Leica TCS SPE User Manual Download Page 3

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Summary of Contents for TCS SPE

Page 2: ...s CMS GmbH Am Friedensplatz 3 D 68165 Mannheim Germany http www leica microsystems com http www confocal microscopy com Responsible for contents Leica Microsystems CMS GmbH Copyright Leica Microsystem...

Page 3: ...illustrations in whole or in part by print photocopy microfilm or other method including electronic systems is not allowed without express written permission from Leica Microsystems CMS GmbH Programs...

Page 4: ...der the following US patents 5 903 688 6 137 627 6 211 988 6 222 961 6 285 019 6 418 153 6 423 960 6 444 971 6 466 381 6 614 526 6 657 187 6 678 443 6 754 003 6 801 359 6 809 815 6 850 358 6 954 306 F...

Page 5: ...icroscope DMI4000 26 9 5 2 2 Microscope DM2500 28 5 2 3 Microscope DM5500 30 5 2 4 Sean head 32 5 2 5 Supply unit 33 m 5 2 6 Mirror housing 35 3 6 Safety devices on the microscope 37 6 1 Transmitted l...

Page 6: ...11 2 Full text search with logically connected search terms 70 11 3 General structure of the user interface 71 12 Introduction to confocal work 73 12 1 Preparation 73 12 1 1 Theobjective 74 12 1 2 Co...

Page 7: ...cquisition and utilization 101 S 12 7 Combinatorial analysis 101 13 Care and maintenance 103 5 13 1 Filter 104 13 1 1 Changing the filter 104 13 2 Moisture 105 5 13 3 Dust cover 105 13 4 Clinging dirt...

Page 8: ...s provided for the individual components and peripheral devices and keep them in a safe place sf These operating nstructions present the first steps for activating the system and describe the basic fu...

Page 9: ...manufacturer assumes no responsibility or liability for any use outside of the intended use or use outside of the specifications from Leica Microsystems CMS GmbH or any risks resulting from such use I...

Page 10: ...efficient prisms The spectral detector ensures the perfect adjustment to the emission spectrum of today s dyes and the dyes of the future The TCS SPE can be equipped with up to four lasers Long lasti...

Page 11: ...ting procedure practice condltion or statement in the operating instructions that must be strictly observed and followed as otherwise you expose yourself to the riskof fatal injury WARNING HIGH VOLTAG...

Page 12: ...ice condition or statement that must be strictly observed to prevent severe system damage or destruction or data corruption or loss This kind of note is a statement that either provides extra informat...

Page 13: ...ing and operation the Supply Unit always has to be set up in upright position The Supply Unit must not be set up other than upright despite a possible lack of space 5 a Repairs and servicing may only...

Page 14: ...re are installed components that can cause fatal injury f handled improperly Opening these components will result n danger to people and the device Therefore only authorized Leica service personnel ma...

Page 15: ...nd other aggressive chemicals To prevent loss of data make regular backups of your data and images on a suitable data carrier Also be sure to back up your data before any service or repair work is per...

Page 16: ...page 111 The system may be connected to a power supply with ground protection conductor only Do not interfere with the grounding function by using an extensi n cord without a ground wire Any interrup...

Page 17: ...nd peripheral devices i The entire system and all components and peripheral devices microscope are not protected against water ingress Water ingress can cause electric shock Do not assemble the micros...

Page 18: ...0V 10 Frequency Power consumption Line input fuse Powersupply on the rear side of the supply unit Ambient temperature Relative humidity Overvoltage category Pollution degree 50 60 Hz max 800 VA 2x T8A...

Page 19: ...oratory Use EMC J Requirements IEC EN 61000 3 2 j Electromagnetic compatibility EMC Part 3 2 Limits Limits for harmonio currents IEC EN 61000 3 3 Electromagnetic compatibility EMC Part 3 3 Limits Limi...

Page 20: ...olution may not be refrigerated but must be stored tightiy closed at room temperature Changes to the ambient conditions affect the glycerin water mixture ratio and thus cause differences in the refrac...

Page 21: ...o assure classification as a Class 3B Illb l ser product according to IEC EN60825 1 and electrical safety compliance regulations all safety devices interlocks and safety systems of the l ser device mu...

