Fluorescence he Vertical Illuminator tee according to Ploem InstructionsDesign subject to alteration without notice. ERNST LEITZ amsBuH D-6330 WETZLAR Subsidiary: Ernst Leitz (Canada) Lid., Midland, Ontario Insert for instructions [SI2B2a/Engl. | Printed in W-Germeny xI1/72/0X/SDInsert for Instructions [=12-2a/EngI. | Fluorescence vertical illuminator Filter combinations for the use with the fluorescence vertical illuminator accord- ing to Ploem with built-in exciting filter turret: the sequence corresponds to that the positions on the switch wheel of the vertical illuminator; position 5 on the ex- citing filter turret has been left blank and permits any desired filter combination. Required Filter for the excliing-titer treet of the Dichroic beam | Suppression Sxctting fivoroscoaee vorleal Huminator (dia Tamm) | spiiting mirtor/| hiker in the radiation builtin hiker ste Suppression | filters | uireviotot | UVR 2mm UG 4 ricaanic oo | 988 460 Blue fitor amm 8G 3 Vietst | Imonterence bend Titer 406 (iyoe AL) | = S409 | TK 488/K 400 | Karo 2 Inter fier Ke 480 setae + Interierence blue filer KP a laeion rier aiue a nna aee Tk sists | Seletio $525 ype AL) 30 Band absorption filler 2mm 86.30 | Green intartoronee band fier S548 (hype AL) | ~ SS 846) TK S80/K Een | ooo For all filter combinations a 2mm KG 1 heat filter (Hg lamps, tungsten filament lamps) or Calflex (xenon lamps) and a 4 mm GB 38 red suppression filter must be inserted in he lamp housing. Filters inserted in the lamp housing are exposed to strong heat. It is therefore recommended to swing them out when not in use. The fluorescence vertical illuminator is supplied with this standard filter set in the exciting filter turret. It is, however, also available with an empty filter turret, to accomodate the conventional glass filters (diameter 50mm) in the lamp housing or to enable the user to build in other types of exciting filter.” * This LEITZ {filter combination corresponds in its use to the dyed.in-the-mass cemented inter ference filters by Schoit. u. Gen, Mainz, celled TAL in the literature. For thie a K 480 (GG 476) filter should be inserted In the lamp housing io eliminate primary fluor escence excited by the UV-portion of the K 490.Fluorescence Vertical Illuminator according to Ploem Instructions 21 22 34 Introduction Operation Visual! observation Photometry __ Practical hints oe Hints for work with the vertical illuminator Range of filters _ References __ 4 woannsan1 Introduction Fig, 1 ORTHOPLAN® universal lvgoiels miroscope and can be obtained for use with the ORTHOPLAN® or ORTHOLUX® 1 and 2 microscopes. The optical path is shown in Fig. 2, The fluorescence-exciting light of suitable wave length is directed to the specimen via one of the 4 dichroic aD ing revolving turret in ing. These beam-s} selectively reflect the exciting light, have the following reflection edges 400, 455, 510 and 580nm. The per- manently built-in suppression fi K 400, K 460, K'515, and K 580 are com- the same sequence on the turret ichrcic beam splitting mi Diagrammatic representation of the: LEITZ Huot minor for the ORTHOLUX 2 In addition further suppression filters are used in the ide slot of the stand 3.16 for the analysis of the fluor- mirror and suppres- ions have boon se- lected so that together with the appro- 9 and the suppres- jon and emission maxima respectively of the most widely jorochromes are accomodated. In this context not only the general UV or blue-light fluorescence method with fluorochromes such as acri- the demonst ces through cytochemical and chemical fluorescence staining (such as catecholamine demons which produces orange or red cence with fuchsin stains of the feulgen type or in the antibody technique with isothiocyanate tetramethylthodamine is possible to employ ences of two or more wave lengths. escences in tho same specimen.2 Operation 2.1 Visual observation Insert filter holder with field diaphragm 4.21 