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PALM MicroBeam January 2008 User Manual

1. 5 11 Foot stepper The foot stepper is used to activate the laser cut ting function manually This option is only avail able in stage mode please refer to PALM RoboSoftware Manual PALM MicroBeam User Manual 0108 5 12 Joystick With the new hardware option you no longer depend on a mouse or keyboard It is controlled by PALM RoboSoftware for fast and precise navigation of the stage for dishes or up to three slides Even PALM RoboMover and PALM CapMover II can be operated via the joystick in the CapCheck position For operation see PALM RoboSoftware manual 25 Preparation of PALM MicroBeam for Application 6 Preparation of PALM MicroBeam for Application 6 1 Switching device on off 6 1 1 Switching on device Figure 15 Switching on device Axio Observer Z1 26 Set the key switch of the ControlUnit to On and if you work with an Axio Observer Z1 switch on the power supply of the microscope Figure 5 No 11 Then switch on the computer The Axio Observer Z1 has an On Off button on the left side of the microscope Press the button and wait until the microscope has started and you see the display on the TFT touch screen A blue LED light will light up on the front of the stand You can then start the PALM RoboSoftware Note If the red system LED F of the PALM ControlUnit see Figure 6 does not go out automatically please switch off the ControlUnit and after 10 seconds switch it on again Should the LE
2. PALM MicroBeam works with a frequency tripled solid state laser wavelength 355nm with a high beam quality It is coupled via the epifluorescence path into a research microscope and focused through the objective Depending on the objective used the laser focus can reach less than one micrometer in diameter In theory the high beam quality of the laser allows you to focus the laser beam to its diffraction limit However the cutting width depends on correct laser focus settings optimal laser energy settings the numerical aperture of the focusing objec tive and PALM MicroBeam User Manual 0108 PALM MicroBeam Overview the absorption behavior of the specimen Note Carl Zeiss provides factory settings for each objective The fine tuning of laser focus and energy setting is carried out by the user please see section 6 5 page 28 and depends on the focusing objective and the type of mate rial you want to handle For cutting the stage is moved and not the laser beam itself PALM MicroBeam User Manual 0108 5 3 Microscope adapter The microscope adapter connects the laser to the microscope The laser beam is guided into the microscope and deflected into the objective a PALM MicroBeam 3 fluorescence illumination lamp option 1 microscope adapter 2 filter wheel filter holder with shutter option Figure 8 Microscope adapter The specially designed beam splitte
3. For further information about checking the cata pulted specimen refer to PALM RoboSoftware manual Caution To prevent the objective from being damaged once you have inspected the specimen move PALM RoboStage back to its initial position after checking the well Proceed ONLY as described in the PALM RoboSoftware manual If you do not have a motorized microscope move the objective to the lowest position and move the collector to the home position for parking 7 4 Using diffusor option RoboMover collectors such as the CapturePlate Collector 96 have an integrated diffusor to opti mize the image quality You will find further infor mation on this in the product information supplied with the collectors and in the PALM RoboSoftware manual 33 PALM MicroBeam with Fluorescence Illumination 8 PALM MicroBeam with Fluorescence Illumination 8 1 Operating microscope with fluorescence illumination The fluorescence attachment is mounted to the microscope adapter with clamping screws please see Figure 10 Please also read the notes on device safety and manuals of the fluorescence lamp Note Make sure that the beam path of the laser does not contain filters that could block laser trans mission The excitation filter wheel is therefore mounted outside the laser beam path directly between the fluorescence lamp housing and the micro scope adapter see Figure 10 No 1 8 2 Simultaneous fluorescence observation
4. 8a Gee w RQA S ee rar Wa b as S naw e are ee wie 20S all 36 PALM ROBOMOVER Z z sy uz yasta Sua oh are e Qasa a a Sup ra aed we 37 NO Video IMAGE u y ruyay A er ee A Z uu Sa 37 Shift between drawing and cutting i ir ee 37 Reflex in VISO MAYEN a a osa 37 Electrical Power o t ge coma ae ae A e eier 37 Maintenance of PALM MicroBeaM c oooococcccnnc nsn 38 MIGFOSGODeS 2 Sa A kuu aus e aa Dal ten ne 38 101 1 Cleaningioptical parts uy cu 220 ee a U O ana Da ae la 38 10 1 2 Cleaning when actually dirty a 38 EONTFOMDOX re A A AAA A Bee Gk ae Geet fast 38 Technical Data uvas a gee ere ee nen 39 Op rati gudata vices it A A AAA en ehe 39 C tting lase sr 58 8 th ee Ba eb A AA AA ea GS 39 PALM ROBOMOVEr esas yuq panaka ee A ahua it 40 PALM C pMoverFIO use 2 202 ss us mase BERS eld A ee S s w k ar a Fe eee ed 40 Light sources ON MICFOSCOPe i s roteare s su a nn 40 PALM MicroBeam User Manual 0108 Important Basic Information 1 Important Basic Information Congratulations With your new generation of PALM MicroBeam from Carl Zeiss you now have the state of the art technology in microdissection and micromanipulation PALM MicroBeam allows you to perform microdissection with virtually every type of research as well as during routine examination with a higher throughput To meet a very high safety standard and to use all the benefits of your PALM MicroBeam you should read t
5. The beam splitter reflector module in the reflector turret is custom made The beam splitter must have a special coating suit able for the simultaneous reflection of the laser light and the fluorescence excitation wavelength as well as for transmission of the emitted flu orescence light Caution Do not use the beam splitters offered by micro scope manufacturers together with the laser under any circumstances as such beam split ters may be damaged by the high energy of the laser If you want to work with fluorescence and use the laser simultaneously you must obtain a special fil ter set available from Carl Zeiss Microlmaging GmbH For further information please contact palm info zeiss de 8 4 Freeze Mode A very special feature is the option of freezing 34 8 3 Settings 8 3 1 Settings for working with motorized microscope Zeiss Axio Observer All elements designed for use under fluorescence illumination can be controlled by PALM RoboSoftware Basic settings are preloaded and can be changed and saved by PALM RoboSoftware please refer to PALM RoboSoftware manual In order to use fluorescence illumination you must switch on the fluorescence illumination and select the desired fluorescence filter unit With this the functions Excitation filter Reflec tor position Microscope light Timer and Shutter are performed via a single command The basic settings of these functions were made during ins
6. The greater the magnification and the aperture of the objective the smaller the focus of the laser and the higher the energy density Figure 17 Position of the laser focus when ablating a or catapulting b 28 PALM MicroBeam User Manual 0108 Preparation of PALM MicroBeam for Application 6 5 1 Laser energy adjustment The laser emits a fixed laser energy For precise laser micromanipulation a precise laser energy adjustment is required A neutral density filter is mounted in the laser beam path which allows you to continuously adjust the laser energy to the individual require ments without beam displacement This graduated coated filter is turned by a high precision DC step ping motor which is conveniently regulated on the monitor screen by means of PALM RoboSoftware please refer to PALM RoboSoftware manual 6 5 2 Laser focus adjustment Precise laser focus alignment provides for laser cutting with an almost diffraction limited laser focus The laser focus must coincide with the opti cal focus of the microscope for precise laser micro manipulation Every wavelength is diffracted differently This is important to know as the spectrum of the visible light of the microscope is at a different wavelength to the cutting laser When performing laser micro manipulation at a greater distance from the spec imen slide or within a thicker or strongly refracting object it is necessary to compensate for the offse
7. 2 SUN ae Re wu ern al gre Ren A E at eat ae A 18 5 35 Microscope adapter u eu an a ae a E ae P yaY 19 5 4 Microscope Zeiss Axio Observer Z1 preparing for laser operation 20 5 5 Fluorescence attachment oooooooors ee sa qutuchay 21 5 67 1 PALM Stage dlrs a y a A A pe ade on Gee AA ay See aie ee 22 5 7 PALM CapMover Tl 2 ewok Sd BE k g ati olen it A 23 5 8 PALMROBOMOVer ce cia 2 Ae ar Medan eee ua ae Chua a we Rea oe 24 5 97 HCAME GAS sa cfd o ad SA GEA wel he eae ere Gu h Sate GaP atta jaca OAS tte E ee N 24 BAL Os COMPUTER a gee Y Ichu Eases A A papa shu ap Faw Shen SR a re FAP a ODO A 25 5 11 FOOt Stepper agas ear ale a A amk ah alee a cama hy we aera Sedan ata aaa Me 25 5 12 Joystick cima ia glia thee tea mea ade a Re Ae as Ah athe ee Be teed 25 6 Preparation of PALM MicroBeam for Application 0000 eee eee ee 26 6 1 Switching device on off u sy uuu a a de E ea Dee ea Re s au ple de aeons 26 6 1 1 Switching on device sick wee aca ba puma RE ee ea Oe ea i 26 6 1 2 Switching off device J 0 0 00 0 sa mn a nn ee 26 6 2 Camera adapter u sasa duuri sa pi a usu ee 26 6 3 Checking laser beam path in MicrOscope ooo 27 PALM MicroBeam User Manual 0108 3 List of Contents 6 4 6 5 6 6 6 7 7 1 7 2 7 3 7 4 8 1 8 2 8 3 8 4 8 5 9 1 9 2 9 3 9 4 9 5 9 6 10 10 1 10 2 11 11 1 11 2 11 3 11 4 11 5 Cutting l ser s ir a0 a na A AAA A