Page 22: ...r Protection Advisor 5 1 L ser and l ser class Wavelength range 635 nm 532 nm 488 nm 405 nm Optional l ser cannot be retrofitted L ser class 3B Illb 3B Illb 3B Illb 3B Illb WARNING L SER RADIATION A A...

Page 23: ...used in the Leica TCS SPE system For the corresponding safety label see Figure 1 In the following illustrations of the safety labels both English and Germ n versions are always provided refer to Figu...

Page 24: ...5 2 1 Microscope DMI4000 The following safety labels are present on the DMI4000 microscope see Figure 2 and Figure 3 Figure 2 Safety labels on the DMI4000 microscope 26...

Page 25: ...V S DANGER USWRA3IA 10HCWSS33 j VQRSICHT WEHx aD CKawoGiSfri n Figure 3 Safety labels on the DM14000 microscope 27...

Page 26: ...5 2 2 Microscope DM2500 The following safety labels are present on the DM2500 microscope see Figure 4 and Figure 5 vis v VIS I Mna Tavoidexpos re I I Figure 4 Safety labels on the DM2500 microscope 28...

Page 27: ...OANGER USt flftClATn CLAS J3 WhEH PtH UORSICHT 33 IICHT8EVSTW UUSSE1ZEK Figure 5 Safety labeis on the DM2500 microscope 29...

Page 28: ...DM5500 microscope see Figure 6 and Figure 7 vis UStHPADIMNN I I ISEKintQ I AVODEXPOSLflE I BESTFHH UO U5 RSIMHU 1D I VEFWDEH L SER RAflUT C 4 VIS aun I avoid bposure II AimRrrrvoN T b tmhlunq I LASEMT...

Page 29: ...Figure 7 Safety labels on the DM5500 microscope 31...

Page 30: ...5 2 4 Sean head The following safety labels are present on the sean head see Figure 8 Figure 8 Safety labels on the sean head 32...

Page 31: ...5 2 5 Supply unit The following safety labels are present on the supply unit see Figure 9 and Figure 10 Figure 9 Safety labels on the supply unit 33...

Page 32: ...Figure 10 Safety labels on the supply unit 34...

Page 33: ...s present on the mirror housing see Figure 11 VIS DANGER L SER RA01ATI0H CIASS 3B WHENOPEW AVOiDEXPOSURETOBEAM i VGRSICHT LASERSTRAHLUNG KLASSE3D WENN AflDEGKUNG GE FFNET HICHT DEM STRAHL AUSSETZEtt...

Page 34: ...pening see Figure 13 must be closed permanently using the cover delivered with the system this is required to protect against the risk of l ser radiation emissions 1 Figure 12 Cover i DANGER L SER BAO...

Page 35: ...and or if there is no cover the l ser may not be switched on as otherwise l ser radiation can be emitted 6 2 Transmitted light lamp housing on the upright microscope If an upright microscope is not eq...

Page 36: ...ly 4 Disconnect the lamp housing from the power supply 5 Remove the lamp housing 6 Perform the intended tasks at the lamp housing 7 When done screw the lamp housing onto the microscope stand I r Ia c...

Page 37: ...to protect against the risk of l ser radiation emissions I V S O 1 1 1 vis E DANGEB i L SER RADIATIOH CIASS 3B 1 WHENOPEN AVOIDEXPOSURETOREAM I VOBSICHT LASERSTBAHLUNG KLASSE 3B WENN ABDECKUNG GEOFFHE...

Page 38: ...ean head at all times The unused output on the mirror housing must be covered with the cover provided Figure 17 Position 3 When the mirror housing is removed the connector on the stand Figure 15 must...

Page 39: ...d the beam collector serve as protection against l ser radiation emission and are located between the condenser base and the transmitted light detector mtuzz fi i Figure 18 Safety beam guide and beam...

Page 40: ...older always be aware that the unused filter holders are swung out of the beam path and the safety beam guide covers the beam path When equipping m ltiple filter holders with filters the order of inst...

Page 41: ...display 1 To switch on the l ser equipment switch on the supply unit at the power switch 2 Once all necessary components and peripheral devices have been nstalled move the l ser keyswitch Figure 19 po...

