Leica DM IRB manual

Leica DM IRB Instructions[...]

2 Issued in 1 998 by: Leica Microsystems Wetzlar GmbH Ernst-Leitz-Strasse D-35578 W etzlar (Germany) Responsible for contents: Marketing MQM, product management, T el. +49 (0) 64 4 1 -29 25 1 9 Fax +49 (0) 64 4 1 -29 22 55[...]

3 Leica DM IRB Instructions[...]

4 Copyrights All rights to this documentation and the software it describes are owned by Leica Microsystems Wetzlar GmbH. Copying of text and illustrations – in full or in part – by printing, photostat, microfilm or other techniques, in- cluding electronic systems, is only permitted subject to the express written consent of Leica Microsystems W[...]

5 Contents Important notes on this manual ....................... 7 General safety information ............................... 8 Intended application .......................................... 10 The microscope and its components ............. 11 Key subassemblies ............................................. 11 Stands .............................[...]

6 Contents Learn mode .......................................................... 43 Installing the objective prisms ......................... 43 Learning the IC objective prisms ..................... 43 Installing the objectives .................................... 44 Parfocality ............................................................ 47 Dry o[...]

7 The manual is multi-lingual. Due to the spiral binding you can turn the language version you want to the front. The DM IR is available both as a life sciences microscope and as a metallographic/industrial microscope. In cases where the function and operation are identical, the same text and illustrations are used in both the separate instruction [...]

8 Make sure that only fuses of the specified type and rating are used as replacements. It is forbidden to use mended fuses or to short- circuit the fuse holder . n. b.: The instruments and accessories described in this manual have been safety-tested and checked for possible hazards. Before modifying the instrument in any way or combining it with no[...]

9 n. b.: The electric accessories of the microscope are not waterproof. If water gets inside them, it may cause electrical shock. Do not put the microscope and its acces- sories too near a water supply or anywhere else where water may get inside them. n. b.: Before changing fuses or lamps, always turn the mains switch off and disconnect the mains c[...]

10 The new DM IRB is the logical further development of the successful inverted research microscope from Leica. It is used for examinations of cells and tissue, for micromanipulation and microinjection tech- niques all the way through to microdissection or confocal microscopy . The DM IRB has universal application potential, incorporating all the c[...]

11 The microscope and its components Fig. 1 – 2 1 Binocular phototube, 2 Eyepiece adapter tube, 3 Eyepieces, 4 T ube mount (tube change interface), 5 T ube port for photo/TV connection, 6 Beamsplitter switch, 7 Mains switch, 8 Brightness adjustment, 9 Lateral TV port, 10 Coaxial coarse and fine drive, 11 Fluorescence module, 12 ICT prism adjustme[...]

12 The stand There are 5 basic versions of the DM IRB stand, which allow over 50 microscope variants to be configured. These 5 basic versions are: – Manual or electronic stand – With or without integrated fluorescence axis – With or without SLR front port or bottom port – Lateral photo port 100 % or 80 % – With or without integrated magni[...]

13 Brightness adjustment A 12 V 100 W transformer is built into the stand for stepless regulation of brightness with the brightness control. Coarse and fine control The coarse and fine focus control allows fast and precise focusing of the microscope image. Focusing is done by a vertical movement of the objective nosepiece. The vertical movement ran[...]

14 Specimen stages and accessories The specimen stage supports the specimens that are to be examined through the mi- croscope. Several options are available to accommodate the wide variety of specimens examined, such as object guides, extension plates, specimen clips, scanning stage, heating stage, etc. Objective nosepiece and objectives The object[...]

15 n. b.: Lamphousings* and power units* must be placed at least 10 cm away from the wall and from flammable objects. Installation site The microscope should be used in a dust-free room which is free of oil and chemical fumes and extreme humidity . Also, the workplace should not be exposed to major temperature fluctuations, direct sunlight or vibra[...]

16 ● First take all the components out of the transport and packing material. ● Put the basic DM IRB stand on a desk which has enough room for it. n. b.: On no account should the microscope be connected to the power socket yet! Unpacking Please compare the delivery carefully with the packing note, delivery note or invoice. W e strongly recommen[...]

17 Fig. 3 Assembly tools 1 Cross-tip screwdriver*, 2 Hexagonal screwdriver , 3 mm, 3 Centring keys, 2 mm*, 4 Centring keys, 1.5 mm*, 5 Allen key , 3 mm*, 6 Allen key , 2.5 mm* (shor t version) Assembly Assembly of the transmitted light illumination column Wipe off the interface surface (4.3) with a dry cloth. T ilt the illumination column (4.1) sli[...]

18 The lamphousing for transmitted light illumination for 12 V 100 W halogen lamps with single-lens aspherical collector and heat protection filter is an integral part of the transmitted light illumination column. The halo- gen lamp is preassembled. The chapter on T roubleshooting includes a description of how to assemble and change halogen lamps. [...]

19 Insert the light rings for Phaco (identified by the code numbers 0, 1, 2, 3 and the intercept distance S of the corresponding condenser top, e. g. 2 S1) and the DF diaphragm (identified by D for darkfield and the intercept distance S of the corresponding condenser top, e. g. D S1, see table “T echnical Description”) in the slots of the disc [...]

