Microspectrophotometric measurements of vertebrate photoreceptors using CCD-based detection technology
Department of Biology, University of Victoria, PO Box 3020 STN CSC, Victoria, British Columbia, Canada V8W 3N5
Present address: Rochester Institute of Technology, Rochester, NY 14623-5603, USA
*Author for correspondence (e-mail: chawrysh{at}uvic.ca)
Accepted April 19, 2001
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Summary |
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Key words: CCD-based microspectrophotometer, photoreceptor, spectral absorption, rainbow trout, Oncorhynchus mykiss.
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Introduction |
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To date, most, if not all, MSP systems used in examining vertebrate photoreceptors are designed to measure the transmitted light flux through photoreceptor outer segment (sample spectrum) and a clear area of the field (reference spectrum), as the spectrum is scanned (at 300800nm wavelength). Absorbance values (logT-1) (T, transmittance) are calculated as a function of wavelength in regular wavelength increments (e.g. 5nm; Harosi, 1987). The measurement time required to traverse the spectrum, usually in both an ascending and a descending series, varies from one apparatus to another. Linear diode arrays have been used to measure the spectral absorbance of photoreceptors with some degree of success (Widder et al., 1987; Hiller-Adams et al., 1988), but spectral absorbance curves presented in these papers consistently show a discordance with Dartnall nomogram templates. With the rapid development of electro-optical technology, CCD (charge-coupled device) chip technology has been used recently with success in a wide variety of spectroscopic applications (Maseide and Rofstad, 1997; Schweitzer et al., 1996; Tang et al., 1994; Tsujita et al., 1997). One of the important features of CCD chip technology is that it is comparatively fast (800ms per scan and only one scan required) relative to wavelength-scanning MSP systems (up to 10s per scan with multiple scans but there are appreciable differences between each MSP apparatus). This is possible because our CCD-MSP system uses one flash of full-spectrum light (300800nm) with a short-duration (800ms). The transmitted flux is measured by a CCD detector used in tandem with a high-resolution (0.4nm) spectrograph, providing the same information as a MSP system in a much shorter period of time. The rapid measurement time may have important implications when measuring fragile retinal tissue. For instance, excised salmonid retina is only viable for approximately 1h. With a shorter measurement time, more cells can be measured at the peak quality of the tissue. Furthermore, photoreceptor movement in retinal preparations, even for the most stable preparations, can account for considerable signal variance, and rapid signal acquisition therefore should help to minimize signal variance.
In this paper, we describe the design of the CCD-based MSP instrument and offer some thoughts on overcoming problems inherent to the technology. We also evaluate the performance of the instrument using rainbow trout (Oncorhynchus mykiss) retinas, previously measured (Hawryshyn and Hárosi, 1994) using the wavelength-scanning MSP system.
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Materials and methods |
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Tissue preparation
After overnight (or at least 2h) dark adaptation, fish were anaesthetized by immersion in Tricaine methanosulphonate (MS-222, Cresent Chemicals) (300mgl-1) for approximately 10min and then killed by cervical transection. The eyes were removed and hemisected under near infrared illumination provided by light-emitting diodes (LEDS) with a peak output at 660nm. The retinae were removed using forceps and placed in a cell culture dish containing freshly prepared, ice-chilled Minimum Essential Medium (MEM; Sigma). 292mgl-1 L-glutamine and 350mgl-1 NaHCO3 were added, the pH adjusted to 7.3 using 1moll-1 NaOH, and the solution filtered through a 0.22µm filter. The dish was kept in a light-tight container placed on ice. Illumination was provided by infrared LEDs (primary wavelength of emission 880nm). A piece of retina was sectioned using a razor blade segment under infrared LEDs and placed on a coverslip. A razor blade was used to cut the retina section into many small pieces. This provided better preparations than could be attained by teasing the tissue with forceps. MSP recordings were collected at a constant temperature of 15°C.
Microspectrophotometer design
Fig.1 shows a schematic layout of the experimental apparatus. The measurement beam light source was provided by a 150W xenon arc lamp (Oriel) equipped with a condenser lens system that was adjusted to focus on the plane of the shutter. A light intensity controller (Oriel) is currently being added because of small transient fluctuations in intensity, which can lead to spurious noise in the signal. To enable the measurement beam to be positioned without undue bleaching of the preparation, an infrared filter (Ealing Far Red, Schott RG850 or RG1000), attached to a swing arm, was moved in and out of the path of the light beam situated in front of the shutter position. A quartz disc diffuser was mounted at the shutter (Uniblitz) plane to blur the arc image. A quartz fibre-optic cable (Voltrex) fed light from the shutter onto an XY variable slit aperture (Leitz), then to a beam-splitter mounted in a cube on the Zeiss Axiovert TV100 inverted microscope above the condenser lens [Ealing 52x, 0.65 numerical aperture (NA) mirror objective]. The condenser lens system formed an image of a variable slit aperture (Leitz). The measurement beam used in the recordings presented in this paper was unpolarised. The background illumination was provided by a 12V, 100W halogen lamp on the Axiovert microscope. An Ealing Far Red, Schott RG850 or RG1000 filter was placed in the background channel to prevent bleaching of photoreceptors. A second CCD camera (Canadian Photonics Laboratory), mounted on the trinocular (not shown in Fig.1), was used for viewing the microscope field and was displayed on the computer monitor. This camera was used to capture infrared images of the preparation.
