Advanced technique of infrared LED imaging of unstained cells and intracellular structures in isolated spinal cord, brainstem, ganglia and cerebellum.
Light-emitting diodes (LEDs) have recently been used for the imaging of unstained living cells in the whole brain and spinal cord preparations, in which one cut was done to remove the overlying white matter. Here we show that in many cases the neurones can be visualized through the white matter in an intact nervous tissue (rats P0-P36 and mice P0-P2). We used an upright microscope with a water immersion objective and a powerful infrared LED (emission peak, 850 nm; maximum radiant intensity, 270 mW/sr) as a source of oblique illumination. In the isolated spinal cord, we were able to visualize lamina I and II neurones as well as motoneurones. In the brainstem, the neurones from the superficial nuclei were successfully viewed. In the sensory ganglion, we obtained images of unstained cells as well as intracellular structures, like endoplasmic reticulum, nucleus and nucleolus. In isolated cerebellum, parallel fibers, Purkinje and granule cells were viewed. Whole-cell recordings were done to fill spinal lamina I neurones, motoneurones and brainstem neurones with biocytin for detailed 2D-3D reconstruction of their dendritic and axonal arbores. Our imaging technique also allowed labelling individual intact neurones by injecting biocytin through the extracellular cell-attached pipette. This imaging technique opens broad possibilities for functional studies of neurones with completely preserved anatomical structures and synaptic inputs. We also show that the application of oblique infrared LED illumination allows a construction of a simple digital videomicroscope for the high-quality living cell imaging in intact nervous tissue.
Szucs
Peter
P
IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal.
Pinto
Vitor
V
Safronov
Boris V
BV
D000818:Animals; D001369:Axons / Q000502:physiology / Q000648:ultrastructure; D048430:Cell Shape / Q000502:physiology; D002490:Central Nervous System / Q000166:cytology* / Q000502:physiology; D004396:Coloring Agents; D003712:Dendrites / Q000502:physiology / Q000648:ultrastructure; D004582:Electronics, Medical / Q000295:instrumentation / Q000379:methods; D007259:Infrared Rays; D008029:Lighting / Q000295:instrumentation / Q000379:methods*; D008239:Lysine / Q000031:analogs & derivatives; D008297:Male; D018715:Microscopy, Video / Q000295:instrumentation / Q000379:methods*; D009445:Neuroanatomy / Q000295:instrumentation / Q000379:methods*; D009474:Neurons / Q000166:cytology / Q000502:physiology; D055095:Optics and Photonics / Q000295:instrumentation / Q000379:methods*; D009924:Organ Culture Techniques; D018408:Patch-Clamp Techniques / Q000295:instrumentation / Q000379:methods; D010775:Photic Stimulation / Q000295:instrumentation / Q000379:methods; D051381:Rats; D013194:Staining and Labeling / Q000379:methods*
D004396:Coloring Agents; C013411:biocytin; D008239:Lysine