20 Fig. 8 Condenser 0.53 S23 1 Condenser base, 2 Condenser top 0.53 S23 (inter - changeable), 3 Condenser disc, 4 Aperture diaphragm, 5 Filter holder , 6 Dovetail guide Fig. 9 Condenser tops for condenser base (8.1) 1 Condenser top 0.53 S23, 2 Condenser top 0.90 S1, 3 Condenser top P 1.40 OIL S1, 4 Spacer ring for assembling 9.2 and 9.3 Fig. 10 6-p[...]

21 Assembly of IC condenser prisms The IC condenser prisms are assembled at the factory . The following steps are only necessary in case of a retrofit: Remove the condenser disc (11.5) by slackening the screw (11.4) on the underneath of the condenser . – Using the centring keys (12.1), slightly un- screw the two centring screws (10.11). – IC co[...]

22 Assembly of the condensers to the illumination column Condenser 0.30 S70 T ilt the TL illumination column to the back (13.1). Insert the condenser 0.30 S70 (13.4) from below into the dovetail guide of the illumination column (13.2), with the condenser top pointing towards the microscope stage. Adjust the height of the condenser until its upper e[...]

23 Fig. 15 Assembly of condenser holder 1 T ransmitted light illumination column, 2 Dovetail guide, 3 Condenser height markings S1, S23 and S70, 4 Condenser holder , 5 Clamp screw for securing the condenser holder , 6 Clamp screw for field diaphragm module, 7 T ransmitted light lamphousing Fig. 16 Assembly of condenser holder 1 Condenser holder in [...]

24 3 1 2 Assembly of field diaphragm T o enable Koehler illumination when using condensers 0.53 S23 and 0.90 S1, a field diaphragm has to be assembled. Insert the field diaphragm module (18.1) into the mount (Fig. 18) from below . The diaphragm adjustment (18.2) should point in the direction of the tube. Secure with clamp screw (18.3). Assembly of [...]

25 2 1 3 3 Assembling the ICT objective prisms Assembling the IC module and IC objective prisms The IC prism disc with the IC prisms ordered by the customer are already assembled in the microscope at the factory . In case you want to retrofit the IC prism disc, please proceed as follows: Remove the front cover (22.2) under the objective nosepiece ([...]

26 Differences between prism D and D1 Prism D is the standard prism with greater shearing and therefore higher detection sensi- tivity for minute topological and refractive index variations in the specimen. Prism D1 has smaller shearing than prism D and a lower detection sensitivity for topological and refractive index variations. However , prism D[...]

27 Inserting the fluorescence module The fluorescence module (Fig. 26) is part of the fluorescence stand, but is also available as a retrofit kit. T o retrofit the fluorescence module, remove the blind cover from the stand. The fluorescence module can be fitted with up to four different filter cubes (26.3). They are inserted into the dovetail mount[...]

28 3 1 2 4 Fig. 30 Mirror housing and illumination telescope 1 Lever for mirror switching, 2 Lateral lamphousing mount with fixing screw, 3 Back lamphousing mount with Allen screw, 4 Illumination telescope for gas discharge lamps Fig. 31 Lamphousing 106 z L 1 Collector adjustment, 2 V ertical lamp adjustment, 3 Horizon- tal lamp adjustment, 4 Mount[...]

29 Lamphousing 107 L Slacken the fixing screw on the cover and lift off the cover (Fig. 32a and 32b). Move the collector to the front and pull the defect 12 V 100 W lamp out of the base towards the front. Without removing its protective cover , put a new lamp into the base, without tilting, as far as it will go. n. b.: Leave the protective cover on[...]

30 Fig. 33 Lamphousing 106 z L 1 Lid, flipped up, 2 Collector , 3 12 V 100 W halogen lamp or gas discharge lamp in holder , 4, 9 Cover fixing screws, 5 Reflector , 6, 7, 8 x-y adjustment screw for reflector , 10 Fixing screws for lamp mount, 11 Socket for contact plug Lamphousing 106 z L Slacken the fixing screw on the lid (33.10). Pull the cut-out[...]

31 Assembling and exchanging incident light lamps Assembling and exchanging Hg and Xe lamps Power units: Hg and Xe lamps are powered by separate power units. Please make sure to read the special manuals for these power units. Lamphousing 106 z L Besides the halogen lamp, the following gas discharge lamps can be used, which each require different la[...]

32 Always insert the burner so that n. b.: 1. the lettering is up rig ht after insertion (dif- ferent diameters of the metal base for the Hg 100 and Xe 75 burners ensure that these are always inserted the right way up). 2. if the bulb has a seal point (Fig. 35), the burner is turned so that this point will be at the side, not in the light path. Put[...]

33 Move the collector to the front position with the focusing knob (36.1). n. b.: Remove the protective cover from the burner . Put the lamp holder with burner inserted into the lamphousing and secure with the screws (10.10). T ry moving the collector (36.1): it must not touch the power lead. When closing the lamphousing, make sure that the pins of[...]

34 n. b.: Hold on to the tube adapter until the clamp screw is tightened. Then insert the HC FSA 25 PE tube in the change mount of the tube adapter and fasten with clamp screw . The following tubes from the Leica DM R range are also adaptable: Bino HC BSA 25 (42.1) T rino HC FSA 25 P and PR (42.2) (P + PR = with and without back reflection) Fig. 37[...]

