PALM MicroBeam User Manual
Contents
1. 1 filter wheel filter holder with shutter 2 fluorescence lamp 3 power supply 4 controller for filter wheel 5 especially designed beam splitter Figure 8 fluorescence attachment The fluorescence lamp consists of lamp housing lamp socket mercury vapor short arc lamp appropriate filter sets PALM MicroBeam User Manual 0202 Z 5 6 PALM RoboStage The computer controlled PALM RoboStage is a motorized specimen stage especially designed for high precision laser micromanipulation in sub micrometer range Its movement is carried out by two stepping motors at a speed of only a few p meters up to several mm per sec and displayed largely vibration free between 1um and approx 70mm The stepping motors are controlled by two encoders that are affixed below the stage 1 stepping motor Y direc 5 specimen slide tion 6 stepping motor X direc 2 microscope table board tion 3 holding frame 7 lever for cup height 4 snap on clip adjustment Figure 9 PALM RoboStage Several special functions are integrated to speed up routine work In PALM RoboStage there is a holding frame into which the specimen slides and or Petri dishes can be clamped Both transversal sides of the object slide are held by a snap on clip One longitudinal side of the slide entirely rests on the holding frame This reduces the risk that extremely thin slides will bend by the adhesion force of the immersion oil C2. kk KK K KIKI KK KIR KI ee 5 1 3 How You Gan Contact Ss a as Ay wo A Ay be ya E l be dvet a d A e a da dek Ae 5 2 Installations arie ii E O a A ds 6 3 Operating ROOM oa ica se aia A Ae ee eee ca 6 3 1 Admissible Ambient Conditions 0 000 kl a al sall eee eee 6 4 Safety ara ee Sige ah a A a 7 4 1 What You Should Do in an Emergency Case saa aaa ee 7 4 2 Observe the User Manual gt saser Ane lala a A a il a 7 453 kaser Safety s a ss Ax A NA 7 4 4 Safety Markings and Type Plate kk sk kk kk a a kU E a Sk kak ak kla kl Lal lk anal lk ka al ka 8 4 5 Qualification of the Staffi saire arua ia AWE n alan A A aya lh s r d Yasar A 9 4 6 Points that must strictly be observed kk kk a kk kK KK a ee 9 4 7 Pay Attention to Remaining Risks and Preventive Measures o ooo e 9 4 7 1 Remaining Risks and Protective Measures Laser ooo ooo 9 4 7 2 R m ining Risks Electricity saa an sya ii wa a hk A ea a T wees 9 4 7 3 Remaining Risks Infectious Material o ooo 9 4 7 4 Remaining Risks EMV E cc e ee Ge a 10 4 7 5 Remaining Risks Fire Extinction kk kk kk ooo 10 4 8 How to Ensure a Safe and Ecological Disposal o o o e 10 5 The PALMO MicroBeam Survey ss k kk kk kk ka kk ka a ak kk a a kk a a kk a a kk kk kk a 11 Sul PALMS Control ay aig san ane 40 Kl Aiy xo hk min n a
3. camper the mechanical manipulator can be used for cap movement via hand driven knobs With the mechanical manipulator you can clamp a take up instrument e g a capillary or a metal rod to hold conventional injection cannulae or the LPC instrument and move it in the x y or z direction For more details see the manipulator manual PALM MicroBeam User Manual 0202 Z The PALMO MicroBeam Survey CCD Video Camera 5 9 CCD Video Camera Pictures taken by a CCD camera are transmitted directly to the computer via a frame grabber The video transferred microscope image is dis played in real time on the monitor screen which allows to simultaneously observe the microscope image with stage or manipulator control functions You can store the transferred images on the hard disk of the computer or on another medium like CD The camera is preset by P A L M Microlaser Tech nologies AG For changing camera parameters please observe the manual of the camera Notice Depending of the Camera Adapter the com puter monitor only shows a section of the area visible in the ocular Caution The camera is aligned in parallel to the stage Don t turn or remove the camera in any way otherwise the calibration of the system is destructed and the laser light might leak out from the camera port Figure 12 CCD Video Camera PALMO MicroBeam User Manual 0202 Z 5 10 Computer The system is equipped with a state o
4. 4ya a h kal hak anln da ae l aka 18 6 2 CCD Video Camera Adapter ames ce 4 sa ala al a lk BVE ee aa an le Ok Ai kr et jk lal ala 18 6 3 Check of the Laser in the Microscope 4 ak a kk ak alla a lk lk a kk ekl ka aa lk ken la ak la 18 6 4 Laser energy and fOGUS 52 c s ya nm kal an d k bln dilan A al d k d l H d an ayak e Wl Wr de an 19 6 4 1 Laser Energy Adjustment a sia noaie k ala a al ld ah a ela a ll e lel lela lk 20 6 4 2 Laser FOCUS COrmection 2004 a neka Wa Aw ee da a ees oP 20 6 5 Adj st Laser FOCUS iay ka ark a y m A yi Mae Vi c an enya a Ge at itis SOG a Ha i a ee db b r la ra La tn 21 6 6 CUTE Speedas s Ak VE bab a biya a dy u A ur A n a BE HEW l j Gi zira Re ks R k een 22 PALM MicroBeam User Manual 0202 Z 3 List of Contents 7 PALMO MicroBeam with Fluorescence Illumination 000eeeeeeeeeeeeeees 22 7 1 Operate the Microscope with Fluorescence Illumination aaa 22 7 1 1 Simultaneous Fluorescence Observation kk kk kk kK KK KIRR K KIRI K KIR KI ee 22 8 Trouble Shooting List l l kk kak kk kk kk kaka sa a a a a a a a a a a a a a a a a aa a axaaa ka 23 8 1 Laser Is NOt CULEING tesina as ya A e BO sr 4 b q l eyane on K X d G Wed Kar aa Goka a Saale aaa o 23 8 2 N video IMAGE sa cs len kala a kad s eG d la a ey j a sal la