8. A a Oa 27 Laser energy and fOCUS ri a nr A A ee aa e tae a 28 6 5 1 Laser energy adjustMent ss ai gon paci u aa a a aa a A 29 6 5 2 Laser focus adjustment saa s ace eee A A ee ee ee 29 Adjusting laser focus and energy sacosa u s a wra a aw en 30 Cutting speed mat saiae 20 Be zu A A w ae ee ee aan Dae eee RO 30 PALM RODOMOVeR ssi ars eia a w a Wie a in 31 Handling and safety advice aas sss son i oia 0 a k wawa pu s w s a ee Wu Cs m ma ee W 31 FAA Intended USE e ws ir au Bas WES Au Quan A a eRe eae a ae S Re alee bes 31 PD Precautions s tr A a AS AE 31 Starting PALM RoboMover miii ace ee 32 Working with PALM RoboMover i iii a asas sas ai inan is nis nis is ni nisi na 32 7 3 1 Preparing collectOrs ma dea ic a US ee ae Be A a ea er 32 7 3 2 Checking catapulted specimens ia sasa ee 33 Using diffusor optin sa caa i064 gees a BREET RAT Ce AR ee 33 PALM MicroBeam with Fluorescence Illumination cee eee 34 Operating microscope with fluorescence illumination nn 34 Simultaneous fluorescence Observati0N ooo 34 SOTO Saca A A A A un q O 34 8 3 1 Settings for working with motorized microscope Zeiss Axio Observer 34 8 3 2 Settings for working with manual microscope Zeiss Axio Observer 34 Freeze MOTE ci a A a een 35 APOTOM Citgo ie a a Ba a aa a a 35 Troubleshooting 000 sis s hw mm nn 36 Cutting laser 22 5 4 8 2
9. Collector 200 CM II SingleCap Collector 200 CM II SingleTube Collector 500 CM II SingleCap Collector 500 CM II Positioning accuracy lt 0 1 mm Repetition accuracy lt 0 1 mm Motor type Stepping motors with 200 full steps per revolution Speed range 9 375 mm sec to 85 mm sec in X Y axis 11 5 Light sources on microscope Mercury vapor short arc lamp HBO 50 W AC max 50 W Mercury vapor short arc lamp HBO 100 X Cite 120 120 W metal halide liquid light guide Cutting laser Technical data see 11 2 Colibri Technical data see relevant manual HAL Technical data see relevant manual HXP 120 Technical data see relevant manual Further technical data can be found in the operating instructions for the individual system components 40 PALM MicroBeam User Manual 0108 EG Konformit tserkl rung EC Declaration of Conformity entsprechend Anhang IV der Richtlinie 98 79 EG according to Annex IV Directive 98 79 EC Carl Zeiss Microlmaging GmbH Standort Bernried 82347 Bernried Germany Qualit tsmanagementsystem zertifiziert nach Quality Management System certified to meet DIN EN ISO 9001 und and DIN EN ISO 13485 Wir erkl ren hiermit die bereinstimmung des genannten Produktes mit den Anforderungen der Richtlinie 98 79 EG ber In vitro Diagnostika Bei nderungen am Produkt die nicht von uns autorisiert wurden verliert diese Erkl rung ihre G ltigkeit We declare the co
10. PALM RoboMover 37 Type plate 11 PALM MicroBeam User Manual 0108
11. data 40 PALM CapMover II 23 PALM ControlUnit 18 PALM MicroBeam Camera adapter 26 Cameras 24 Computer 25 Fluorescence attachment 21 Foot stepper 25 Joystick 25 LaserUnit 18 Maintenance 38 Microscope adapter 19 Microscope Zeiss Axio Observer 20 Overview 17 PALM CapMover II 23 PALM ControlUnit 18 PALM RoboMover 24 PALM Stage II 22 Preparing for application 26 Switching off 26 Switching on 26 PALM RoboMover 24 31 Handling 31 Inserting collector 33 Intended use 31 Precautions 31 Safety advice 31 Starting 32 Technical data 40 Troubleshooting 37 PALM Stage II 22 Inserting SlideHolder 22 Power connection 9 44 Precautions PALM RoboMover 31 Preparation for laser operation 20 Preparing collectors 32 Preparing for application 26 Preventive measures 14 S Safety 10 Disposal 16 Electricity 15 EMC 15 Fire extinguishing 15 Further risks 14 Infectious material 15 Laser safety 10 Moving parts 15 Preventive measures 14 Safety markings 11 Staff qualifications 14 Safety markings 11 Settings Fluorescence attachment 34 Simultaneous fluorescence observation 34 SlideHolder Inserting 22 Inserting slides 23 Specimens Checking 33 Staff qualifications 14 Switch off Cutting laser 27 Switch on cutting laser 27 Switching off PALM MicroBeam 26 Switching on PALM MicroBeam 26 T Technical data 39 Cutting laser 39 Operating data 39 PALM CapMover 40 PALM RoboMover 40 Troubleshooting 36 Cutting laser 36
12. earthing contact Use a mouse pad that is suitable for an optical mouse Note Please also consider vibrations caused by your routine lab equipment No device of any kind may be mounted within the travel area of PALM RoboMover PALM RoboMover does not block the left hand port of the microscope allowing devices such as stan dard cameras to be mounted Nevertheless the available space is limited 3 2 Admissible ambient conditions Transport in packaging The device may be transported at the following ambient condition Admissible ambient temperature of the air 20 C to 50 C 4 F to 122 F Storage The device may be stored at the following ambient conditions Ambient temperature of the air 10 C to 40 C 50 F to 104 F Air humidity 20 to 70 not condensing Operation The device may be operated at the following ambi ent conditions Ambient temperature of the air 15 C to 30 C 59 F to 86 F Air humidity 20 to 70 not condensing To ensure highest precision of the stage tem perature stability during operation must be 1C Altitude at usage location max 2000m Atmospheric pressure 800 to 1060hPa Degree of soiling 2 PALM MicroBeam User Manual 0108 Operating Room 3 3 Making power connection The power connection to the instruments is made by connecting the power cables supplied to the two power strips also supplied Depending on the country o
13. the original The relevant information is printed on the back of the control box Other types of fuse do not meet the safety requirements There are no further serviceable parts in the control box For servicing other modules see individual manu als PALM MicroBeam User Manual 0108 Technical Data 11 Technical Data 11 1 Operating data Usage location Closed rooms Table dimensions 152cm x 9icm Weight 200kg complete system Protection class I Ingress protection IP 20 Electrical safety In accordance with DIN EN 61010 1 under consideration CSA UL regulations Laser safety In accordance with DIN EN 60825 1 ICE 60825 1 Overvoltage category II Susceptibility As per EN 61000 4 Electromagnetic compatibility In accordance with DIN EN61326 2 6 12 2006 and DIN EN 55011 08 2003 Ambient temperature of the air 15 C to 30 C 59 F to 86 F Air humidity 20 to 70 not condensing Input voltage 100 230 V 10 Frequency 50 60 Hz Frequency fluctuations 2 Hz Power consumption of the complete sys tem in the standard configuration 11 2 Cutting laser Wavelength 1000VA 115V 8 7A or 230V 4 35A approximate distribution power strip 1 PC 550VA 115V 4 8A or 230V 2 4A power strip 2 microscope 450VA 115V 3 9A or 230V 1 95A 355 nm Pulse energy gt 90 u Pulse duration Pulse fre
14. 2 Admissible ambient conditions 2 222 w Cus a sa C mie Cus n W n QU E Wu w Sua s sua s Q E 8 3 3 Making power connection iis ae v s s s a nn 9 4 Safety tii wi arusasa ea ie Qu w A Sus a A A as a a en 10 4 1 What you should do in the event of afault i ii is ninas ninin i ninas s s i is it 10 4 2 Observe User Manual u san ne e RE pe wa b Q anil s a W Re a 10 4 3 Laser safety anne eed Sia Haw Seon w OR Et es we A EN A A a are 10 4 4 Safety markings and type plate u ce sosa da emanis aa iaa wa s a e ea A q Cas q a a 11 4 5 Staff qualifications masie dd era Chusquea Se O re ae Bae A a A 14 4 6 Points requiring Strict compliance con 14 4 7 Consideration of further risks and preventive measures i es 14 4 7 1 Further risks and protective measures laser nn 14 47 2 Further risks electricista a ae 15 4 7 3 Further risks infectious material ooo 15 4 7 4 Further risks EME u guage a a qis a Ya ee Po ee eee oo 15 4 7 5 Further risks extinguishing fire es 15 4 7 6 Further risks moving parts ses ieat neea ua nn 15 4 8 DISPOSAL yuq tua suls tupu Sus ea ae EQ ER ae A At at NOG ASS 16 4 8 1 Ensuring safe and ecological disposal ooo 16 4 82 FOr EU SstatesronlVyy yum ata a lu a Ea Hb oa elie eins 16 5 PALM MicroBeam Overview nn nn nn nn nn 17 5 1 PALM CONtrOlUNIE 2 0 24 wate re ad ea Ae a S s aF usus 18 5
15. D not go out please contact palm info zeiss de 6 1 2 Switching off device It is important to exit PALM RoboSoftware before you switch off the PALM system Exit PALM RoboSoftware Exit WINDOWS Switch off the ControlUnit PC etc Switch off the monitor 6 2 Camera adapter Caution Do not touch the camera If the camera pos ition is changed recalibration will be necessary please refer to PALM RoboSoftware manual for how to calibrate your system Note Depending on the camera adapter the com puter monitor only shows a section of the area visible in the ocular PALM MicroBeam User Manual 0108 Preparation of PALM MicroBeam for Application 6 3 Checking laser beam path in microscope The laser is coupled into the microscope via the epifluorescence beam path Before using the laser check that any filter slide is set to a free slot see Fig ure 9 No 4 that the reflector turret under the objective is moved to the correct position Laser see Fig ure 9 No 3 Caution Only use objectives authorized by Carl Zeiss Microlmaging GmbH for laser operation Objectives designed for oil immersion or water immersion must be used with the respective immersion medium or the objectives might be damaged Caution Do not turn the objective turret during simultaneous laser operation Never use the laser without an objective in the appropriate position Never insert reflecting objects in t
16. IN EN 60825 1 2003 involving no special safety requirements if operated according to the instructions given in this User Manual The accessible laser beam is safe for the naked unprotected eye when the laser casing is closed A Laser of class 3B is incorporated When the laser casing is open it presents a potential risk Any direct or reflected beam must not strike the unpro tected eye do not look directly into the beam For technical data of the laser see chapter 11 Laser class 1M Laser products that are safe for the naked unaided eye including long term direct intrabeam viewing The MPE maximum permissi ble exposure may be exceeded with injury to the eye possibly occuring following exposure with one of the two categories of optical viewing instru ments under certain conditions Laser class 3B Laser products that are normally hazardous when intrabeam ocular exposure occurs including accidental short time exposure Viewing diffuse reflections is normally safe Class 3B lasers which approach the AEL accessible emission limit for class 3B may cause minor skin injuries or even pose a risk of igniting flammable materials How ever this is only likely if the beam has a small diameter or is focused During servicing the device must be operated only by the service staff or an authorized representa tive of Carl Zeiss Microlmaging GmbH even with the laser casing closed Warning The housing of the LaserUnit may only be
