a specimen, such as the tendency of living tissues toward stimulus-evoked or spontaneous movement or light-scattering change . While limitations of the latter class often prove the most serious , they are difficult to discuss in any general way. Differential absorbance recording is usually helpful , but solutions to individual problems are otherwise likely to be specific to the specimens and investigators involved. Instrumental resolution limits, on the other hand, are governed by well-known physical laws. Some of these limits will be discussed here . More complete treatments of some the topics raised can be found in general instrumentation texts, such as that of Strobel ( 1973), and in the excellent monograph on calcium measurement by Thomas ( 1982).

Instrumental noise sources can be considered in two categories: dark no ise and light no ise . These are, quite simply, the apparent output noise when the illuminating source is off and the extra noise when the source is on , respecti vely. Dark noise is characteristic only of the photodetector device and its associated amplifier, whereas light noise is associated with the illuminating light itself and possibly other factors. Light noise may have two components. One component , which can (at least in theory) always be reduced or compensated for, is due to such factors as fluctuations in lamp output , mechanical instabilities of the optical arrangement, or fluctuations in amplifier gain . The other component of light noise is due to the graininess or quantum nature of light. Arrival of photons at a detector and , more importantl y, the individual photoelectric events they cause are random events with the stati stics o f a Poisson process: In any given period of time the number of such events will have a variance equal to the mean number of such events.

The instrument described here was optimized for operation in a region where the statistical photon noise is the primary resolution-limiting factor. With the 45- j..Lm fiber-tip aperture and 20-nm bandwidth , photodiode currents are on the order of I 0 nA, or approximate ly 108 photoelectrons/ms . At the millisecond time resolution level , the corresponding ratio of mean effective photon count to r.m .s. effective photon noise, or shot no ise, is therefore I 04

. All sources o f dark noise are approximately a factor of I 0 be low thi s level . Optimization was therefore based on attempting the largest possible photocurrent , since the ratio of photocurrent to shot noise is proportional to the square root of the photocurrent. Illumination brightness is

 

260 POLYCHROMATOR FOR RECORDING OPTICAL ABSORBANCE CHANGES

limited by the surface brightness of the incandescent filament. Other sources such as arcs or lasers could illuminate the target with much greater brightness but would entail other complexities and greater cost. Large photocurrents were therefore sought by maximizing detector efficiency.

The dispersive spectral detector described here achieves large average photocurrents from a multiplexing advantage: Signals at all wavelength bands are integrated continuously. In comparison, the chopped-beam type of spectral detector (Chance et a!., 1975), integrates each wavelength for only a small fraction of each measurement interval and is therefore less suitable for the smaller optical apertures where photon flux is limited . The polychromator arrangement shown in Fig. I provides an overall efficiency for in-band light of about 30%. In comparison, a chopped-beam detector, where a given interference filter of 50% efficiency is in the beam about 10% of the time, would yield an overall efficiency closer to 5%.

Within limits, it is possible to trade off between temporal and spatial resolution limits to absorbance measurement. Longer integration times will thus allow reduction in size of the optical aperture and improved spatial resolution, and vice versa. This trade-off may fail with integration times of many seconds or longer, however, owing to I if noise in preamplifiers. Under these circumstances, beam chopper stabilization or photomultiplier tube (PMT) detectors might be desirable in spite of the consequent losses in photoelectron count (PMTs have only about I 0-20% the quantum efficiency of silicon photodiodes, even though they offer vastly quieter preamplification) .

If excellent accessibility for micromanipulation is not required , temporal and spatial resolution might be improved by increasing the numerical apertures of illumination and light collection. This could be accomplished , for instance, by placing the specimen between oil immersion condenser and objective , and coupling transmitted light collected at the objective image plane, either directly or by an optical fiber, to a polychromator like that described here .

REFERENCES

Brown, J . E. , P . K. Brown, and L. H. Pinto, ( 1977) Detection of light-induced changes of intracellular ionized calcium concentration in Limu/us ventral photoreceptors using arsenazo III . J. Physio/. (Lond.), 267, 299-320.

Chance, B., V. Legallais. J. Sorge, and N. Graham, ( 1975) A versatile time-sharing multichannel spectrophotometer, renectometer and nuorometer. Anal. Biochem., 66. 498- 5 14.

Smith, S. J. and R . S. Zucker, ( 1980) Aequorin response facilitation and intracellular calcium

accumulation in molluscan neurones. 1. Physiol. (Lond.), 300, 167- 196.

Strobel, H. A. (1973) Chemica/ Instrumentation: A Systematic Approach. Addison-Wesley, Reading. MA.

Thomas, M. V. (1982) Techniques in Calcium Research. Academic , London.