5. Therefore the excitation filter is mounted out side the laser beam path directly between the fluorescence lamp housing and the laser coup ling in the filter wheel 7 1 1 Simultaneou luorescenc bservation The beam splitter in the fluorescence slide switch directly in front of the objective in the reflector slide or in the fluorescence cube 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 the transmission of the emitted fluo rescence light Caution Do not use at any rate the beam splitters deliv ered by microscope manufacturers along with the filter because such beam splitters would be destroyed by the high energy of the laser If required please contact P A L M Microlaser Technologies AG PALM MicroBeam User Manual 0202 Z Trouble Shooting List Laser is not cutting 8 Trouble Shooting List 8 1 Laser is not cutting check energy and focus settings as described in the user manual if necessary take factory settings check red LED on Laser unit LED is on o k LED is off check key switch on laser unit check power connection of laser check green LED on Laser unit LED is on o k LED is off check function of safety switch in illumination arm of the microscope check position of adjusting aid for the fluorescence lamp see section 5 5 page 14 left position o k right position
6. be opened This can be done with the button FL on off When the shutter is open the sign FL appears on the display of the microscope e The filter revolver has to be in the position in which the beam splitter reflects the laserbeam 14 adjustment aid in left position filters and diaphragms open a filter f 5 revolver UZ Ze set to ed P Laser a 4 gt gt 1 FL shutter open gt FL on off O HAL on off 7 adjustment aid 8 two diafragms 9 filterslider This position is labeled with Laser In case of installed fluorescence attachment option it is possible to use also the installed beam splitters of the fluorescence filtersets only in case of original used P A L M beam splitters The two diaphragms in the epifluorescence channel must be open The adjustment aid for the fluorescence illumi nation has to be in the left position otherwise the laserbeam will be blocked Do not insert any filters in the filterslider in the epifluorescence channel They might block the laser beam Notice If the illumination arm of the microscope is pushed back the laser will be switched off immediately This is a security device which protects you from looking into the laserbeam PALM MicroBeam User Manual 0202 Z The PALM MicroBeam Survey PALMO RoboStage 5 5 1 Fluorescence attachment The epifluorescence attachment expands the microscope to a fluorescence microscope
7. electronic scrap Electronics as electronic scrap Cables as reusable material 10 PALM MicroBeam User Manual 0202 Z The PALM MicroBeam Survey How to Ensure a Safe and Ecological Disposal 5 The PALM MicroBeam Survey Modular Survey The PALMO MicroBeam Device consists of the parts shown in figure 3 1 microscope 2 CCD video camera 3 Imicroscope stage option 5 filter wheel option 6 fluorescence illumination 7 laser interface and laser 8 control unit 4 CapMover option Figure 3 Survey of the PALM Micro Beam PALM MicroBeam User Manual 0202 Z 9 monitor 10 power supply fluo illum 12 These parts and their function are described in detail in the following chapters 13 foot stepper 14 keyboard control unit filter wheel 15 joystick CapMover option mouse 16 computer 11 5 1 PALM ControlUnit The PALMS ControlUnit is the main device which runs and controls the movement of the PALMO RoboStage energy and focus unit and the laser The key switch also operates the mains for the single components of your system If you want to run single components as the computer for exam ple it is possible to connect these components directly to the mains The actual status of the control unit can be observed by LED s on the front panel LED color green yellow red indication status ready busy error OUT FO 10 30 0 50 70 Qo 9 J gt N 40V O 2