17. am may only be installed by Carl Zeiss service engineers or their representa tives Should you want to move an installed unit please contact palm info zeiss de for assistance The PALM Laser Systems are essentially mainte nance free With any malfunction of the device please contact our Service department see section 1 4 page 6 PALM MicroBeam User Manual 0108 7 Operating Room 3 Operating Room 3 1 General To ensure proper functioning and problem free use of your new PALM CombiSystem the following requirements should be taken into consideration The system should be installed in a well venti lated room which is sufficiently large about 6 square meters and well aired There should be clearance of approx 90cm above the table and all shelves wall cupboards etc should be removed PALM RoboMover greatly extends the dimensions of a PALM System to the left It projects beyond the PALM MicroBeam base plate by 12 centimeters on the left Figure 1 shows a standard configuration The dimensions may vary depending on the configura tion Figure 1 Dimensions Type of floor low vibration if not special working table recommended and flat The aluminum plate requires a table measuring at least 152x91cm for set up of the system and a table load carrying capacity of gt 90kg m The total weight is approx 200kg depend ing on the configuration Two properly installed socket outlets with
18. arl Zeiss Microlmaging GmbH You can determine the type and number of cells to be catapulted into each col lection vessel position You can easily pre select how you want the specimen or concentration series to be distributed The CapCheck function instantly allows you to visually examine the sam ple in the collection vessel The diffusor ensures the best possible imaging for you Any purposes other than the collection of matter in combination with PALM MicroBeam or PALM CombiSystem are not recommended by Carl Zeiss Microlmaging GmbH and are not supported by PALM RoboMover or its software 7 1 2 Precautions PALM RoboMover can travel freely up to its stops in the space above the PALM MicroBeam platform Consequently there is always a risk of PALM RoboMover colliding with other neighboring hard ware or even with users themselves It is therefore very important that users stand back from the device while it is moving Users must also ensure that the area around PALM RoboMover is clear of any hardware that could col lide with it PALM RoboMover has several integrated features to minimize these risks The forces that could be applied by PALM RoboMover while traveling in a given X Y axis are not very great so that the associated risks of damage or injury are correspondingly low The Z axis includes a safety clutch to limit the forces that can be transferred A collision in any axis will be detected by the controller res
19. c tion vessels with maximum precision PALM RoboMover for work at higher through puts This unique robotic collection device pro vides for the computer driven sampling of catapulted specimens from multiple spots into a variety of collecting devices PALM MicroBeam User Manual 0108 The fluorescence attachment expands the microscope into a fluorescence microscope All functions can be controlled by PALM RoboSoftware to facilitate operation and con trol of PALM MicroLaser Systems The lasers used are interfaced with a research microscope and focused through objectives with high numerical aperture to a minimum possible spot size The effective laser energy is concen trated solely on this minute focal spot and most biological objects are transparent for the applied laser wavelengths It is therefore possible to work even inside a living cell without interfering with its viability Applications PALM MicroLaser Systems are deployed in a wide range of research biological and industrial appli cations Our one of a kind laser technology makes it possible for microscopic specimens to be pos itioned microsurgically processed and captured with absolute precision and unlike other systems without risk of contamination due to our patented non contact Laser Pressure Catapulting process lifting the desired objects into a vessel for further analysis only by the force of laser light It is intended for the collection of precisel
20. cation 26mm x 52mm One per system Front of the Cutting LaserUnit housing left side upright Size Quantity Location Edge length Meaning Location Size Location 26mm x 52mm One per system On the coupling tube 10mm Warning of hazardous points Next to the screw for the condenser and the screw for the HAL 8mm x 30mm Next to the screw for the condenser Size Quantity Location 26mm x 52mm One per system On the right side of the microscope stand below the reflector turret 12 PALM MicroBeam User Manual 0108 Safety Description PALM MicroBeam Made in Germany Manufactured Serial No XXXXXX XXXX MBXXXXX C Ratings 100 240 VAC 50 60 Hz max 1000 VA Carl Zeiss Microlmaging GmbH Location Bernried Am Neuland 9 12 82347 Bernried Germany Phone 49 8158 9971 0 wwwzeiss de microdissection PALM MicroBeam User Manual 0108 Identification label Edge length 12 5mm Meaning Risk of hand injury Location To the right side of the CapCheck opening of the stage 13 Safety 4 7 Consideration of further risks and preventive measures 4 5 Staff qualifications The device may only be operated by trained and specially instructed staff of the legal minimum age 4 7 1 Further risks and protective who have carefully studied this User Manual measures laser Staff undergoing teaching
21. cence attachment The filter wheel is accessed by PALM RoboSoftware It is mounted outside the optical path of the laser To change the bulb please refer to the microscope manual For the correct settings of the power supply and the controller for the filter please consult the PALM RoboSoftware manual For detailed information about operating the microscope with fluorescence illumination please see section 8 page 34 Depending on specific customer requirements Carl Zeiss offers two different fluorescence attach ment options PALM MicroBeam User Manual 0108 Basic fluorescence attachment for PALM MicroBeam with Zeiss Axio Observer Simultaneous fluorescence observation and laser functions are not possible or desirable Depending on the type of microscope the filter revolver for fil ter change and cutting position is handled manu ally Fluorescence hardware components Fluorescence lamp HXP 120 X Cite Colibri or HBO 100 Uni blitzshutter Any basic fluorescence filter set The basic fluorescence attachment is suitable for fixed samples only Advanced fluorescence attachment for PALM MicroBeam with Zeiss Axio Observer Simultaneous laser functions and fluorescence illumination are provided for Fluorescence hardware components Fluorescence lamp HXP 120 X Cite Colibri or HBO 100 Motorized filter wheel Selected advanced fluorescence filter sets Depending on the type of micros
22. connection between camera and computer S VHS plug must be plugged in frame grabber board Check position of switching mirror in microscope 9 4 Shift between drawing and cutting Check position of laser marker Check alignment of camera the camera must be adjusted parallel to the stage movement Test by moving the stage with the arrow keys at a slow speed Recalibrate as described in PALM RoboSoftware manual make sure that you are using the settings for the active objective Check LEDs on encoder of moving stage LED is green o k LED is orange or red please contact palm info zeiss de 9 5 Reflex in video image Incidence of light through the ocular may cause reflexes in the video image in case of reflexes close shutter at the microscope 9 6 Electrical power outage Switch off all units of the system microscope ControlUnit etc Remove all projecting consumables like culture dishes microtiter plates etc from the stage Turn the objectives down in case of a motorized microscope switch on the microscope again and turn down the objectives Reboot the computer and start PALM RoboSoftware PALM MicroBeam User Manual 0108 37 Maintenance of PALM MicroBeam 10 Maintenance of PALM MicroBeam Caution Before starting maintenance disconnect all system components from the mains 10 1 Microscope For further details see microscope manual 10 1 1 Cleaning optical parts e Clean all easily accessible optical par
23. cope motorized or manual shutter and filter wheel different adjustment will be necessary Note If you use a Zeiss ApoTome advanced fluor escence is not possible Warning Gas discharge lamps such as the HBO 100 emit ultraviolet radiation which may cause burns to eyes and skin Never look directly into the light and avoid direct skin exposure See microscope manual for safety instructions 21 PALM MicroBeam Overview 5 6 PALM Stage II The computer controlled PALM Stage II is a motor ized stage especially designed for high precision laser micromanipulation in sub micrometer range It is moved by two stepping motors at a speed ranging from just a few micrometers to several mm per sec and displayed largely without vibrations between 1yum and approx 60mm per second The stepping motors are controlled by two encoders that are mounted underneath the stage The max imum travel path is 76mm in the Y direction and 114mm in X direction Caution Do not move the stage manually Do not touch the moving stage Fingers can be caught in CapCheck open ing Avoid contaminating the guide of the stage with oil or chemicals Avoid any kind of mechanical stress e g placing heavy parts or leaning on it as it is a precision instru ment 5 SlideHolder 6 specimen slide 1 stepping motor Y direction 2 stepping motor X direction 3 microscope table board 4 CapCheck Figure 11 PALM RoboStage II For high thro