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CCD detector
After light has passed through the specimen, it enters the spectrograph. There, it is diffracted by an ultraviolet blazed grating, which acts to spread the light beam spectrally across the X-axis (columns) of the CCD chip. The CCD array used a chip with a pixel array of 1340 columns and 400 rows. Each pixel can be considered as a well in which charges accumulate when photons are absorbed. The voltage potential of each pixel rises as the number of charges increases. When the CCD is read, the charges are moved towards one side of the chip (electronically). It is possible to fetch each (of 400) rows of pixels individually or to let the charges accumulate in the exit row. The number of rows summed in the exit row (before it is dumped) can be set. This procedure is called vertical binning. Its usefulness is limited by the 16-bit (65536 level) resolution of the A/D converter, which allows a maximum value of 65535. The A/D converter converts the analog potential at the CCD exit row to a digital number.
A second characteristic of CCD chips is called the region of interest (ROI). The measurement beam is approximately 1 µm x 2µm in size (adjusted by an X-Y variable slit system, Leitz) when it traverses the photoreceptor. The grating in the spectrograph spreads this rectangle across the CCD according to wavelength. This image does not cover the whole height of the CCD array. To eliminate noise originating from less well illuminated pixels of a column it is possible to restrict the dump from the CCD to specified rows or strips of rows. The rows selected belong to the ROI (region of interest). It is also possible, at a loss of wavelength resolution, to average columns. The ROI is specified by the range of rows to be dumped and by the number of columns to average.
Parameters of measurement
To measure the spectral absorbance of a photoreceptor two spectra were compared: a reference measurement was performed by placing the measurement beam in a clear tissue-free area and a photoreceptor measurement was performed by placing the measurement beam within the photoreceptor outer segment (background level was subtracted from all spectra collected). Absorbance was calculated as log10 of the ratio of the intensity of the reference spectrum over the intensity of the sample measurement spectrum.
To achieve the best signal-to-noise ratio (S/N), the exposure time is adjusted to provide pixel counts in the ultraviolet region of the spectrum that are well above background noise. For instance, with the use of vertical binning we have determined that a 0.8s exposure time produces about 6000 counts at 340nm and 400000 counts at 700nm. This exposure time is set to minimize photoreceptor bleaching. For the experiments described here, exposure times from 0.8 to 1.2s in one flash proved optimal.
In the wavelength range below 350nm, low source output was the main source of signal noise. We have added a light intensity controller (Oriel) to reduce the variability of the signal amplitude across the spectrum.
Calibration
Wavelength calibration of the CCD-MSP system was performed using spectral lines generated from both a xenon light source (Oriel) and a fluorescent lamp (RadioShack). This wavelength calibration is performed every 2 weeks. Optical density calibration was performed by comparing neutral density filters of known density with density measurements determined by the CCD-MSP system.
Software
The drivers for the CCD camera were provided by Roper Scientific as a dynamic link library (DLL). The graphical user interface (GUI) was written in Visual Basic (Microsoft). For plotting and mathematical analyses, a run time version of IDL (Research Systems, Inc) as an Active X control (software library/toolbox accessible from multiple software applications) was used.
Photoreceptors are refractive and thus, any displacement of the image of the measurement beam on the CCD detector can lead to a shift either along the wavelength axis or within the region of interest (leading to a shift in amplitude), or both. A shift along the wavelength axis between the reference and the measurement spectra introduces distortion into the absorbance spectrum. Because all recorded spectra exhibit spectral spikes or lines, the measurement spectrum can be shifted to coincide with the reference spectrum. This procedure of correcting for spectral pixel-shifting is required for approximately 30% of the measurements performed.
Acceptance criteria for spectra
The long-wavelength limb baseline was used as the first criterion. Since the photoreceptors are small and the absorbance weak, MSP data are inherently noisy, and strategies of analysis have been developed. For this publication, criteria of acceptance and methods of analyses were guided by Hárosi (Hárosi, 1987), MacNichol (MacNichol, 1986) and Levine and MacNichol (Levine and MacNichol, 1985).
