Use of UV excitation in confocal laser scanning fluorescence microscopy |
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| Affiliation: | 1. Physics IV, The Royal Institute of Technology, S-100 44 Stockholm, Sweden;2. Max Planck Institute for Medical Research, Jahnstr. 29, D-6900 Heidelberg, Germany;3. Department of Mathematics, The Royal Institute of Technology, S-100 44 Stockholm, Sweden;1. Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark;2. Interdisciplinary Nanoscience Center, Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus, Denmark;1. Department of Pathology, School of Basic Medical Sciences, Xi''an Jiaotong University Health Science Center, Xi''an, China;2. Second Division of In Vitro Diagnostic Reagents, National Institute of Food and Drug Control, Beijing, China;3. Department of Pathology, Xi''an Medical University, Xi''an, China;4. Therapeutic Vaccines Engineering Center of Shaanxi Province, Xi''an Jiaotong University Health Science Center, Xi''an, China;5. Institute of Cancer Research, School of Basic Medical Sciences, Xi''an Jiaotong University Health Science Center, Xi''an, China;1. Department of Mechanical Engineering, State Polytechnic of Jakarta, Depok 16424, Indonesia;2. Institute of Photonics System, National Chiao Tung University, Tainan 71150, Taiwan;3. Department of Materials Science and Engineering, National Formosa University, Yunlin 632, Taiwan;4. Center for Condensed Matter Sciences, National Taiwan University, Taipei 10617, Taiwan;5. Department of Materials Science and Engineering, National Dong Hwa University, Hualien 97401, Taiwan;1. Department of Anatomical Pathology, University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX, 77030, USA;2. Department Gastrointestinal Medical Oncology, University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX, 77030, USA;3. Department of Surgical Oncology, University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX, 77030, USA;4. Department of Radiation Oncology, University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX, 77030, USA;5. Department of Translational Molecular Pathology, University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX, 77030, USA;1. EDF R&D, 6 quai Watier, 78400 Chatou Cedex, France;2. Institute of Research and Development on Photovoltaic Energy (IRDEP), UMR 7174 CNRS-EDF- Chimie ParisTech, EDF R&D, Chatou, France;3. NextPV, CNRS-RCAST Joint Lab, Tokyo University, Tokyo, Meguro-ku 153-8904, Japan;4. IPVF, Institut Photovoltaïque d’Ile-de-France, 8 rue de la Renaissance, 92160 Antony, France |
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| Abstract: | By making only minor modifications, we adapted a conventional confocal beam-scanning laser microscope for the recording of UV-excited fluorescence. The major, and most expensive, change is that we coupled an external UV argon ion laser, providing the wavelengths 334, 351 and 364 nm, to the microscope scanner. We also replaced some optical components to obtain improved transmission and reflection properties in the UV. Only easily obtainable and inexpensive off-the-shelf components were used. The most serious problem encountered was the chromatic aberration of the microscope objective when using both UV and visible wavelengths. This is of no consequence in conventional microscopy where good imaging properties are important only in the visible region. In confocal microscopy on the other hand, good imaging properties are necessary for both the exciting and fluorescent light. Rather than having new optics designed, we tried with simple means to reduce the effects of the chromatic aberration to a tolerable level. This was done by mechanical adjustments in the ray-path. In addition we also tested two mirror objectives, which are inherently free from chromatic aberrations. However, such objectives have rather limited numerical apertures and are not of the immersion type. Their value in biomedical applications is therefore limited.The objective most frequently used in our experiments was a 63/1.25 oil-immersion fluorite. Without any compensation this objective had a depth resolution in UV-excited confocal fluorescence that was an order of magnitude worse than when using visible-light excitation. The useful field of view was also very small due to lateral chromatic aberration. By simple means we managed to improve the depth resolution by a factor of 4.4, and at the same time increase the useful field of view substantially. Still, the depth resolution was worse than what is obtained using visible light excitation. We think this is due to the fact that after compensation the objective is working with an incorrect tube length.Using the modified instrument, we recorded specimens labelled with AMCA and Fluoro-Gold, obtaining 1.5 μm thick optical sections. |
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