24. d with software control both when collecting and checking for the catapulted specimen Please con tact palm info zeiss de for further information Caution Moving parts like CapMover II and Stage II must not be touched during operation In the event of a collision PALM CapMover II must be referenced see software manual 1 Cap 3 CapMover II 2 SingleCap Collector Figure 13 PALM CapMover II 23 PALM MicroBeam Overview 5 8 PALM RoboMover PALM RoboMover is the state of the art device for modern cell harvesting and sorting different types of microdissected specimens at higher through puts It enables you to automatically position col lecting devices above your specimen For more details see chapter 7 PALM RoboMover Figure 14 PALM RoboMover with Stage II 24 5 9 Cameras There are several camera ports available to incor porate up to 3 cameras With the newest PALM MicroBeam generation you can incorporate different digital cameras AxioCam MR and ICc 1 You have the option of switching between the different cameras To use one of the cameras you have to select the desired camera and make your selections using PALM RoboSoftware Pictures taken by a camera are directly transmit ted to the computer via a frame grabber The video transferred microscope image is dis played in real time on the monitor screen which allows you to simultaneously observe the micro scope image during stage laser or col
25. formation 5 Applications 5 Contact information 6 Intended use 5 Legal notes 6 C Camera adapter 26 Cameras 24 Cleaning optical parts 38 Collectors Preparing 32 Computer 25 Contact information 6 Control box Maintenance 38 Cutting laser 27 Interlock 27 Switch off 27 Switch on 27 Technical data 39 Troubleshooting 36 Cutting speed 30 D Diffusor option 33 Dimensions 8 Disposal 16 Electricity 15 EMC 15 PALM MicroBeam User Manual 0108 F Fire extinguishing 15 Fluorescence attachment 21 Advanced 21 Basic 21 Settings 34 Fluorescence illumination 34 Simultaneous fluorescence observation 34 Foot stepper 25 Freeze Mode 35 Further risks 14 Electricity 15 EMC 15 Fire extinguishing 15 Infectious material 15 Laser 14 Moving parts 15 Fuses Changing 38 I Infectious material 15 Inserting collector 33 Installation 7 Intended use 5 PALM RoboMover 31 J Joystick 25 L Laser Further risks 14 Safety 10 Laser beam path Checking 27 Laser energy 28 Adjustment 29 Laser focus 28 Adjustment 29 Laser safety 10 LaserUnit 18 Legal notes 6 43 Maintenance 38 Control box 38 Microscope 38 Microscope Checking laser beam path 27 Maintenance 38 Operation with fluorescence illumination 34 Microscope adapter 19 Microscope Zeiss Axio Observer 20 Moving parts 15 O Operating room 8 Ambient conditions 8 Power connection 9 Overview PALM MicroBeam 17 P PALM CapMover Technical
26. ge RoboMover or CapMover II and the LaserUnit The actual status of PALM ControlUnit is indicated by LED s on the front panel OUT sawn OIOO OO OOOO 00000000 20000000 000 000 000 200 200 000 000 000 5 key switch Figure 6 PALM ControlUnit Note Shortly after PALM ControlUnit is switched on the red LED F goes out automatically Other wise switch off the ControlUnit and after 10 seconds switch it on again Should the LED not go out please contact palm info zeiss de The type of ControlUnit may differ depending on the individual device configuration 18 5 2 LaserUnit The Laser Unit is a one box device which houses the laser and all the necessary optics to guide the laser into the microscope to make the laser focus coincide with the optical focus of the microscope at the object plane and to adjust the laser energy a PALM Microbeam Figure 7 LaserUnit This is a prerequisite for precise laser micromani pulation with maximum precision Furthermore the laser position remains stable which allows you to routinely work with the system without any time consuming search for the laser beam focus Note The adjustment of laser focus and energy set tings is performed by the user see chapter 6 5 also observe PALM RoboSoftware manual Caution Do not open the housing of the LaserUnit Your unprotected eyes might be exposed to invisible laser class 3B radiation
27. gooz Asenuec WeaSgolInNn WTVd This document is supplied only to persons who are trained and authorized by Carl Zeiss Microlmaging GmbH No part of this document may be reproduced or distributed in any form or by any means without the prior written consent of Carl Zeiss Microlmaging GmbH The information in this document is subject to change without notice Carl Zeiss Microlmaging GmbH assumes no responsibility for any errors that may appear Printed January 2008 Eight edition Regulatory Notice PALM MicroLaser Systems are intended for research use only and are not approved for medical applications in all countries Published by Carl Zeiss Microlmaging GmbH Location Bernried Am Neuland 9 12 82347 Bernried Germany Phone 49 0 8158 9971 0 Fax 49 0 8158 9971 249 E mail palm info zeiss de www zeiss de microdissection O Carl Zeiss Microlmaging GmbH 2008 All rights reserved 2 PALM MicroBeam User Manual 0108 List of Contents Contents Chapter Page 1 Important Basic Information 2220 mnu nn nn nn nn nn 5 1 1 Intended use of PALM MicroLaser Systems 000 cet a 5 1 2 egal notes urn aaa a A A a aod eed A aoe Bie at asa 6 1 3 Conventions for User Manual v esia widre ee ee 6 1 4 HOW to contact S ias aa a a a w eee a ee eB ae S e eae a ee 6 2 Installation 3 62 ee ease Pe e A a eee ee ee eee w N 7 3 Operating ROOM nun a 8 341 Gener le ae een ee Boe ae pa a A A ae 8 3
28. he LED 2 will be dark When the illumination arm is positioned back in the working position both LEDs will light up green To reactivate laser emission the respective button in the PALM RoboSoftware can be used or the laser ON OFF button pressed Switch off the laser e To deactivate laser emission press the LASER ON OFF button 3 The red LED 2 will go out 27 Preparation of PALM MicroBeam for Application 6 5 Laser energy and focus To be able to precisely cut the material and reliably catapult it the laser focus has to be accurately set to optimal values under various conditions The diameter of the cuts depends on the numerical aperture of the objective used the position of the laser focus with regard to the microscope focus the applied laser energy and on the absorption properties of the mate rial to be cut Ablation takes placce within the area of the laser focus please see figure 17a The smallest avail able cutting size can only be achieved within the center of the laser focus using as little energy as possible Catapulting is performed by placing the laser focus underneath the specimen to be catapulted please see figure 17b a ablation b LMPC The laser beam is guided through the epifluores cence channel of the microscope and focused through the objective used Therefore the energy and the size of the laser focus depend on the objective used The following generally applies
29. he central position again e Press the Loadposition button and wait until the Load Position is reached e Withdraw a holder possibly already assem bled and insert the SlideHolder into the open ing provided The sharp edge has to be positioned on the right at back This edge means that the holder can only be inserted in one direction The software now prompts you to select the cor rect holder e In order to return to the work position press the Loadposition button again Note The objectives have to be focused on the sam ple or lower to avoid a collision between the objective and the stage PALM MicroBeam User Manual 0108 PALM MicroBeam Overview Inserting of the slides in SlideHolder e Choose the appropriate slides e Push the slider the metal clamp on the lower side of the holder to the left and insert the slide e Make sure that the slide fits exactly into the three edges e Release the slider carefully Note When you change from one slide to another you will have to focus manually If you are using an 100x oil objective lower the objective when changing from one slide to another PALM MicroBeam User Manual 0108 5 7 PALM CapMover II The motorized PALM CapMover II was developed for fast positioning with high precision and fast exchange of the collection device for sample col lection Different collectors for caps are available This means that the cap is positioned and move
30. he frame Please con tact palm info zeiss de for a collector tailored to your specific needs PALM MicroBeam User Manual 0108 PALM RoboMover Inserting the collector into PALM RoboMover e Prepare to insert the collector as described in the PALM RoboSoftware manual e Insert the collector into PALM RoboMover as shown in Figure 22 Figure 22 Inserting the collector into PALM RoboMover PALM RoboSoftware has to scan the new collector type Please refer to the PALM RoboSoftware man ual for detailed information Place an insert in the frame e Place an insert in the appropriate frame refer to Figure 23 the figure shows PALM CapturePlate Collector 96 Figure 23 Inserting CapturePlate 96 into the frame PALM MicroBeam User Manual 0108 7 3 2 Checking catapulted specimens After catapulting the contents of the wells can be observed through the objective To do this you have to move PALM RoboStage to CapCheck for further information refer to PALM RoboSoftware manual CapCheck Caution Set the objective to the lowest position possible before you move PALM RoboStage either to or back from CapCheck Otherwise PALM RoboStage will collide with the objective This could cause serious damage to the system and to your specimen in systems with motorized microscopes such as Z1 the objective is moved down automatically If you do not have a motorized microscope move the objective to the lowest position