If it was well defined, the spectrum was given further consideration. If it showed a clear linear trend (tilt) it was linearly detrended (Hárosi, 1987) and a template was then fitted to the data. For A1-based visual pigment cones (vitamin A1-based visual pigments are also called rhodopsin visual pigments; vitamin A2-based visual pigments are also called porphyropsin visual pigments), an eighth-order polynomial (Bernard, 1987) was fitted to the absorbance spectrum and for the rods a long-wavelength limb chart (Munz and Beatty, 1965), characterized by max and half-maximum bandwidth (HBW), was fitted to the long-wavelength limb of the absorbance spectrum. If the short- or long-wavelength limb of the spectrum was narrower or wider than the template, the spectrum was rejected. Lastly, for most photoreceptors it was also possible to acquire a bleached spectrum by exposing photoreceptors to the measurement beam for 120s. This was used as an additional criterion for acceptance.
Cones
The fit of the eighth-order polynomial template provided max and HBW for each spectrum. The spread in
max of the ultraviolet-, blue- and green-sensitive cones and their number of measurements were not large enough to try to group them according to porphyropsin content. This does lead to a less precise estimation of the HBW for these cones. The red-sensitive cones could be separated on the basis of visual pigment composition (i.e. those clustered around 570nm for the A1-based visual pigment and those clustered around 620nm for the A2-based visual pigment). Each group was normalized and averaged.
Rods
max and HBW for rods containing rhodopsin, porphyropsin and mixtures of both, were known (Munz and Beatty, 1965). For each rod spectrum these two values were fitted. After normalizing, the rod data were divided according to porphyropsin content relative to rhodopsin concentration: less than 10%, more than 80% and approximately equal concentrations (4060%). Fish that contained a predominance of porphyropsin had a lower body mass in the size range used. The averages for each group were calculated and compared with published values (Munz and Beatty, 1965).
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Results |
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Rods
We measured 23 rod photoreceptors, and in this sample we were able to differentiate rhodopsin, porphyropsin and mixed A1 and A2 visual pigment spectra. In Fig.4AC we show the absorbance spectra for three classes of rod photoreceptors (see Table1 for spectral data). Table1 shows three mean max values of the
-band of rods: 504±2.1nm (N=10, rhodopsin-dominated rods), 522±8.2nm (N=5, porphyropsin-dominated rods) and 510±2.1nm (N=8, rhodopsin porphyropsin mixed rods). Table1 also shows that the half-maximum bandwidth (HBW, cm-1) of the three absorbance spectra vary in a predictable manner, with the porphyropsin absorbance curve being wider than the rhodopsin absorbance spectrum and the mixed chromophore rods having the broadest absorbance spectrum. All spectra were fitted with long-wavelength limb templates derived from Munz and Beatty (Munz and Beatty; 1965) (100% A1, Fig.4A; 50% A1: 50% A2, Fig.4B and 100% A2 Fig.4C) and the HBWs were determined using eighth-order polynomial templates (rhodopsin) and Fourier transform (porphyropsin and A1/A2 mixture absorbance curves). The absorbance spectra for the rods are slightly elevated in the short-wavelength portion of the spectrum due to photoproducts resulting from red light contamination within the dark room. This source of contamination has been identified and eliminated.
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Discussion |
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Prior to a seaward migration, anadromous salmonids (migrating from fresh water to sea water) change their visual pigment composition from a porphyropsin (freshwater visual pigment) to rhodopsin (marine visual pigment). Measurements of changing visual pigment composition of rods over time, together with changes in other physiological characteristics (silvering, modification of gill epithelia for purposes of osmoregulatory competence), are now being examined as tools for assessing the smoltification status of anadromous salmonid fishes. One aspect of our future research is to use visual pigment composition as an index of smoltification in an application that may have important implications for Salmonid Enhancement Programs (hatchery stocking) and the aquaculture industry.
While we are satisfied with the performance of the CCD-base MSP instrument described here, its development was challenging in a number of respects. The initial phase of development utilized an InstaSpec IV CCD system (Oriel), which lacked the sensitivity required to measure the low absorbance typical of vertebrate photoreceptors. We subsequently retrofitted a photointensifier (Generation II, Science Tech, London, Ontario); however, at low photon flux, we discovered that the multichannel plate intensifier introduced problematic levels of noise into the signal (Shot Noise). The back-illuminated CCD system (Roper Scientific) that we ultimately chose has very high sensitivity over the range of 300800nm and chip-cooling maintains the noise at a low level. Such CCD systems now used in spectroscopy are more expensive than photomultipliers used in wavelength-scanning systems but, as with most electro-optical technology, we expect that CCD spectroscopy systems will be more cost-effective in the near future. The development of CCD-based MSP technology has been limited, to some extent, by developments in the electro-optical industry, but we are now satisfied with the performance of the system described here. Rapid data acquisition offers the advantages of less variance due to movement of target photoreceptors during measurement, reduced spectral distortion due to photoproduct interference and an ability to measure fast, transient changes in absorbance as bleaching proceeds. These factors contribute to low signal variance and offer CCD-based MSP as an attractive alternative to wavelength-scanning MSP.
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Acknowledgments |
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