ИСТИНА

Various sophisticated techniques are available for microscopic examination of individual neurons in experimental animals. For human specimens, however, the options are much more limited. In particular, there are fluorescence methods for visualizing processes of individual neurons in laboratory animals, while in humans, attempts at such methods have met with limited success. Thus, the field has continued to rely upon Golgi methods, which extensively stain individual neurons, though sparsely and apparently at random (Ramon y Cajal, 1894). Golgi stains have always been viewed by bright field transmission microscopy, usually in thick sections (100-200 microns). Analysis of such material is complicated by the superimposition of signal from out-of-focus planes. Here we report that superior images of metal stained cells can be obtained with the use of a pulsed infrared (multi-photon; 2P) laser. Unexpectedly, several metal stains luminesce in visible wavelengths under low power 2P excitation. The luminescence shows a quadratic dependence on excitation power, effectively limiting signals to the plane of focus, which results in excellent axial resolution (~1 micron). Resolution is further improved by deconvolution based on an empirically determined point spread function. Striking improvement over bright field imaging was obtained with mercury-based (Golgi Cox) and silver-based (Golgi-Kopsch) methods for visualizing dendrites and spines. The use of neuron tracing software (essentially developed for fluorescence microscopy) is greatly facilitated. The advantage over bright field microscopy for Golgi stains of mouse brains were similar. The method also worked well with several other metallic stains of brain: With Bielschowsky silver stain, it allowed 3-dimensional tracking of closely spaced axons in white matter. Bright luminescence was also obtained with Gallyas (silver stain with gold toning) and Von Braunmuhl (silver) methods, apparently increasing their sensitivities in revealing pathological structures. The mechanism of this 2P luminescence is not yet elucidated, as its photophysical characteristics are inconsistent with both fluorescence and reflectance. While we are still working to determine the mechanism, the method is already adding startling detail to our picture of human neurons.