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A method for automated collection of various specific data from an entire microscopical preparation and their quantitative evaluation is described. Its application to the study of neuronal connections is discussed in some detail. Brain sections are scanned using a computer-controlled microscope for reflectance, fluorescences or absorbance signals. Two illuminating beams are used, one of them being amplitude modulated. By means of a synchronous detection the two signals are recorded simultaneously: for example, in an autoradiograph, the reflectance (measuring the density of the silver grains in emulsion) and the absorbance (allowing to localize the underlying counterstained cells). The data are stored in a computer. Various off-line processing schemes allow the reconstruction of the data with respect to the corresponding spatial coordinates. Thus pseudo-three-dimensional, analogue or digital, graphic displays may be obtained in which the patterns of neuronal connections can be recognized and interpreted. A method for the detection of weakly labelled nerve fibres based on digital filtering is presented. The whole processing for a frontal section of the mouse brain (7 X 10 mm area) takes less than 1 h. In addition to the evaluation of microscopically labelled material (grains of autoradiographs, horseradish peroxidase, nucleic acids) the technique described has been successfully used for the study of naturally fluorescent intracellular components in living tissue cultures.
A computerized microspectrophotometer was developed to provide rapid and accurate sequential measurements on cells, including nuclear DNA content, by fluorescence staining, photometric grain counting from autoradiographs, and nuclear size estimated by measurement aperture area. To alleviate bulk and vibrations caused by additional stepper motors, the excitation light and filters as well as the emission filters and photomultipliers were detached from the main microscope frame. The light was directed to and from the microscope through fibre optic bundles. This configuration provided an excellent light gathering faculty and made optical alignment easier. The machine provided highly accurate sequential measurements on a relatively large numbers of cells by static photometry.
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Microspectrophotometric measurements of individual dark-adapted rhabdoms of the prawn Palaemonetes vulgaris reveal the presence of two light-sensitive pigments. A pigment with maximum absorbancy at 555 nanometers is converted by light to a long-lived intermediate with wavelength of maximum absorbancy at 496 nanometers. A second pigment with wavelength of maximum absorbancy at 496 nanometers bleaches in the light, seemingly without forming detectable products at wavelengths longer than 375 nanometers. Both pigments occur in each layer of microvilli.
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Two methods, which relate grain number to cell cycle phase in Feulgen-stained autoradiographic preparations, have been developed and compared. Both methods automate grain number estimations, one by taking integrated absorbance measurements at different wavelengths, the other by measuring absorption at a single wavelength before and after chemical removal of silver grains. With tritium-labelled tobacco mosaic virus as a probe, a quantitative analysis has been made of the binding of virus particles to plant protoplasts in different compartments of the DNA replication and partition cycle. The preliminary results indicate that the quantity of virus bound by protoplasts is related to their cell cycle phase. Whilst in this case, the methods have been used with plant cells, both techniques are equally applicable to animal cells.
Microspectrophotometric examination of the retina of a procellariiform marine bird, the wedge-tailed shearwater Puffinus pacificus, revealed the presence of five different types of vitamin A(1)-based visual pigment in seven different types of photoreceptor. A single class of rod contained a medium-wavelength sensitive visual pigment with a wavelength of maximum absorbance (lambda(max)) at 502 nm. Four different types of single cone contained visual pigments maximally sensitive in either the violet (VS, lambda(max) 406 nm), short (SWS, lambda(max) 450 nm), medium (MWS, lambda(max) 503 nm) or long (LWS, lambda(max) 566 nm) spectral ranges. In the peripheral retina, the SWS, MWS and LWS single cones contained pigmented oil droplets in their inner segments with cut-off wavelengths (lambda(cut)) at 445 (C-type), 506 (Y-type) and 562 nm (R-type), respectively. The VS visual pigment was paired with a transparent (T-type) oil droplet that displayed no significant absorption above at least 370 nm. Both the principal and accessory members of the double cone pair contained the same 566 nm lambda(max) visual pigment as the LWS single cones but only the principal member contained an oil droplet, which had a lambda(cut) at 413 nm. The retina had a horizontal band or 'visual streak' of increased photoreceptor density running across the retina approximately 1.5 mm dorsal to the top of the pecten. Cones in the centre of the horizontal streak were smaller and had oil droplets that were either transparent/colourless or much less pigmented than at the periphery. It is proposed that the reduction in cone oil droplet pigmentation in retinal areas associated with high visual acuity is an adaptation to compensate for the reduced photon capture ability of the narrower photoreceptors found there. Measurements of the spectral transmittance of the ocular media reveal that wavelengths down to at least 300 nm would be transmitted to the retina.
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