35 Fig. 41 T ubes from the DM R range 1 HC FSA 25 PE, 2 Side port for optical overlay , 3 T ube adapter IR HC, 4 Clamp screw for mounting the adapter to the stand, 5 Clamp screw for mounting the tube to the adapter , 6 Photo/TV port Fig. 42 Leica DM R HC tubes 1 HC BSA 25, 2 HC FSA 25 P + PR, 3 Beamsplitter switch rod, 4 Mount for photo adapter tub[...]

36 Adaption of the slide overlay device and the macro dual system With the Leica DM IRB inverted microscope, the slide overlay and macro devices can only be adapted onto the FSA 25 PE tube. This tube has a side flange (44.1) for mounting the reflection optics. These reflection optics are used for mechanical and optical adaption of the slide overlay[...]

37 Screw the reflection optics (46.3) to the tube flange with the coupling ring (46.2). Align the macro adapter (46.5) to the macro dual zoom and secure with the screw ring (46.6). Screw the macro adapter and macro dual zoom to the reflection optics with the coupling ring. Check that the guide pin engages in the groove. Inserting the eyepieces The [...]

38 Inserting the photoeyepieces* The HC PLAN observation eyepieces (slot-in diameter 30 mm) are designed for direct visual observation. For the adaption of photo-micro- graphic equipment with a fixed magnification factor , e. g. DM LD and MPS systems, and for special TV adaption systems, special eyepieces with a slot-in diameter of 27 mm and the en[...]

39 3-plate x/y stage The 3-plate x/y stage no. 19, size 247 x 230 mm, x-y adjustment range 60 x 40 mm, is delivered in separate packaging and assembled as follows: This stage is usually delivered with the DM IRM, so the description of its assembly has been taken from the DM IRM manual! 1. Screw the 3 Allen screws out of the stage support surfaces a[...]

40 Rotary stage and insert frame for coverslips The rotary stage is secured with 3 screws. The rotary mount has to be moved to make all the screw holes accessible. Align the screws (51.2). n. b.: W ashers (51.3) should be used as well for the back drill holes. Only screw the screws in lightly , as the rotary stage first has to be pressed into the c[...]

41 The E version DM IRB/E Features of the Leica DM IRB/E The Leica DM IRB/E offers the following addi- tional functions: – Motorised, sextuple objective nosepiece – Electronic focusing – Coding of the IC objective prisms* – Motorised fluorescence filter cube change with electrically operated dark flap* – Control panel for remote control o[...]

42 Lower Z position Focus position Focus stepwidth (S0, S1, S2, S3, SC) Assembly and initial installation The assembly of the individual components, such as transmitted light illumination column, condenser , etc. has been completed. n. b.: The objectives should not be screwed in at this point. The best time to do this is when executing the learn mo[...]

43 Input menu of learn mode After switching on, the microscope is in the nor - mal operation mode. Normal operation mode The learn mode is switched on with the “LEARN” key . The objective nosepiece rotates through 180 ° so that the current objective is in the most accessible position (furthest to the right on the outside). This function also s[...]

44 T ur n the IC turret (situated under the objective nosepiece) until it clicks into the brightfield position (H). Operate the focus handwheel until the letter H appears on the display panel as well. T urn the IC turret by a quarter of a rotation into the next clickstop area. The message “IC prism 2” appears in the display . Read the marking o[...]

45 Now screw the objective with the lowest magnification into the nosepiece opening which is furthest to the right. Display: Objective magnification By turning the focus handwheel, select the number in the electronic display that corre- sponds to the magnification of the objective. Display: Phase contrast By pressing the “CHANGE” key , select t[...]

46 Operating modes: Dry/Immersion T o ensure simple yet safe objective change, the objectives have to be classified in one of the following three categories: 1. Dry objectives (D) = all dry objectives with a short working distance (< = 3 mm). 2. Immersion objectives (I). 3. Combined objectives (C) = dry objectives with a long free working distan[...]

47 Oil immersion objectives Once the parfocality has been learnt for all the dry objectives it can be done for the immersion objectives. Please remember that if the specimen is very small and lightweight it must be fixed onto the stage to prevent it being moved by the adhesive force of the oil. Select the oil immersion objective with the objective [...]

48 Z-drive Objective Nosepiece move- keys keys ment when the Chosen function right handwheel is turned clockwise right left up right left down left right up left right down Possible combinations for user adjustment By turning the focus wheel, choose the one of the four possible combinations that suits you best. The standard setting made at the fact[...]

49 Learning other filters: Select the next filter cube position by pressing one of the “FLUO” keys on the control panel of the FLUO module. Insert the filter cube and select the corresponding name in the display . Repeat the procedure for any other filters. Unoccupied positions are given the code “-”. T o conclude the Learn mode for the flu[...]

50 Left side of microscope The upper key is pressed to increase the magnification, the lower key to decrease the magnification. Short pressure on the key switches to the next lower or higher magnification. If you sustain the key for longer then 0.3 sec., the display jumps to the next higher or lower magnification every 0.5 sec. The nosepiece is not[...]