in the upper light aperture of the ORTHOPLAN. Swing out the hinged lens 4.22 in the ORTHOPLAN stand. insert the vertical illuminator with ives in the slide guide of the mi- ‘croscope and secure it with the clamp- Ing screw 4.18 on the right. Switch on the fluorescent light source and centre it according to the directions contained in the instructions Lamp Housing 250 (514-72) and Lamp Hous- ing 100 and 100 Z (514-19) Select the exciting filter and set the appropriate position on the exciting- filter turret For other than bi ier combinat- ions insert the ex: Iter in the filter slot of the lamp housing of in filter holder 3.12 depending on its diameter. Insert the desired beam-splitting mirror/ suppression filter combination in the beam path by rotating the selector wheel 3.17, The figure appearing in the round window after a rotation of the se- lector wheel indicates the combination ically focus the surface of the specimen, Set the field diaphragm 4.20 so that it imits the field of view. If necessary centre the field diaphragm with the screws 4.19, The stray-light diaphragm 5.23 in the fluorescence vertical illuminator for the ORTHOLUX 2 can be closed to elimin- ate disturbing glare effects. inaa 612 OftHowux 2 13 Bxoting diapheages 2.2 Photometry For photometry the lateral reflection of the vertical illuminator is used. By means of the changing lever 3.13 visual ‘observation can be changed to lateral reflection. 3 Practical hints 3.1 Particularly recommended for work with the fluorescence vertical illumin- ator: Low-power eyepieces, e.g. PERI- PLAN? or Huygens 6.3x eyepieces. Higher-power eyepieces can be used, but image brightness will be reduced GW or GG eyepieces on the ORTHO- PLAN aro unsuitable, as their enlarged field of view is not fully illuminated 32 Use objectives of large aperture, since the fluorescence intensity in- creases as the square of the aperture For medium and highest magnifications objectives with builtin iris diaphragm are best, as any stray light can be sup- pressed with the iris diaphragm. 3.3 Use ultra-high-pressure mercury lamps, high-pressure xenon lamps, and, it suitable, tungsten halogen lamps. ‘These light sources have the necessary Intensity, Consult insert 12-92 for the useful filter combinations,4 Range of filters for the fluorescence vertical illuminator Exciting filters Green exciting titer 88 546, ‘Violet exciting fier $ 400 ter K 40 Rianne es suppres $ Suppression tere K 490,480, 470 Suppression titers K 480, 510, 520 ‘Suppression fitters K 7, 58, 590, 610 Dlehrole beam spiting mirrors TK 400,485, 580 bull ino the Muorescence vertcal natorReferences: BECKER, H., _,Fluoreszenzhistochemie Amine", Mikroskopie Bd. 26 1870 159-168. EICHLER, J. und F. WALTER, Fluoreszenzmikroskopie des _Kni LEITZ-Mitt. Wiss. u. Techn. IV (1963) 110. biogener KRAFT, W., Dio Technologie dos LEIT2-Fluoras nzopak", LEITZ-Mitt. Wiss. u. Techn., Bd. IV (1963), 239, NITSCH, 8. MURKEN, 1. 0. u. BRUCK, HJ. Determining Feulgen-DNA 0! Individual Chromo somes by Fluorescence Cytophotometry with Inci dent Light", Histochemie 23, 254-285 (1970) Springer- Verlag 1970. PLOEM, J. S., Die Méglichksit der Auflichttluo- reszenzmethoden’ bei Untersuchungen von Zellen in Durchstrémungskammem und Leightonrohren", Acta histochemica Supplementband VII (1967) PLOEM, J. S., ‘Tho Use of a Vertical Illuminator with Interchangeable Dichroic Mirrors tor Fluores- cence Microscopy with Incident Light” Z. Wiss. Mikrosk. 68, 4 (1967), 129 PLOEM, J. S., .Ein neuor illuminator-Typ fir die Auflicht-Fluoreszanzmikroskopie", LEITZ-Mitt, Wiss. u. Techn. Bd, IV (1963), 225, WALTER, F., ine Auflicht-Fluoreszenz-Einrichtung fur dio Routinediagnoso", LEITZ-Mitt. Wiss. u. Techn., Ba Iv, Ne, 6, 186-187,ta WETZLAR = Registered Trademark Design subject to alterations without notice, ERNST LEITZ cmsun D-6330 WETZLAR Subsidiary: Ernst Leitz (Canada) Ltd., Midland, Ontario Printed in W.-Germany X,72;DX/S0