Page 42: ...d on and the l ser equipment has been switched on using the l ser keyswitch The l ser emission display contin es to be illuminated even though the l ser equipment has been switched off using the l ser...

Page 43: ...Action The transmitted illumination holder is tipped back for example to change specimens The deflection mirror to the scanner is motorized Result The path of the l ser beam is interrupted The path of...

Page 44: ...hus the emission of stray light will be prevented when the microscopist switches from confocal mode to study of the specimen through the eyepieces Figure 21 Interlock Position of interlock connector F...

Page 45: ...can be used to connect the remote interlock if needed Remote interlock devices such as those connected to the room the door or other onsite safety interlock systems can be connected to the remote int...

Page 46: ...ring the sean process Do not look into the eyepieces when switching the beam path in the stand Never look directly into a l ser beam or a reflection of the l ser beam Avoid all contact with the l ser...

Page 47: ...e removing the scanner the system must be completely switched off Do not use an S70 microscope condenser The large working distance and the low numeric aperture of the S70 microscope condensers could...

Page 48: ...damage to the eye Because of this cautious handling is essential as soon as l ser equipment is switched on Figure 23 Specimen rea of the laser scanning microscope Never look directly into a l ser beam...

Page 49: ...anging the specimen Do not change the specimens during scanning Procedure for changing the specimen Upright microscope 1 Finish the sean process 2 Ensure that no l ser radiation is present in the spec...

Page 50: ...ish the sean process 2 Ro ate the objective turret so that the objective to be changed is swiveled out of the beam path and points outward 3 Exchange the objective All unoccupied positions in the obje...

Page 51: ...see Microscope Stand operating instructions 4 Remove the filter cube beam splitter 5 Insert the desired filter cube beam splitter 6 Reattach the cover to the front of the fluorescence module Inverted...

Page 52: ...ppiy unit see Figure 25 The suppiy unit with the integrated workstation PC and lasers are at zero potential only when the system has been disconnected from the power suppiy Switch off the On Off switc...

Page 53: ...pright left and inverted microscope right Position in Figure 26 1 2 3 4 5 6 7 8 Designation Monitor Mouse Supply Unit Keyboard Microscope upright Sean head with the upright microscope Sean head with t...

Page 54: ...use outside of the specifications from Leica Microsystems Wetzlar GmbH or any risks resulting from such use In such cases the EU Declaration of Conformity shall be invalid 8 1 2 Supply unit with works...

Page 55: ...nformation refer to the chapter Filter on page 104 To prevent loss of data make regular backups of your data and images on a suitable data carrier Also be sure to back up your data before any service...

Page 56: ...he sean head and its components can cause severe and permanent damage to eyes and skin Therefore only authorized Leica service personnel may open or work on the sean head If you have any further quest...

Page 57: ...e 103 See also the instructions for care in the microscope operating instructions The Leica TCS SPE system may be used indoors only SETTING UP THE SUPPLY UNIT For safe handling and operation the Suppl...

Page 58: ...A upright B inverted Note that the rear and right side must be unobstructed and available for service and maintenance tasks Therefore keep t at least 10 cm away from the wall and other obj cts 9 1 1 A...

Page 59: ...least 10 cm away from the wall and from flammable substances 9 2 nstallataon of the system The system is installed and made ready for operation by authorized Leica service personnel SETTING UP THE SU...

Page 60: ...tion Then switch on the peripheral devices 3 After all the necessary components and peripheral devices have been switched on and the supply unit with the operating system has been booted the software...

Page 61: ...afety page 23 Figure 29 Laser keyswitch Now the Leica TCS SPE system is ready for operation Optimal optical performance of tha system when using standard objactivas and standard immarsions can ba achi...

Page 62: ...ser keyswitch see Figure 29 3 Exit the LAS AF 4 Shut down the operating system and the workstation PC 5 After shutting down the workstation with the operating system switch off the supply unit at the...

Page 63: ...men Click on the Help men on the men bar The men drops down and reve is among others the following search related options Contents This dialog field contains the table of contents in form of a directo...