8. objective in the appropriate position Never insert reflecting objects in the laser beam path PALM MicroBeam User Manual 0202 Z How to Prepare the PALMO MicroBeam for the Application Laser energy and focus 6 4 Laser energy and focus To be able to precisely cut the material and reliably catapult it the laser focus has to be set accurately properly to optimal values under various condi tions The diameter of the cuts is dependent 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 The ablation occurs within the area of the laser focus please see figure 14a The smallest avail able cutting size can only be achieved within the center of the laser focus using as little energy as possible The catapulting is effected by a laser beam wid ened to the appropriate diameter with its focus Figure 14 Position of the laser focus when ablating or catapulting PALMO MicroBeam User Manual 0202 Z placed underneath the specimen to be catapulted please see figure 14b The laser beam is guided through the epifluo rescene channel of the microscope and is focused with the objective of the microscope Therefore the energy and the size of the laser beam depend on the kind of objective which is used with the device Generally the following is va
9. staff instructed in laser safety is allowed to stay in the hazardous area when the laser cas ing is open Warning The housing of the laser interface may only be removed by the service staff or an authorized representative of P A L M Microlaser Technolo gies AG Safety Safety Markings and Type Plate 4 4 Safety Markings and Type Plate Make sure that the following safety markings and the type plate exist and are legible Replace miss ing or illegible safety markings or type plate DANGE INVISIBLE LASER RADIATION AVIOD DIRECT EXPOSURE TO BEAM 337 nm 6 mw MAX AVG POWER 400uJ MAX ENERGY PULSE CLASS IIIB LASER PRODUCT Complies with 21 CRF 1040 10 and 1040 11 Figure 2 Safety Markings and Type Plate Caution The safety markings on the device must always be clearly visible and legible and must strictly be observed P A L M aser Technologies MicroBeam Manufactured Serial No o A y al A P A L M Microlaser Technologies AG Am Neuland 9 12 D 82347 Bernried Phone 49 8158 9971 0 www palm microlaser com Identification label PALMO MicroBeam User Manual 0202 Z Safety Qualification of the Staff 4 5 Qualification of the Staff The device shall only be operated by trained and specially instructed staff having the legal minimum age and having carefully studied this user manual Staff in the course of being taught trained instructed or being in a genera
10. switch to left check shutter for FL see section 5 5 page 14 shutter open FL on display o k shutter closed open shutter check position of filter revolver filter revolver on position laser o k any other position except you have switch to laser fluorescence equipment with P A L M simultaneous cutting check both sliders with diaphragm see section 5 5 page 14 diaphragm open o k check position of filter slider see section 5 5 page 14 one of the three open positions o k check position of the correction ring if available of the objective according to the thickness of the slide PALM MicroBeam User Manual 0202 Z 23 Trouble Shooting List No video image 8 2 No video image check image in oculars no image check illumination of microscope check position of main switch on camera s power supply if applicable check connection between camera and camera power supply if applicable check connection between camera and computer S VHS plug must be plugged in framegrabber board check position of switching mirror in microscope 8 3 Shift between drawing and cutting check alignment of camera the camera must be adjusted parallel to the stage movement test this by moving the stage with the arrow keys in a slow speed g check position of laser marker Calibrate new as described in the User s Manual make sure that you are using the settings for the active lens check LED s on encoder of moving stage LED is g
11. yal lar Dev a ea da Wun tea ew A i 24 8 3 Shift between drawing and cutting o ooo e KK KI KIR KK KK KK KK KK KK KK 24 9 Maintenance of the PALM MicroBeam kk kk kk kk kk kk a a a a a a a kk a a a kk a kk aa 25 Lo Ma AS r rc cc rc D 25 9 2 MiCEOSCODB 3 pru 5 3 3 a als She a k kul y A Ble A o Rig wen 25 9 2 1 Cleaning of the Optical Parts ooooooooorrrrr U ak lal ar al a tak Hel k kulk ala 25 9 2 2 Cleaning When Actually Dirty kk kK kk kK KI KK KI KK KIRI KK KK KI KK KK KK KK KK KK KK 25 10 Technical Data gas xa a cee eis a dk la lk Za al ar l je ea al a d j dak 26 10 1 Electricity 2 i a nar dia Ka aa nd nik YA A ar sn Eya A A ke E Se nadin bette ae A 26 10 2 Nitr gen LASER o a EO A A A ee ad 26 4 PALMO MicroBeam User Manual 0202 Z Important Basic Information Legal Notes 1 Important Basic Information Congratulations With your new PALM Micro Beam you obtained the State of the Art Technol ogy in microdissection and micromanipulation The PALMO MicroBeam is a high precision instrument which allows to perform microdissection in nearly every kind of research as well as in routine exam ination with a high throughput To meet a very high safety standard and to use all the benefits of your PALMY MicroBeam you should read this user s manual carefully and observe all instructions given in this manual Please follow also the instru