51 Operating modes Contamination of the dry objectives is prevented by the fact that the objective nosepiece is always lowered before objectives are changed. When switching from one operating mode to the other , please proceed as follows: Starting in the DRY mode: (Display at the bottom right in the LD display: D) Press the keys “lower z position[...]

52 The procedure for switching to the Dry mode is analogous: Again, press the keys “lower z position” and “focus position” on the control panel of the microscope simultaneously to switch from “Immersion” to “Dry”. The objective nosepiece is lowered and the message “Change Objective” appears in the display . Y ou now have the opp[...]

53 Brightness adjustment Instead of a potentiometer , Leica DM IRB/E microscopes are equipped with an incremental transducer for brightness adjustment. This means that the adjustment wheel is automati- cally moved from clickstop to clickstop and therefore has no end stops. After it is switched on, the microscope is in nor - mal operating mode. In t[...]

54 Electronic focus The controls of the electronic focus are: – The focusing handwheels, conventionally positioned on both sides of the microscope. – T wo keys (focus keys) for fast lowering of the objective nosepiece and returning to the focal plane. The keys are in a convenient position in front of the right handwheel. If both focus keys are [...]

55 – Key for defining the “lower Z position”. Pressing the key for longer than 1 sec. deletes the threshold; another press of the key for longer than 1 sec. sets the current Z position as “lower threshold”. – Key for defining the “upper Z threshold” (= focus position). Pressing the key for longer than 1 sec. deletes the threshold, a[...]

56 Motorised fluorescence filter cube change (option) A control panel is connected to the Leica DM IRB for motorised filter cube change. Three keys are used for operation: The two “FLUOR” keys are used to switch to the adjacent filter cube. If you switch one of these keys twice, you switch by two filter cube po- sitions. The “SHUTTER CLOSED?[...]

57 When using a Leica DM IRB microscope, the unit must be powered by an external power unit. The switch on the back of the unit must therefore be at EXT , and voltage is supplied via an ordinary 7 V plug-in-power supply . (Mains unit: 7␣ –␣ 15 V , pole direction ␣␣␣␣ , I = > 100 mA). The supplied connecting cable is connected to th[...]

58 Keys on the control unit The control unit is operated by keys. Keys on the control unit The keys can be used to operate the following microscope components: Component Name on the unit Filter FIL TER ((in preparation) Lamp LAMP Condenser COND Field diaphragm FIELD Aperture diaphragm AP Objective change MAG Incident light fluroescence axis FLUOR Z[...]

59 The control unit is operated in a similar way to the key-mode operation of the Leica DM IRB microscope. Significance of individual keys: LAMP Brightness can be adjusted with the arrow keys: Up arrow → brighter , down arrow → darker . The ON/OFF key is used to switch the lamp off and on. When it is switched on again, the brightness that was s[...]

60 FOCUS Here the arrow keys control the movement of the Z drive. The movement speed for the fine focusing is selected with the STEP key . For safety reasons, the movement range is limited to 400 µ m above the focus position and 400 µ m below the lower Z position. If both arrow keys are pressed at the same time, the system switches to coarse focu[...]

61 Examples for the use of the footswitches Example 1 Switching between two magnifications with a footswitch: Set a magnification and switch to a second magnification you would like to use. Assign the LAST function for magnification change to the left, by first pressing key 1 and then the LAST key (at MAG). Now switch between the two magnifications[...]

62 Switch position AUTO The dark flap is opened when you look through the eyepiece. When you move away , it will be closed again after about 3 seconds. Switch position LIGHT ON The dark flap remains open (e. g. for photo- micrography). If the flap is still closed, it must be opened first manually . It will then remain open. Switch position SENSOR O[...]

63 The display gives information on the following functions: – Z position in µ m or mm. – Set stepwidth for the fine focusing (S0, S1, S2, S3 and coarse focusing = SC, can be switched on and off by simultaneously pressing both focus keys). – Lower Z position set (symbol visible = threshold set). – Focus position set (symbol visible = thres[...]

64 16 24 25 13 19 22 23 2 3 5 1 20 4 28 Fig. 54␣ –␣ 55 1 Binocular phototube, 2 Eyepiece tube, 3 Eyepieces, 4 T ube mount (tube interface), 5 T ube port for photo/TV connection, 6 Beamsplitter switch, 7 Mains switch, 8 Brightness adjustment, 9 Lateral TV port, 10 Coaxial coarse and fine drive, 11 Fluorescence module, 12 ICT prism adjustment, [...]

65 Adjustment specimen For initial microscope adjustment we recom- mend you use a specimen that has both high and low contrast areas. It is easier to focus incident light fluorescence specimens in transmitted light first. Focusing the specimen (For the DM IRB version, please read the section about the operation of the E focus and objective nosepiec[...]

66 Checking of various microscope components Engage or disengage the filters (54.16) accord- ing to the required brightness. If necessary , disengage the Bertrand lens by turning the knurled knob (54.22), pos. 1. Disengage the analyser (55.21), if necessary , by pulling it out partway . Disengage the filter systems, if necessary , by rotating the t[...]

67 Eyeglasses with multirange lenses (bifocal and progressive) must be removed for microscopy . ● Focus the specimen through the eyepieces. Only when one ey ep iece is without an adj ustable eyelens: ● Exactly focus the specimen through this eye- piece first (close your other eye). ● Then focus the image by adjusting the eye- lens of the seco[...]