Page 64: ...nd search term you would like to associate with the first search term behind the operator Examples Pinhole AND sections Pinhole OR sections Pinhole NEAR sections Pinhole NOT sections Result This phras...

Page 65: ...l structure of the user interface The user interface of the LAS AF is divided in four reas Figure 30 LAS AF user interface 1 Men line The various menus for calling up functions are available here n Fi...

Page 66: ...e image recording Beam Path Setting the individual components of the beam path Experiments Directory tree of the files stored in memory HDD Tools Directory tree with all the functions available in the...

Page 67: ...ing down Background information has also been provided to explain the reasons behind various settings These are not descriptions of the individual functions and controls of the instrument and graphica...

Page 68: ...on objectives ensure that an adequate quantity of immersion m dium is applied between the front lens of the objective and the specimen Immersion oil glycerol 80 and water may be used as immersion medi...

Page 69: ...flect or fluoresce Fluorescent specimens are most common In many cases specimens with m ltiple dyes will be examined Reflective specimens can also provide interesting results however Filter cubes Fig...

Page 70: ...BFP GFP CFP D E4 FI RH G R GFP H3 13 K3 L5 M2 N2 1 N3 Y3 Y5 YFP Excitation filter 400 430 500 415 435 565 420 455 455 455 500 505 500 510 510 510 505 580 580 565 565 560 515 HflHH Table 4 Selection of...

Page 71: ...the entire image can be reconstructed n paraliel To cr ate a two dimensional image the spot must be moved over the entire surface and the associated signal recorded on a point by point basis This is p...

Page 72: ...an optical section created The term confocar1 is strictly technical and does not describe the effeets of such an arrangement That will be described in greater detail here As already described in12 1...

Page 73: ...ameter of zero one would theoretically receive the thinnest optical section for the wavelength and numerical aperture used A range apparently exists at 1 Airy which does not yet offer the thinnest opt...

Page 74: ...Acquire Fig Figure 36 For descriptions of the individual functions please see the online help This section will describe the aspects affecting the configuration of the most important acquisition para...

Page 75: ...set up an image of the result mav be captured Functions such as accumulation and averaging are supported The third data acquisition situation is the acquisition of a series in which the preselected p...

Page 76: ...s the acquisition of a series in which the preselected parameters are changed incrementaily between the capture of the individu senes lambda senes and z stacks can be acquired in this manner Fig F 9ZT...

Page 77: ...line The intensity setting of the slider is realized steplessiy via an acousto optic tunable filter AOTF The intensity at which a sufficiently noise free image of the specimen can be obtained must be...

Page 78: ...than necessary and attenuation to 10 is generally sufficient for good images although that depends very strongly on the specimen s dye of course One can thus also excite the fluorescence on the blue s...

Page 79: ...above or for fluorescence and reflection below It is possible to move the entire bar back and forth to adjust the average frequency or move the limits independently Using the excitation lines and disp...

Page 80: ...o wide as the image would contain excessive blurred shares of the specimen from above and below the focal pla e The relationship of the thickness of the optical section to the diameter of the pinhole...

Page 81: ...background in the images with very large diameters A pinhole diameter of 1 Airy is therefore the default setting Switching objectives also automatically adjusts the diameter of the pinhole accordingl...

Page 82: ...zoom into details without the need for additional optics As the sean angle can be adjusted very quickly and continuously over a wide range magnification increases of around 16x can be achieved simply...

Page 83: ...pixels Magnification Numerical Aperture Optical Resolution 465nm Intermed ate Image Edge Field Edge Resel Field Resolution 2x Oversampling 3x Oversampling jim mm jim 63 1 4 0 16 11 174 1048 2095 3143...

Page 84: ...the approphate zoom and pan val es This function is very fast and thus easy on the specimen An lindo Zoom function returns you to your starting point for quickly concentrating on a different cel in t...

Page 85: ...of the information they originally contained This image information is then lost Fig Figure 46 Correctly setting the zero point is also important This can be accomplished by shutting off the illuminat...

Page 86: ...In the case of extremely sensitive specimens and in situations in which rapid changes in intensity in living specimens is of interest images with more noise can be acceptable However this compromise d...