31. he laser beam path 6 4 Cutting laser The cutting laser is integrated in the LaserUnit Switch on the laser e Switch on the main power supply of the PALM controller Only the left LED 1 will light up yellow indi cating that a supply voltage is present The left LED 1 will light up green after one second The right LED 2 does not light up during the warm up period After a warm up period of approx 5 minutes the left LED 1 and the right LED 2 will light up green The laser is now ready for operation and can be activated e To activate or deactivate laser emission press the LASER ON OFF button 3 It is also possible to activate or deactivate the laser using PALM RoboSoftware If the laser is activated the green LED 2 will change color and light up red The LED 1 stays green After a delay of approx 1 second laser emission is ready to start e To start laser emission start the laser function in the PALM RoboSoftware PALM MicroBeam User Manual 0108 The laser works with output pulse energy regula tion After a total warm up period of less than 7 minutes the laser parameters will be within the specifications FOS RNA Makea tt N 1 LED 1 2 LED 2 3 ON OFF button Figure 16 Laser Unit Interlock When the microscope illumination arm is moved back the laser safety interlock is activated Laser emission will stop immediately and the LED 1 will light up yellow and t
32. his User Manual carefully and observe all instructions given in the manual Note Please also consider the manuals provided for your peripheral devices You will find a list of all manuals supplied with your system Features of the peripheral devices listed in the manuals may vary in combination with PALM MicroLaser systems The information given in this manual prevails over the manuals for the peripheral devices Please also follow the instructions for the single components of your PALM MicroBeam especially for the microscope the fluorescence attachment the video cameras and the laser Carl Zeiss Microlmaging GmbH will not be responsible for errors and possible consequences caused by fail ure to observe user manuals 1 1 Intended use of PALM MicroLaser Systems PALM MicroLaser family of products is a modular tool for reliable and precise non contact laser based microdissection and micromanipulation and consists of PALM MicroBeam PALM MicroTweezers and as a combination of both PALM CombiSystem It can be supplemented by different hardware options that can be adapted to individual needs The microscope stage PALM RoboStage II can be equipped with different target holders for slides Petri dishes capillaries and even for work with serial sections The following options are available for position ing different collecting vessels The newly designed software controlled PALM CapMover II for fast positioning of single colle
33. icrolmaging GmbH is not liable for damage due to failure to observe this User Manual either partly or in full The warranty shall lapse where the customer opens the laser housing or uses the device for pur poses other than those specified If used parts are replaced by similar parts which are however not identical to those recommended by the manufacturer in this manual Carl Zeiss Microlmaging GmbH shall only be liable where the product supplied was already defective before the parts were replaced For details concerning the manufacturers war ranty please refer to the contractual conditions 1 3 Conventions for User Manual Special markings on the left margin indicate infor mation you should observe without fail Warning This warns you about the possibility of extremely serious irreversible injuries to per sons or even death Caution This indicates the possibility of personal injury of moderate severity and of damage to the device Note This indicates information that calls for your special attention 1 4 How to contact us Business hours 9 00 am 3 00 pm CET Mo Fr Phone 49 0 81 58 99 71 0 Fax 49 0 81 58 99 71 249 E mail palm info zeiss de Service address Carl Zeiss Microlmaging GmbH Location Bernried Am Neuland 9 12 82347 Bernried Germany palm techsupport zeiss de www zeiss de microdissection PALM MicroBeam User Manual 0108 Installation 2 Installation Your PALM MicroBe
34. ithin the optical path The position of the ApoTome must not be changed when the laser is running Note If you want to upgrade your PALM System with an ApoTome ask for the ApoTome safety kit from Carl Zeiss Microlmaging GmbH 35 Troubleshooting 9 Troubleshooting In general if malfunctions occur with the laser system first check that all connections to power supply and laser head as well as to existing exter 9 1 Cutting laser No Error 1 The laser system switches off automati cally during operation The LED1 is yellow 2 Red LED Laser On fails to light up even though the On Off but ton was pressed LED2 fails to light up 3 LED1 fails to light up even though the power switch was pressed 4 LED2 is red but no laser emission 5 LED1 and LED2 not functioning 36 Possible Cause Out of the operating tem perature range e g the laser head is overheated Room temperature too high INTERLOCK signal is missing Internal electrical fault in laser head or in PALM ControlUnit See No 1 The LED2 fails to light up which means laser system is not ready for operation Internal electrical fault in laser head or in control device Power supply is not activated PALM ControlUnit is defective External or internal trigger is not activated or does not work properly Control unit or booster is defective Power supply is not activated LEDs are deactivated Power s
35. lecting device functions You can store the transferred images on the hard disk of the computer or on CD The camera is preset by Carl Zeiss Microlmaging GmbH When changing camera parameters please con sult the manual of the camera Note Depending on the camera adapter the com puter monitor only shows a section of the area visible in the ocular Caution The camera is aligned parallel to the stage Do not turn the camera at all or calibration of the system will be invalidated All camera ports are protected with UV blocking filters PALM MicroBeam User Manual 0108 PALM MicroBeam Overview 5 10 Computer The system is equipped with a state of the art mini workstation or a workstation with two CPUs The computer itself is provided with a CD burner With the integrated network card you have the option of connecting your computer with your local network If you want to connect it please speak to your local area network administrator Please con tact palm info zeiss de for details Note You must not change or modify software or hardware components on the computer Carl Zeiss Microlmaging GmbH is not responsible for any damage resulting from failure to com ply with these points Any modification of the computer hardware software is subject to the prior written consent of Carl Zeiss Microlmaging GmbH At all events Carl Zeiss Microlmaging GmbH is not liable for any loss due to computer viruses of any kind
36. lication conditions and performance data defined in this User Manual may not be altered For technical data see chapter 11 page 39 industrial protection when using laser devices must be observed The operator of the unit must also observe all relevant statutory acci dent prevention regulations Further risks The laser works with invisible light When the casing is open and the interlocks overridden the laser falls under the laser protection class 3B The powerful radiation cause dimmed sight of the eye The casing must always be kept closed and locked and may only be opened by the ser vice staff of Carl Zeiss Microlmaging GmbH Unintentionally mirrored reflections must be avoided by an awareness of the risks involved by staff For reasons of laser safety all ports must either be equipped with the appropriate device fluorescence lamp camera UV block ing filter objective or covered with blind plugs or covers The devices blind plugs or covers may not be removed by the customer as laser radiation might be emitted In this case a direct or reflected laser beam could strike and injure the unprotected eye Laser safety concept A UV absorption filter is used with the eye piece of the microscope to absorb hazardous UV laser radiation It is contained in the microscope stand This prevents the user coming into direct contact with laser radia tion even when the binocular tube or eye pieces are removed laser clas
37. mpliance of the product with the requirements of the Council Directive 98 79 EC about In vitro Diagnostica Any modification to the product not authorized by us will invalidate this declaration Produktbezeichnung Product name PALM Mikrolaser Systeme PALM CombiSystem IV PALM MicroBeam IV PALM Tweezers IV Normen Standards DIN EN 61010 1 DIN EN 61010 2 101 DIN EN 60825 1 DIN EN 61326 1 DIN EN 61326 2 6 DIN EN 14971 Grundlage Konformit tsakte Nr Basis Record of Conformity No MI 076 08 IvD Das Produkt ist gekennzeichnet mit The product is marked with Pr fung Test GS und EMV Labor Kompetenzzentrum Qualit t Product Safety and EMC Laboratory Competence Centre Quality Carl Zeiss Jena GmbH Registriert Registered CZ MIB M CE MB MT CS IV 02_2008 Bernried 03 03 2008 Carl Zeiss Microlmaging GmbH Standort Bernried Bernried Location Dr Richard Ankerhold Dr Hermine Hitzier Gesch ftsf ld Mikrddissektion Qualit tsmanagement Business Group Microdissection Quality Management Gew hrleistung und Haftung sind in unseren Allgemeinen Verkaufsbedingungen geregelt Conditions of guarantee and liability are dealt within our General Conditions of Sale PALM MicroBeam User Manual 0108 41 42 PALM MicroBeam User Manual 0108 Index A Adjustment Laser energy 29 Laser focus 29 Ambient conditions 8 Operation 8 Storage 8 Transport 8 ApoTome 35 Application 5 Basic in
38. n to find out details for the disposal or recycling of your device The product must not be disposed of with house hold waste or be given to the municipal disposal collection In the event of resale the seller is obliged to point out to the buyer that the product has to be disposed of according to the guidelines 16 PALM MicroBeam User Manual 0108 PALM MicroBeam Overview 5 PALM MicroBeam Overview Modular overview PALM MicroBeam device consists of the parts shown in Figure 5 1 TFT display 5 microscope stage 2 microscope see section 5 4 see section 5 6 page 22 page 20 6 LaserUnit and laser 3 video camera see section 5 2 page 18 see section 5 9 page 24 7 filter wheel 4 RoboMover 8 fluorescence illumination see section 5 5 page 21 Figure 5 Overview of PALM MicroBeam with PALM RoboMover PALM MicroBeam User Manual 0108 These parts and their function are described in detail in the following chapters 9 control unit filter wheel 10 power supply fluo illum 11 power supply microscope only Axio Observer Z1 12 monitor 13 ControlUnit see section 5 1 page 18 14 mouse 15 foot stepper see section 5 11 page 25 16 keyboard 17 joystick 18 computer see section 5 10 page 25 17 PALM MicroBeam Overview 5 1 PALM ControlUnit PALM ControlUnit is the main device which actu ates and controls the movement of PALM RoboSta