68 Fig. 59 HCI 3T22, trinocular tube with 45 ° viewing angle Light path: 100 % vis – switch rod 1 50 % – 50 % – switch rod 100 % – photo – switch rod Field of view no. up to 22, eyepiece diameter 30 mm for HC PLAN 10x/20 or 22 eyepieces, interpupillary distance set- ting: 55 – 75 mm 1 Clamp screw, 2 T ube port, 3 Siedentopf binocular p[...]

69 Operation of the side photo/TV port The delivery comprises two alternative outfits for the lateral photo/TV exit (Fig. 59a). One outfit has a beam split of 1 100 % visual 8 0 % side 2 1 20 % visual 80 % side The second version has a beam split of 1 100 % visual 11 0 % side 2 11 0 % visual 100 % side If the switch rod (60.2) for the side port is [...]

70 Immersion objectives OIL: Only use DIN/ISO standard immersion oil. n. b.: Observe the safety information on the im- mersion oil! W: Water immersion. The special water im- mersion objectives with ceramic front part can be used for all hydrous solutions. IMM: Universal objective for water , glycerine and oil. Colour coding of objectives → “T e[...]

71 Operation of transmitted light Setting the aperture diaphragm The aperture diaphragm determines the lateral resolution, field depth and contrast of the microscope image. The best contrast is obtained when the apertures of the objective and the condenser are roughly the same. When the aperture diaphragm is stopped down to be smaller than the obje[...]

72 Fig.␣ 61␣ ␣ Koehler illumination␣ a Field diaphragm closed, not focused, not centred, b Field diaphragm focused, but not centred, c Field diaphragm focused and centred, but diameter too small, d Diameter of field diaphragm = diameter of field of view (Koehler illumination) Setting Koehler illumination T ur n a 10x objective into the ligh[...]

73 n. b.: The aperture diaphragm in the illumination light path is not for adjusting image intensity . Only use the brightness adjustment knob or neutral density filters for this. An aperture diaphragm in the objective is nor - mally opened fully . Nar rowing it reduces the intensity and increases field depth reduces coverslip sensitivity creates a[...]

74 Operation of phase contrast Set the light ring (64.2) in the condenser disc that corresponds to the objective engraving (PH2). Open the aperture diaphragm (= pos. PH). Move the Bertrand lens into the light path = pos. B by turning the knurled knob and focus the annular structures with the lever (Fig. 64). Insert the two supplied centring keys in[...]

75 Setting phase contrast with condensers 0.53 S23 and 0.90 S1 Phase contrast observation is possible with condenser 0.53 S23 with objective magnifica- tions from 5x to 100x, with condenser 0.90 S1 from 10x to 100x. For both condensers, phase contrast is set as described as for the 0.30 S70 condenser . However , before the centration process itself[...]

76 Operation of transmitted light darkfield Darkfield observation Darkfield observation is not possible with condenser 0.30 S70, with condenser 0.53 S23 it is possible from 5x objective magnification, the max. usable objective aperture is 0.40. With condenser 0.90 S1, DF observation is possible from objective magnification 10x, the max. usable obje[...]

77 DL polarisation Polarisation contrast for examining birefringent specimens is possible with condenser 0.30 S70 with objective magnifications from 2.5x to 40x, with condensers 0.53 S23 or 0.90 S1 from 5x or 10x to 100x. A P 1.40 OIL S1 condenser top is also available for extremely hgh resolution. Crossing the polarisers First: Set Koehler illumin[...]

78 TL interference contrast TL interference contrast observation is possible with condenser 0.30 S70 with objective mag- nifications from 10x to 40x, with condensers 0.53 S23 or 0.90 S1 from 10x to 100x. For objective 100x there is also a condenser top P 1.40 OIL S1 for extremely high resolution. Crossing the polarisers Remove the Bertrand lens and[...]

79 Centration of the condenser prisms If you have ordered a complete microscope, this adjustment will already have been made at the factory . However , it is advisable to check the centration from time to time, particularly after transport: disengage the objective-side IC prisms (pos. H). Remove an eyepiece from the eyepiece tube. Engage the conden[...]

80 Fig.␣ 67␣ Setting ICT contrast Choice of prisms Choose the objective-side prism with the letter indicated in the top line of the objective en- graving, e. g. C for pupil position C, by rotating the turret. Choose the condenser -side prism that corre- sponds to the magnification of the objective used, e. g. pos. 40 for objective 40x, by rotat[...]

81 The BG 38 filter should always be used for photography . When not looking through the microscope, always block the incident light path to prevent specimens fading. Push the switch rod in all the way . The 3 clickstop positions of the switch rod mean: Switch rod Stop v Incident light path blocked (light stop) BG 38 BG filter engaged V Incident li[...]

82 Centration of the 12 V 100 W , Hg, Xe lamps Lamphousing 107/2 for 12 V 100 W halogen lamp This lamphousing is permanently set and does not require centration. However , it is essential that the lamp is aligned straight in its mount. Lamphousing 107 L for 12 V 100 W halogen lamp (Fig. 68) 3 alternative centration methods: Method 1: Centration wit[...]