Page 87: ...cing through a cake permitting impressions to be gained online about the contents of the specimen Camera based systems also confocal systems can only compute such profiles out of complete stacks Fig F...

Page 88: ...is not always the case however there are naturally also dyes with differing emissions that can be excited by the same wavelengths A distinctive example would be a botanical specimen with a FITC dye a...

Page 89: ...lasers are usually installed in the instrument for this purpose 12 2 2TO act vate a second excitation line simply set the desired slider for the second wavelength as described in for simple excitatio...

Page 90: ...ser lines are used for exatat on and this is generally the case sequential capture provides significantly improved separation as only one dye is excited at a time and the emissions are thus solely fro...

Page 91: ...nd 600nm so 500nm would be a suitable valu for a rough est mate Choosing the pinhole diameter 1 Airy will result in section thicknesses between 0 5 m and 2 5nm for apertures from 0 7 to 1 4 These are...

Page 92: ...normal image with 512 x 512 pixels one channel and a standard 8 bit grayscale resolution weighs in at 0 25MB One hundred such images i e a specimen thickness of 20 nm at high resolution already requi...

Page 93: ...further option for displaying the full range of information with losses compressed into two dimensions is to compute projections of the entire series The most common method is the so called m ximum pr...

Page 94: ...series shown above The SFP simulated fluorescence projection method uses a more complex approach to achieve impressive images with shadow projections The quantification must always be checked with car...

Page 95: ...eing the location the selected line and time as the second dimensi n 12 5 3 Planes Standard capture processes generally at 512 x 512 pixels will also be used for long term experiments in which the ima...

Page 96: ...he image are then chosen interactively for evaluation Their average intensity is then graphed as a function of the wavelength a spectrum at the selected point 12 7 Combsnatorial analysis Many of the m...

Page 97: ...omponents or peripheral devices during cleaning Check the ventilation filter on the front panel of the supply unit for cleanliness at regular intervals Replace the filter every 24 months at the latest...

Page 98: ...s installed behind the screen Replace the filter every 24 months at the latest Article number 15001000000001 Designation Dust filter TCS SPE 10 pcs Figure 52 Ventilation screen 13 1 1 Changing the fil...

Page 99: ...s installed behind the screen Replace the fiiter every 24 months at the latest Article number 15001000000001 Designation Dust fiiter TCS SPE 10 pcs Figure 52 Ventilation screen 13 1 1 Changing the fil...

Page 100: ...h use 13 3 Dustcover Allow the entire system to cool down to room temperature before covering the system with a dust cover Most dust covers are not temperature resistant Also condensation water would...

Page 101: ...he protection of the parts Acetone xylene or distillations containing nitro can harm the microscope and thus may not be used 13 5 Cleaning the specimen stage Remove light colored spots on the stage by...

Page 102: ...the chapter on 13 6 Cleaning glass surfaces The upper lens is cleaned by being blown off with a squeeze blower 13 8 Removing immersion oil Follow the safety instructions for immersion oil First wipe o...

Page 103: ...clean cloth slightly moistened with water Ensure that no fluids enter the individual components or peripheral devices during cleaning 13 12 Cleaning the monitor Remove dust and lo se dirt particles u...

Page 104: ...If you have any questions related to disposal please contact the Leica branch office n your country see Chapter 15 Contact 109...

Page 105: ...nockburn Illinois Tel 61 2 9879 9700 Tel 43 1 486 80 50 0 1 905 762 2000 Tel 45 4454 0101 Tel 33 1 473 Tel 49 6251 136 0 Tel 39 0257 486 1 Tel 81 3 5421 2800 Tel 82 2 514 65 43 Tel 31 70 4132 100 Tel...

Page 106: ...safety 18 Filter 104 Changing the filter 104 Glycerin 22 Handling 22 Remarks 22 Storagelife 22 Immersion oil 107 Installation location 61 Installation of the system 63 Intendeduse 10 Interlock 47 48 I...

Page 107: ...Safety labels 25 26 27 28 29 30 31 32 33 34 35 Safety Notes L ser class3B lllb 18 23 Meaning 13 Safety switches 46 Specimen rea 51 Standards 21 Starting up 65 Supply voltage 20 Switching off 67 Syste...

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