39. ne of the two power strips may be a so called master slave power strip This is clearly marked with a label and or color coding of the individual outlets The master outlet is normally red or yellow The power supply wiring for the system instru ments must comply with one of the two variants A or B refer to Figure 2 or Figure 3 It is valid only for the standard configuration If you want to con nect additional devices the installation may vary Consult palm info zeiss de Wall sockets PC Zeiss microscope 5 N N y Slave Master Q fi Q A Q fi fi Q N Q y y y N o ControlUnit Fluorescence lamp peed p Cutting laser power supply Slave Mm 7 gt oO e N y Slave Slave Slave r Ma E Figure 2 Variant A PALM MicroBeam User Manual 0108 Wall sockets Zeiss PC microscope Monitor 6 d po ControlUnit Fluorescence Cutting laser lamp power supply Filter wheel HUD Figure 3 Variant B Each of the two power strips must be connected to a dedicated wall outlet with working protective earth conductor No other devices should be con nected to the power strips Caution If the instructions above are not followed in some circumstances the requirements for equipment safety will n
40. o longer be met The information in the technical data should be observed regarding the permitted supply voltage Safety 4 Safety 4 1 What you should do in the event of a fault e Switch off the device immediately by turning the key switch to the OFF position see Figure 6 and make sure that it cannot be switched on again e Disconnect the system from mains e Call the service staff in charge 4 2 Observe User Manual This User Manual contains important information about safe operation of the device Only if the User Manual is strictly observed accidents can be avoided and trouble free operation of the device can be guaranteed By observing the User Manual downtimes and repair costs will be reduced and the service life of the device increased This User Manual must be stored close to the device at an accessible location known to staff where it is available at any time This User Manual must be read understood and observed in all points by the operating staff responsible Please observe all safety instructions and other information given in the User Manual Please read and observe all instructions given in the attached manuals Caution Any use other than the purposes defined in this User Manual might lead to physical injury or material damage for which Carl Zeiss Microlmaging accepts no liability 10 4 3 Laser safety PALM MicroBeam is a laser device belonging to laser protection class 1 M according to D
41. oon as the laser focus is shifted up or down slightly The cutting edges must show identical symmetry when the object is moved into X or into Y direction With increasing energy the membrane margins should still look symmetrical Note For each type of sample laser focus adjustment must be performed individually The individu ally determined values can be stored in a sep arate file please refer to PALM RoboSoftware manual Note For objectives with an aperture diaphragm adjust the diaphragm to the thickness of the object slide or the dish used to ensure optimum presentation focusing contrast For objectives with a short working distance special dishes and 0 17mm slides are necess ary 6 7 Cutting speed The laser pulse repetition rate of the cutting laser is 100 pulses per second To achieve a fine cutting line it is therefore necessary to optimize the laser energy in relation to the cutting speed PALM MicroBeam User Manual 0108 PALM RoboMover 7 PALM RoboMover 7 1 Handling and safety advice 7 1 1 Intended use PALM RoboMover is a software controlled device for the automated collection of matter which has been microdissected and catapulted via LMPC from a substrate on PALM MicroBeam You can choose from a variety of collection vessels for different types of experiments individual caps or tubes AdhesiveStrips even microtiter plates in their own collectors These collectors are only provided by C
42. ot be abrasive or strongly basic or acid as this would attack the protective coating 4 7 4 Further risks EMC As regards interference resistance and emission the system meets the requirements of DIN EN 61326 2 6 12 2006 It is categorized as belong ing to class A according to DIN EN 55011 08 2003 Class A devices are intended to be used in industrial environments In other environments the electromagnetic compatibility may be affected 4 7 5 Further risks extinguishing fire To extinguish fire use sand or carbon dioxide 4 7 6 Further risks moving parts Some of the devices like PALM Stage and PALM RoboMover move during operation Please keep away from these parts during operation to avoid injuries or physical damage 15 Safety 4 8 Disposal 4 8 1 Ensuring safe and ecological disposal Dispose of the following materials in an environ mentally friendly way Microscope lamps with a built in bulb or individ ual mercury vapor short arc lamps as hazard ous waste Laser as electronic scrap Electronic components as electronic scrap Cables as reusable material 4 8 2 For EU states only PALM MicroLaser devices are produced according to the EU directive 2002 95 EC RoHS where applicable We have set up a process for disposal and recy cling within the member states subject to the directive 2002 96 EC WEEE Please contact your relevant Carl Zeiss Service department or sales organizatio
43. quency lt 2 nsec max 100 pulses per second Beam divergence Laser control Pulse frequency of laser control 3 mrad Laser control can be changed by impulse packages external by earth free foot stepper or via RS232 interface automatic permanent impulses Pulse width 0 1 ms Adjustable via software Laser class 3B laser class of system is 1M Energy output at outlet opening objective a AEL accessible emission limit PALM MicroBeam User Manual 0108 lt 3 2 mW AEL lt 0 5 mW 39 Technical Data 11 3 PALM RoboMover Dimensions approx 50 x 25 x 27 cm Weight approx 4 9kg Collectors For PALM CapurePlate 96 CapturePlate Collector 96 For up to eight 500 ul tubes TubeCollector 8x500 For up to eight 200 ul tubes TubeCollector 8x200 For up to twelve AdhesiveStrips 8C CapCollector 12x8 For AmpliGrid AG480F SlideCollector 48 For single tubes CollectorSet Single Tube II 200 RM CollectorSet Single Tube II 500 RM For microtiter plates from Sarstedt PlateCollector S96 For microtiter plates from Greiner PlateCollector G96 Customer specific holders on request Positioning accuracy lt 0 1 mm Repetition accuracy lt 0 1 mm Motor type Stepping motors with 200 full steps per revolution Speed range 9 375 mm sec to 85 mm sec in X Y axis 11 4 PALM CapMover II Dimensions approx 29x 13 x 7 cm Weight approx 1 5kg Collectors SingleTube
44. r allows you to connect the fluorescence illumination lamp and the filter wheel to the system for simultaneous usage of fluorescence illumination with laser application Note A specially designed beam splitter must be used for simultaneous fluorescence illumina tion due to the high power laser For more information please contact palm info zeiss de Caution Do not touch or remove the screws on the microscope adapter This causes misalignment of the system and if the mirror is removed your unprotected eyes might be exposed to invisible radiation belonging to laser class 3B 19 PALM MicroBeam Overview 5 4 Microscope Zeiss Axio Observer Z1 preparing for laser operation 1 illumination arm 2 fluorescence attachment option Figure 9 Overview of the microscope The high optical and mechanical quality Zeiss inverse microscope Axio Observer is part of PALM MicroBeam It comes in two specifications Axio Observer D1 with encoded semi motorized design Axio Observer Z1 with fully motorized design incl motorized z transmission These microscopes Axio Observer Z1 D1 can also be used as normal research microscopes Please comply with the Axio Observer user manuals The laser beam is guided through the microscope adapter see section 5 3 page 19 into the epiflu orescence channel of the microscope It is reflected by a special coated beam splitter in the reflector
45. removed by the service staff or an authorized representative of Carl Zeiss Microlmaging GmbH Warning Do not place any optical part into the laser beam path Warning Please note wavelength is invisible to the human eye 355nm Objectives must not be removed Otherwise the device has to be classified as laser class 3B involving all special savety requirements according to this laser class PALM MicroBeam User Manual 0108 Safety 4 4 Safety markings and type plate the type plate are present and legible Replace The safety markings on the device must always missing or illegible safety markings or type plate be clearly visible and legible and must be Make sure that the following safety markings and Caution strictly observed RUN Macweaw Figure 4 Safety markings and type plate PALM MicroBeam User Manual 0108 11 Safety Description Edge length Quantity Location Size Quantity Location for Europe 50mm One per system Rear right on the laser unit s housing 26mm x 52mm One per system Rear right on the laser unit s housing Size Quantity Location 80mm x 30mm One per system Next to the identification label a 4 DO Size Quantity Location Complies with 21 CFR 1040 10 and 1040 11 80mm x 15mm One per system Next to the identification label Size Quantity Lo