83 5. Using an Allen key , adjust the screw for hori- zontal adjustment (68.3) until the pale stripe of the filament image is in the centre of the pupil. 6. Then adjust the screw for vertical adjustment (68.2) to align the filament image vertically in the centre of the pupil. Method 3: Centration in the plane of the specimen stage 1. Put a piece of[...]

84 Hg 100 W and Xe 75 W lamps Using the centring buttons (70.1, 70.5) move the direct image of the arc to the middle of the centration area, with the bright tip of the arc, the focal spot of the cathode, just off centre. Then focus the reflection (70.3) and, using the centring buttons of the reflector adjustment, move the reflection until it is sym[...]

85 Fig. 71 Schematic diagram of the lamp centration in lamphousing 106 z (in reality the lamp images are not as sharp) a direct lamp image, focused, but decentred b direct lamp image in correct position c indirect and direct lamp image in correct position Halogen lamp Hg 50 lamp Hg 1 00 / Xe 75 lamp[...]

86 Centring the aperture diaphragm T urn a low to medium objective magnification 10x/20x into the light path and focus a specimen with the coarse and fine drive. Remove an eyepiece from one of the two eyepiece tubes and look into the empty tube or move the Bertrand lens into the light path. Regulate the light intensity so that the rear objective pu[...]

87 Possible errors W eak fluor escence, insuf ficient brightness: Wrongly stored, overaged or faded specimens. Fast fading of the specimens (e. g. for FITC). Unspecified filter combination. Numerical aperture of the objective too low . Eyepiece magnification too high. Spent lamp. Room too bright. T rinocular tube: wrong beamsplitter setting. Stray [...]

88 Light filters Up to max. 3 light filters can be inserted in the filter holder (1.16). They can be switched in and out the light path as required. Filter Use Grey filter Grey filters (neutral density filters) are used to attenuate the light without influencing the colour temperature. The engraved value, e. g. N16, indicates the attentuation value[...]

89 The original is imaged 2␣ :␣ 1 in the intermediate image plane of the microscope. A distance of e. g. 5 mm in the slide overlay is enlarged to 10 mm in the intermediate image plane of the microscope. The overlay is only possible in beamsplitter position 50/50 (switch rod) in the middle position of the tube (FSA 25 PE). The framed slide is in[...]

90 Stand lamps, cold-light illuminators and fibre- optic lamps, etc. are suitable sources for micro- scopy . The image is observed in the microscope tube and focused by turning the knurled ring (73.10). The magnification can be changed continuously in a range of 1 : 4 by adjusting the zoom ring (73.7). When changing the magnification with the zoom [...]

91 V iewed with a 10x eyepiece, this intermediate image of 0.1x gives a total magnification of 1x in the microscope eyepiece (0.1 x 10 = 1x). The total magnification of the film plane of a camera is derived from multiplying the inter - mediate image magnification M 1 by the magnifications of the photo eyepiece and camera attachment, e. g.: intermed[...]

92 The total magnification can be roughly calculated with the scale divisions on the macrodual zoom: The following factors have to be multiplied for this: – Magnification factor of the working distance (scale (73.9), e. g. 0.11x) – Zoom factor (scale (73.8), e. g. 1x) – Correction factor of the reflection optics (without engraving 1.17x) – [...]

93 Length measurements The following components are required for length measurements: – Graticule with scale in eyepiece (Fig. 76) or in the slide overlay device (Fig. 74). – T ransmitted light stage micrometer for cali- bration. Before measurement, the micrometer value of the objective/eyepiece combination must be known, i. e. the distance in [...]

94 Recorded picture diagonal in mm with 1 inch 2 / 3 inch 1 / 2 inch 1 / 3 inch Order no. camera camera camera camera Without zoom magnification, for 1 chip cameras c-mount adapter 1x HC 1 6 1 1 ␣ 8 ␣ ␣ 6 .5 541 510 c-mount adapter 0.63x HC +) ␣ – 1 7 .5 1 2.7 ␣ 9.5 541 5 37 c-mount adapter 0.5x HC ␣ – ␣ ␣ – 1 6 1 2 .5 541 511[...]

95 Calculation of the magnification on the monitor For all TV exits the magnification on the monitor can be calculated with the following formula: M TV = objective magnification x tube factor x TV monitor diameter x –––––––––––––––––––– adapter magnification chip diameter of camera Beamsplitting for photomicr[...]

96 n. b.: Long-term video microscopy The solid and therefore stable basic body of the stand takes a while to stabilise thermally after the microscope is switched on. For investiga- tions taking over > 30 min. therefore, the microscope should be switched on about 1␣ – 2 hours beforehand. Fig.␣ 80␣ ␣ Leica DM IRB, equipped with three TV [...]

97 Operation of LMC Leica modulation contrast (LMC) is a special form of oblique illumination based on the principle of Hoffmann modulation contrast. In this technique, the phase gradients of an unstained specimen are converted into dif- ferences in amplitude with the aid of a modu- lator . This gives a three-dimensional impression simi- lar to an [...]

98 The principle Leica modulation contrast (LMC) is based on the principle of Hoffmann modulation contrast. This imaging technique is particularly suitable for unstained, colourless objects with little image contrast. Such objects change the phase of the light when it passes through them. The conversion of these phase gradients into differences in [...]

