Imaging of fluorescent neurons labelled with fluoro-gold and fluorescent axon terminals labelled with AMCA (7-amino-4-methylcoumarine-3-acetic acid) conjugated antiserum using a UV-laser confocal scanning microscope.
This paper describes the implementation of an ultraviolet (UV) laser (Spectra Physics 171-18 with 3 lines: 334, 351 and 364 nm in UV) as light source for fluorescence confocal scanning microscopy. With this instrument it is possible to use fluorophores not previously available for confocal laser microscopical imaging of fluorophores such as fluoro-gold and AMCA. In the study we show confocal laser microscopical imaging of fluorescent motoneurons labelled by retrograde transport of fluoro-gold and AMCA-fluorescent axon terminals labelled with antisera against immunogenes as thyrotropin-releasing hormone (TRH) and calcitonin gene-related peptide (CGRP). These two fluorophores may be recorded simultaneously or separately by using a filter that suppresses the emission of one of the fluorophores. The described instrument should also be useful in applications involving detection of monoamines by the Falck-Hillarp technique, as well as measurements of cytosolic free calcium by indicators such as Fura-2 and Indo-1. Measurements performed in reflected and fluorescence light indicated that the resolution along the optical axis improved by about 25% when UV (351 nm) is used instead of visible light (514 nm). This figure is close to that expected on theoretical basis. There are, however, also serious problems related to the use of UV excitation. Firstly, objectives must be selected based on their UV transmission properties. Secondly, chromatic aberration may cause a substantial focal shift between illuminating and emitted light, calling for a flexible instrumental design in order to allow for compensation. As shown here, this problem can be circumvented by using reflecting objectives but at a price of lower resolution compared with high-aperture refracting objectives.
Ulfhake
B
B
Department of Anatomy, Karolinska Institute, Stockholm, Sweden.
Carlsson
K
K
Mossberg
K
K
Arvidsson
U
U
Helm
P J
PJ
D000818:Animals; D001369:Axons / Q000648:ultrastructure*; D015740:Calcitonin Gene-Related Peptide / Q000276:immunology; D003374:Coumarins; D005453:Fluorescence; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D007150:Immunohistochemistry; D008860:Microscopy, Ultraviolet; D009411:Nerve Endings / Q000648:ultrastructure*; D009474:Neurons / Q000648:ultrastructure*; D051381:Rats; D011919:Rats, Inbred Strains; D013116:Spinal Cord / Q000166:cytology / Q000648:ultrastructure*; D013266:Stilbamidines; D013973:Thyrotropin-Releasing Hormone / Q000276:immunology
C049774:2-hydroxy-4,4'-diamidinostilbene, methanesulfonate salt; D003374:Coumarins; D005456:Fluorescent Dyes; D013266:Stilbamidines; D013973:Thyrotropin-Releasing Hormone; D015740:Calcitonin Gene-Related Peptide; C051029:7-amino-4-methylcoumarin-3-acetic acid