46. s 1M The TV ports are therefore also protected with filters designed to absorb UV light Objectives not enabled for cutting and unas signed objective positions can be blocked for laser operation using the software Access is only possible in service mode of PALM RoboSoftware Service mode is password protected In addition free objective slots in the objec tive revolver are closed off with metal caps These metal caps are screwed into the objec tive revolver A tool is required to remove these metal caps The front port cannot be used to ensure laser safety PALM MicroBeam User Manual 0108 Safety Securing objectives Objectives approved for use with MicroBeam and enabled via the PRS 4 2 are prevented from being unscrewed with a coupling ring Only Zeiss technicians and trained staff are permitted to remove and replace objectives A customer interface is not involved here Securing reflector revolver The lever used to open the reflector revolver flap is fixed with an arm to prevent the lock ing device from being released The fixing arm is screwed to the microscope with a threaded connection The lever is thus dis abled Only Zeiss technicians and trained staff are permitted to remove the reflector revolver and replace or modify filter sets A customer interface is not involved here Caution The objective revolver and reflector revolver are not customer interfaces Protective measures taken by
47. ser focus indi cates good focusing of the glass surface With a higher energy setting cutting is possible not only within the laser focus but also above or below If the laser focus is just inside the glass smaller or larger pieces of glass will be blown out Figure 20 Zigzag line cut with the laser using different energy and focus settings Find the correct focal plane in the following ways please refer to PALM RoboSoftware manual to use the graphic tools the automated laser functions and how to adjust energy and focus settings for the laser 30 e use a plain membrane slide without specimen tissue e draw a zigzag line with the freehand graphic tool spanning the entire monitor screen e select an appropriate stage speed not too fast e start automatic cutting with the Cut function e reduce or increase laser energy using the Page down or Page up key on the keyboard or the slider for laser energy on the monitor screen until only a faint cut can be seen e move the laser focus up and down with the keys home and end on the keyboard or the corresponding slider for laser focus on the mon itor screen to locate the smallest cutting size e reduce the laser energy and repeat the proce dure described above a second or third time until the smallest available focal spot is found The perfect laser focus setting is achieved when using a minimum of laser energy produces a fine cutting line which immediately disappears as s
48. sted clear surface and a rim which is not higher than 2mm available from Carl Zeiss Microlmaging GmbH Figure 19 Laser focus below specimen plane The smallest laser focus diameter can be obtained when using objectives with a high numerical aper ture When using objectives with a lower numerical aperture the minimum achievable diameter of the laser focus increases accordingly When using objectives with a short working dis tance the thickness of the object slide must be adapted to the objective used If the objective is manually adjustable please check that the objec tive is adjusted to the thickness of the object slide actually used 29 Preparation of PALM MicroBeam for Application 6 6 Adjusting laser focus and energy As the laser beam itself is not visible only the laser effect ablation can be used for control To visualize the changes of the laser focus posi tion you must cut ablate an object whereby the laser is permanently pulsating and the object is being moved simultaneously We recommend using a membrane coated slide for this purpose Note Make sure that the membrane is always focused within the microscope focus or the laser focus setting might be incorrect Simultaneous movement of the stage will create a cutting line within the sample This line will be fuzzy or fairly thick as the laser focus setting is not yet sufficient Star like glass ablation around the la
49. t of the laser focus position This is especially impor tant for living cells The same applies when using different objectives To compensate for the so called chromatic aber ration the laser focus position has to be changed independently of the microscope focus The laser focus correction is motorized A high precision DC stepping motor moves the corre sponding lens Focus setting is conveniently con trolled and regulated on the monitor screen by PALM RoboSoftware The display allows you to change preselected laser focus settings Laser energy and focus is predefined by Carl Zeiss Microlmaging GmbH to override the factory set tings for each objective please refer to PALM RoboSoftware manual Only the sample specific values need to be defined by the individual user These user specific values can be stored in a spe cial file for each user and or application Note The factory settings for laser focus and energy have been adjusted to the objectives supplied by Carl Zeiss Microlmaging GmbH Please note The factory settings apply to the standard membrane slide without any tissue Itis not permitted to remove objectives or the reflector revolver Ask palm info zeiss de PALM MicroBeam User Manual 0108 For catapulting and microdissection a LD 40x objective has proved successful in most applications For the visualization of catapulted specimens with the LD 40x objective you need a cap with a flat non fro
50. tallation of your PALM system PALM RoboSoftware provides you with the capability of saving various combinations of these parameters and retrieving them when needed Note Close the shutter before switching on the flu orescence lamp and before finishing or inter rupting work This will prevent unintentional exposure of your sample to fluorescence light 8 3 2 Settings for working with manual microscope Zeiss Axio Observer To use fluorescence illumination you must switch on the fluorescence illumination select the desired fluorescence excitation filter only necessary when using a filter wheel please refer to PALM RoboSoftware manual swivel to desired reflector position in the micro scope switch the microscope light on or off as neces sary activate the timer if you want to preset a time for which the excitation light should illuminate your sample refer to PALM RoboSoftware man ual and open the shutter please refer to PALM RoboSoftware manual PALM RoboSoftware controls all functions of the fil ter wheel and the shutter please refer to PALM RoboSoftware manual The microscope settings are made manually the fluorescence image on the screen with the PALM RoboSoftware tool Freeze Mode please refer to PALM RoboSoftware manual Thus PALM MicroBeam User Manual 0108 PALM MicroBeam with Fluorescence Illumination bleaching effects of the sensitive samples can be avoided while defini
51. the manufacturer On the microscope lighting arm and the con denser if it can be swiveled out of the beam paths there is a safety switch that shuts down the laser as soon as the lighting arm is swiveled The travel path of the laser beam as well as those of the stage RoboMover and CapMover are equipped with limit switches to avoid damage 4 7 2 Further risks electricity The unit must be connected to two properly installed socket outlets with earthing contact via the mains cables supplied Extensions or additional socket outlets are not permitted PALM MicroBeam complies with the requirements of the standard DIN EN 61010 1 IEC 61010 1 Safety requirements for electrical equipment for measurement control and laboratory use DIN EN 61010 2 101 Particular requirements for IvD medical equipment and DIN EN 60825 1 2003 IEC 60825 1 2003 Safety of laser products The system complies with requirements 1 in the directive 98 79 EC and bears the CE marking Before cleaning make sure that the power has been switched off PALM MicroBeam User Manual 0108 4 7 3 Further risks infectious material Danger of infectious particles Work on infectious material e g viruses must be carried out under a sterile protective cover When working with infectious material the relevant safety regulations applicable must be observed Clean the working area of the microscope after use if necessary The cleaning agent should n
52. tion of the laser cutting lines is possible e g freehand select elements In Freeze Mode the fluorescence is deactivated as well as all functions to move the stage After han dling the frozen picture on the screen you can leave Freeze Mode to continue with other areas under fluorescence or carry out laser functions Note Before you switch on a laser function you have to turn the reflector revolver of the microscope into a position which has a suitable coating for this wavelength For basic fluorescence attach ments this is the first position only In Freeze Mode you can use the new software modules multichannel fluorescence and extended focus With the first module you can combine different fluorescence images and extract a calculated image on the user interface With the second module you extract an image out of a Z stack exposure For details see RoboSoftware manual PALM MicroBeam User Manual 0108 8 5 ApoTome ApoTome the innovative insert module from Carl Zeiss Microlmaging GmbH provides for very fast production of extremely high quality optical sec tions through fluorescence labeled biological spec imens with two and three dimensional visualization ApoTome offers better image qual ity contrast and resolution For instructions see Zeiss ApoTome User Manual Note If you use a Zeiss ApoTome advanced flu orescence is not possible Caution Make sure that the grid of the ApoTome is not w
53. tion vessel PALM RoboMover enables you to automatically position collection vessels above your specimen Various collectors are available containing one or more collection vessels up to 96 wells with PALM CapturePlate Collector 96 If you use collectors with more than one collection vessel you can automatically lift elements from your specimen into different collection vessels To do this you only need start the laser function once Note The following illustrations descriptions and instructions relate to PALM CapturePlate Collec tor 96 You can work in exactly the same way with all other collectors with more than one col lection vessel For collectors with only one col lection vessel the functions for collectors with multiple collection vessels will not apply Control of PALM RoboMover is incorporated in PALM RoboSoftware Refer to the software manual for PALM RoboSoftware for starting and quitting the program 7 3 1 Preparing collectors A collector system consists of at least two parts the frame and the insert in the example used here the CapturePlate 96 with 96 wells You will find a more detailed description of the collector you are using in the product information supplied with the collector There are different collectors available refer to Chapter 11 3 PALM RoboMover The caps or wells can be supplemented with different fillings for RNA DNA or protein extraction Choose the right collector to insert into t