99 Components The components LMC consists of the following components: S40/0.50 LMC condenser The condenser (order no. 521 225) is supplied with a condenser disc to accommodate 3 LMC diaphragms, plus two phase contrast light rings and a brightfield position (3x LMC, PH1, PH2, brightfield). Adhesive labels are enclosed for labelling the individual p[...]

100 Assembly /adjustment Assembly When taking the following steps, consult the manual for the Leica DM IRB/E manual. n. b.: Before installing the LMC components, remove the field diaphragm. Also remove any filters, prisms and interference contrast components. Screw the LMC objectives into the objective nosepiece. Replace the condenser on the micros[...]

101 The light slit diaphragm is now adjusted until the bright stripe of the slit image is fully inside the grey stripe of the modulator . The light slit diaphragm can be rotated and moved in x and y direction. For the 10x objective the image of the modulator and the light slit are virtually the same size. Adjust the light slit diaphragm until the b[...]

102 Areas of application On the Leica DM IRB/E microscope, LMC is particularly suitable for life science applica- tions. Use of birefingent materials T ransparent, living cultures in petri dishes can be observed in three dimensions, for example. Use of a micromanipulator The long free working distance of the S40/0.50 LMC condenser offers plenty of [...]

103 n. b.: Before cleaning and maintenance work, re- member to disconnect from the mains! Protect electric components from damp! Microscopes in warm and humid climates need special care to keep them free of fungus. The microscope should be cleaned every time it is used and the microscope optics should be kept immaculately clean. Dust protection n. [...]

104 Removal of immersion oil n. b.: Read the safety information for immersion oil! First wipe the immersion oil off with a clean cotton cloth and then wipe several times with ethyl alcolhol. Acids, alkaline solutions Particular care should be taken when working with acids or other aggressive chemicals. n. b.: Always avoid direct contact between suc[...]

105 Electric errors These may be: 1. The lamp on the microscope does not work. 2. There is no power . Check the following possible causes: The on/off switch does not respond (no illumination): ● Check that all mains cables are properly con- nected. ● Make sure that there is power at the sockets you are using and that they are not deactivated by[...]

106 Rep lacing the 12 V/ 100 W halogen lamp n. b.: Remember to disconnect from the mains! Leave the protective cover on until the lamp is inserted. Avoid making fingermarks, or wipe off immediately . ● Switch off the microscope and the power unit (if used). ● Disconnect the appliance cable of the microscope and the power unit. ● Disconnect th[...]

107 Rep lacing the mains fuse on the power unit* n. b.: Remember to disconnect from the mains! ● Switch off the microscope and the power unit. ● Disconnect the appliance cable of the micro- scope and the power unit. ● Remove the defect fuse from the fuse holder . Replacement fuses of IEC 127-2 standard and/or UL 198 G and/or company type: Par[...]

108 n. b.: Always disconnect external transformers and the microscope from the mains when carrying out assembly work! ● Switch off the microscope and the power unit. ● Disconnect the appliance cable of the micro- scope and the power unit. ● Slacken the clamp screw on the microscope and remove the lamphousing. ● Slacken the screw (85.1) on t[...]

109 ● Switch off the microscope and the power unit. ● Disconnect the appliance cable of the micro- scope and the power unit. ● Slacken the clamp screw on the microscope and remove the lamphousing. ● Slacken the screws (88.4 and 88.9) on the lid with a cross-tip screwdriver . ● Pull the cut-out plug slightly out of the socket (88.11) and f[...]

110 Changing the Hg and Xe lamps on lamphousing 106 z n. b.: ● Always disconnect the power unit from the mains before carrying out assembly work. ● W ait for the lamphousing to cool down for at least 15 minutes as otherwise it may explode. ● Never touch glass parts of the burner with your bare hands as finger perspiration burns in. ● Wipe o[...]

111 Fig. 90 Lamp holders for gas discharge lamps 1 Upper clamp, 2 Seal point of the burner , 3 Lower clamp, 4, 6 Drill holes for fixing the lamp holder , 5 Sockets for cut-out plug, 7 Protective cover Hg 50 1 2 3 4 5 6 Xe 75 1 3 7 1 3 Hg 100 1 3 Hg 100 Stab. ● Put the lamp holder with burner inserted into the lamphousing and secure with the screw[...]

112 Storage Protect your microscope from dust by putting on the cover after each work session. The microscope must be kept in a cupboard in which the temperature is ≥ 5 ° C above room temperature. The cupboard must have ven- tilation holes which are plugged with cotton wool, for example, to keep dust out. If this type of storage is not possible,[...]

113 All techniques, not only in microscopy , are subject to limits of performance due to basic physical laws and principles of eye physiology . The following information should therefore be remembered when using the microscope. Performance data of objectives The Leica DM IRB microscope is based on tube length ∞ (infinity) and a focal length of th[...]

114 10x/0.22 Magnification and aperture. The aperture (pick-up angle) influences resolution, field depth, contrast and brightness. Objectives with built-in iris diaphragm have an engraving showing the maximum and minimum aperture, e. g. 0.85 – 0.55. n. b.: Objectives with built-in iris diaphragm. The knurled ring may only be used for adjusting th[...]

115 Colour coding of the objectives The magnification of each objective is indicated as per DIN/ISO standard by a colour ring: Black Oil or IMM (= universal for oil, water , glycerine) White W ater Orange Glycerine Fig. 91 Immersion objectives 1, 2 Oil immersion objectives (OIL), 1 in working position, 2 locked in shortened position, 3 Water immers[...]

116 Performance data of eyepieces Leica eyepiece Magnification/ Eyepiece port +) type fov HC PLAN 10x/20 M HC PLAN 10x/20 HC PLAN 12.5x/16 M HC PLAN 10x/20 MF HC PLAN 10x/22 M HC PLAN 11x/20 MF Eyepiece tube diameter: 30 mm +) = With removable or push-back anti-glare protection for use with or without eyeglasses. M = Ajustable eyelens (dioptre comp[...]

117 Example: Eyepiece 10x/20 Objective PLAN 4/0.10 Magnification factor 1x T otal magnification 10 x 4 x 1 = 40x Field performance of objectives The field performance of objectives is not engraved on the objectives. It may vary within the same class, e. g. low objective magnifi- cations may well exhibit slightly higher values than the average value[...]

118 Filter performance data Filter Use Grey/neutral density filter N Grey (neutral density) filters are used for light attenuation without influencing the colour temperature. The engraved value, e. g. N16, indicates the attenuation value. So N16 means a reduction to 1/16 = 100/16 = 6.25 % transmission. Green filter , GR, panchromatic For general co[...]

119 T ube performance data T ube changing is the same as for the upright microscopes. The tubes are interchangeable. Binocular tube HCI B22 The binocular tube consists of a basic part with the tube change ring at the bottom. The tube lens has the factor 1x. The Siedentopf binocular part allows adjustment of the interpupillary distance from 55 mm to[...]

120 HC FSA 25 PR Binocular observation and photo tube, viewing angle 30 ° , with back reflection. Controllable dark flap of the binocular port for photography and microphotometry . 3 clickstop positions of the beamsplitter in the tube: Switch rod Observation Photo VIS 100 % 0 % 50/50 1 50 % 50 % PHOTO 11 0 % 100 % Back reflection only at the 50 % [...]

121 HC FSA 25 PE Binocular observation and photo tube, viewing angle 30 ° , with provision for optical overlay for documentation of transparencies (slide overlay device) or opaque macro objects (macro de- vice). 3 clickstop positions of the beamsplitter in the tube: Switch rod Observation Photo VIS 100 % 0 % 50/50 1 50 % 50 % PHOTO 11 0 % 100 % Po[...]

122 T ogether with condenser top 0.53 S23, max. FWD 30 mm, culture vessels can be examined microscopically up to liquid levels of 25 mm. For contrasting techniques brightfield (HF), phase contrast (Phaco/PH), transmitted light inter - ference contrast (ICT) and polarisation contrast, objectives with magnifications up to 100x can be used. For transm[...]

123 Lamphousing performance data Lamphousing 106* Lamphousing 106 is equipped with a 12 V 100 W halogen lamp. The lamp holder is centrable in x and y direction. The aspherical collector can be focused. Lamphousing 106 is fitted with a diffusing disc and heat protection filter , but does not have a reflector . Lamphousing 106 z* Like lamphousing 106[...]

124 Non-centrable lamphousings LH 106 LH 107, left LH 107/2 LH 35/2 6 V/35 W 504 088 12 V/100 W , 0.55 m 504 058 504 086 504 080 12 V/100 W , 2.0 m 504 059 12 V/100 W , 2.0 m, 504 085 shielded Centrable lamphousings LH 106, right-hand op. LH 106, left-hand op. 4-lens 6-lens 6-lens 12 V/100 W , 0.55 m 507 070 504 087 12 V/100 W , 2 m 504 071 12 V/10[...]

125 General technical data General technical data For indoor use only Mains voltage: 90␣ –␣ 250 V ~ Frequency: 50␣ –␣ 60 Hz Power consumption: DM IRB max. 160 W Fuses: T 4 A Ambient temperature: 10␣ –␣ 36 ° C Relative humidity: 0␣ –␣ 80 % up to 30 ° C Overvoltage category: II Contamination class: 2[...]

126 T echnical data of the power unit General technical data For indoor use only Mains voltage: 90␣ –␣ 250 V~ Frequency: 50␣ –␣ 60 Hz Power consumption: 160 W Fuses: T 4 A Ambient temperature: 10␣ –␣ 36 ° C Relative humidity: 0␣ –␣ 80 % up to 30 ° C Overvoltage category: II Contamination class: 2 T echnical specifications [...]

127 Main wearing and spare parts, tools Order No. Part no. Component Used for Sp are lamp s 500 974 Halogen lamp 12 V 100 W Lamphousing 105 500 137 Ultra high pressure Hg lamp 50 W Lamphousing 106 z 500 138 Ultra high pressure Hg lamp 100 W Lamphousing 106 z in preparation Ultra high pressure Hg lamp 100 W Lamphousing 106 z (103 W/2) 500 139 High p[...]

128 EU Conformity declaration W e hereby declare that the product specified below conforms in its design and construction as well as the model we have put on the market to the relevant safety and health regulations laid down by the European Union. This declaration will cease to be valid if the instrument is modified without our consent. Product nam[...]

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