54. training instruction or general education should only be permitted to Note work with the device while constantly supervised WHO recommendations concerning health and by an experienced person 4 6 Points requiring strict compliance 14 Caution Service works on the laser may only be per formed by Carl Zeiss Microlmaging GmbH or its representative When service is needed please contact palm techsupport zeiss de Protective equipment may not be circum vented removed or made ineffective under any circumstances All requirements regarding the condition of the device the qualification and behavior of the staff set forth in the User Manual have to be met Make sure that unauthorized persons cannot use the device Persons under the influence of alcohol other drugs or substances that affect their ability to react are not allowed to operate the device The device may only be used used when in per fect condition In the event of malfunctions or problems with the power supply and or dam age to the electric equipment the device must be shut down immediately Any malfunctions or damage indicated by the system or otherwise detected must be immedi ately reported and eliminated The device may not be modified Protective equipment e g protective filters or covers of laser beam outlets may not be dis mantled The device may only be operated if the entire protective equipment is functioning perfectly The app
55. ts once a week e Carefully wipe the optical parts with a cleaning tissue moistened with alcohol of 70 to 80 10 1 2 Cleaning when actually dirty Actual dirt e g caused by fingerprints or immer sion oil must be removed immediately or the optical functions will be permanently impaired Dirt that has been burnt onto the surface of lenses or filters by laser light can no longer be removed e Lightly moisten a cleaning tissue with pure alcohol 70 80 or spirit and wipe the dirty parts very carefully e Clean dry objectives and oculars gently with a cotton bud or a toothpick wrapped around with cotton wool Only use surgical cotton from the pharmacy e Remove cotton lint and fluff by blowing e g with an enema syringe Caution Moisten cleaning tissue only slightly do not soak Excessive solvent might dissolve the cement of the lenses Do not use acetone for cleaning under any cir cumstances 38 10 2 Control box Changing fuses There are two fuses located in the control box Je A Jo A 9 Fi F2 e O y a y e Co ee O Figure 24 Fuses Warning Before replacing the fuses the control box must be disconnected from the mains to pre vent electric shocks e Remove the fuse holder with a screwdriver e Replace the inserted fuses e Reassemble the holder Caution The replacement fuse must be identical to
56. turret and focused by the objective It is necessary to observe the following instructions or the laser beam might be blocked and performing microdissection will not be possible 20 3 reflector turret 4 filter slider Reflector turret turned to Laser The reflector turret must be positioned so that the beam splitter reflects the laser beam This position is labeled with 1 Laser see indication plates on the front of the microscope and the reflector turret must be set to position 1 accordingly If the fluorescence attachment option is installed it is possible to also use the installed beam splitters of the fluorescence fil ter sets only with advanced filter sets Do not insert any filters in the filter slider in the epifluorescence channel They might block the laser beam therefore put to a free slot left most position Note If the illumination arm of the microscope is pushed back the laser will be switched off immediately interlock This is a protective safety device which prevents you from looking into the laser beam PALM MicroBeam User Manual 0108 PALM MicroBeam Overview 5 5 Fluorescence attachment The fluorescence attachment expands the micro scope into a fluorescence microscope 1 filter wheel filter holder with shutter 2 fluorescence unit 3 control unit filter wheel 4 power supply fluorescence illumination Figure 10 Fluores
57. ughput applications up to 3 micro scope slides can be investigated at the same time something that is specially useful for serial sec tions Also different stage inserts like the SlideHolder 50x1 0 or 35mm and 50mm DishHolders for working with living cells or capil laries can be inserted by using stage inserts opti mized for microdissection Even microtiter plates according to the ANSI SBS 2004 standard can be 22 inserted into Stage II please contact palm info zeiss de for details For easy insert change a so called Load Position is defined In this position the stage insert or microtiter plate can be withdrawn without using force and any kind of holder then inserted A separate CapCheck position located on the right of Stage Il allows you to check the catapulted and captured samples with all collection devices Inserting a SlideHolder into Stage II The surface of the stage is flat and not reduced in size by motors or spindles There are two multiple SlideHolders available for up to 3 slides 1mm black anodized and 0 17mm achromatic anod ized that can be used in combination with PALM RoboStage II The slides have to meet the require ments of DIN ISO 8037 1 i e 25 26mm width and 75 76mm length PALM RoboSoftware 4 2 or a higher level is required Figure 12 Inserting a SlideHolder into Stage II e For loading start PALM RoboSoftware and wait until referencing of the stage has finished and it has reached t
58. ulting in an immediate stop of all motors and the issue of an error message PALM MicroBeam User Manual 0108 ns MA Figure 21 PALM MicroBeam fitted with PALM RoboMover What to do in an emergency oe e Click on the Stop button in the control soft ware or e switch off PALM ControlUnit In both cases movement is immediately aborted 31 PALM RoboMover 7 2 Starting PALM RoboMover Caution Before switching on the PALM ControlUnit please ensure that there is no solid object i e a condenser in the travel path of PALM RoboMover It is the user s own responsibility to take all appro priate precautions to minimize any danger from the use of PALM RoboMover Similarly PALM RoboMover can be damaged by improper use Caution Do not lean on any part of PALM RoboMover Do not use loads exceeding a weight of approximately 500g Moving parts must not be touched during operation During start up PALM RoboMover will be initial ized Note After switching on PALM RoboMover a collector that is already being used is not recognized automatically by the control software PALM RoboSoftware After switching on the device you must therefore run a scan to identify the collector Refer to chapter 7 3 1 Preparing col lectors for instructions on how to do this 32 7 3 Working with PALM RoboMover The LMPC laser functions allow you to lift dissec tions from your specimen into a collec
59. upply unit is defective PALM ControlUnit is defective nal or internal control and safety systems inter lock enable key switch in position 1 are plugged in Correction Let laser system cool down and switch on again Check reason for high temperature and make sure room cools down Check if INTERLOCK connection on Inter lock and the external control and safety systems are connected properly Contact Technical Service See No 1 Wait for 3 minutes until LED2 turns green and try again Laser diode driver is out of order Switch off and on PALM ControlUnit Try to start the laser again Contact Technical Service Check input voltage and power supply cable Check fuse beside the main switch Contact Technical Service Check external trigger pulses Contact Technical Service Check input voltage and power supply cable Check key switch position of PALM ControlUnit Contact Technical Service Contact Technical Service PALM MicroBeam User Manual 0108 Troubleshooting 9 2 PALM RoboMover No Error Correction 1 Error 14 collision in X Y axis Remove any solid part from the stage 2 Error 18 collision in Z axis Reference PALM RoboMover 9 3 No video image Check image in oculars no image check illumination of microscope Check position of main switch on camera power supply if applicable Check connection between camera and camera power supply if applicable Check
60. y defined cells living or fixed cell areas or subcellular par ticles from all kind of samples as used in pathol ogy oncology forensics botany or as needed for proteomics and cytogenetics as well as genetic engineering or other fields of biological medical or pharmaceutical research Microprocessing of non biological matter as for example laser beads or glass is also possible Important Basic Information 1 2 Legal notes LMPC is a trademark of Carl Zeiss Microlmaging GmbH PALM is a registered trademark of Carl Zeiss Micro Imaging GmbH Third party products are cited for information purposes only and this does not repre sent approval or recommendation of these prod ucts Carl Zeiss Microlmaging GmbH accepts no liability for the performance or use of such prod ucts Patent rights The instruments instrument components or meth ods described in this manual are protected by the following patents Laser catapult technology Laser Pressure Catapulting LPCPt Patents US 5 998 129 EP 879408 B1 JP 3311757 and others Three dimensional laser beam positioning system Patents US 5 689 109 EP 679325 Bi and others Element List Patent US 6 930 764 RoboMover Patent DE 102 54 229 5 Additional patents pending Carl Zeiss Microlmaging GmbH location Bernried is certified according to quality management sys tem ISO 13485 2003 and quality management system for medical products ISO 9001 Carl Zeiss M

PALM MicroBeam January 2008 User Manual

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