12. 4V O 5V O key switch Figure 4 PALM Control Unit After switching on the system with the keylock switch the three diodes for the voltage ranges 40V 24V und 5V should be illuminated Notice Shortly after switching on the PALM Control Unit the red LED turns off automatically Other wise switch off the PALMO ControlUnit and after 10 seconds switch it on again Should the LED not turn off please contact P A L M Microlaser Technologies AG 12 5 2 Laser Interface The laser interface is a one box device which houses the laser and all the necessary optics to guide the laser into the microscope brings the laser focus coincide with the optical focus of the microscope at the object plane and to adjust the laser energy Laser interface Figure 5 Laser Interface This is a prerequisite for precise laser micromanip ulations with highest precision Furthermore the laser position stays stable which allows to routinely work with the system without time consuming search for the laser beam focus Notice The correct laser focus and energy settings are done by the user please observe Software Manual It is also dependent on the thickness and absorption behavior of the specimen Caution Do not open the housing of the laser interface Invisible radiation of a laser class 3B might be guided unprotected to your eyes PALMO MicroBeam User Manual 0202 Z The PALM MicroBeam Survey Laser 5 3 Laser Th
13. CIN XW N A Figure 1 Dimensions Nature of the floor vibration free if not spe cial working table recommended and even Load carrying capacity for steel or granite table lt 400 kg Minimum space needed in total for steel or granite table about 140 x 90 cm or for the alu minum plate a table measuring at least 120 x 90 cm on which the system will be set up and with a load carrying capacity of 90 kg Power supply 115 230VAC 16VA safety fuse Notice Please consider also vibrations raising from your routine lab equipmemt 3 1 Admissible Ambient Conditions The divice may be operated at the following ambi ent conditions Ambient Temperature of the air 15 C to 25 C Air humidity 30 to 80 not condensing PALM MicroBeam User Manual 0202 Z Safety What You Should Do in an Emergency Case 4 Safety 4 1 What You Should Do in an Emergency Case e Switch off the device immediately and make sure that it cannot be switched on again e Unplug the multiple socket e Call the service staff in charge 4 2 Observe the User Manual This user manual contains important information concerning the safe operation of the device Only when strictly observing the user manual accidents can be avoided and a 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 use
14. Laser Focus The smallest laser focus diameter is obtained when using objectives of a high numerical aperture When using objectives with a lower numerical aperture the diameter of the laser focus increases correspondingly When working with objectives providing a short working distance the object slides must be thin ner 6 5 Adjust Laser Focus Notice For objectives with aperture diaphragm adjust the diaphragm to the thickness of the object slide or the Petri dish used to ensure an opti mum presentation focusing contrast Adjust the laser focus to the object setting accordingly With high numerical aperture use only the Petri dishes and object slides that are defined in this User Manual Notice Make sure that the membrane is always focused within the microscope focus otherwise the laser focus setting might be incorrect As the laser 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 to use an LPC object slide membrane coated for this purpose Notice Preferably use a LD 40x objective Make sure that the membrane in this area is tightly mounted onto the glass slide Look for areas where Newton s rings are visible rain bow colored striae Newton s rings indicate that the membr
15. Laser Focus Correction is done motorized A high precision DC stepping motor moves the cor responding lens Focus setting is conveniently con trolled and regulated on the monitor screen by means of the of the PALM RoboSoftware The dis play allows relocating selected laser focus settings Laser energy and focus is predefined by P A L M Microlaser Technologies AG over the factory set tings for each objective please refer to the PALMO RoboSoftware Manual Only the sample specific values have 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 Notice The automated laser focus and energy set tings have been adjusted to the objectives supplied by P A L M Microlaser Technolo gies AG all of a comparable lens configura tion Please contact P A L M Microlaser Technol ogies AG prior to the use of other objec tives For the catapulting and microdissection a LD 40x objective has proved successful in most of the applications 20 For the visualization of catapulted specimen with the LD 40x objective you need a cap with a flat non frosted clear surface and a rim which is not higher than 2mm available from P A L M Microlaser Technologies AG Figure 17 Laser focus below specimen plane PALM MicroBeam User Manual 0202 Z How to Prepare the PALMO MicroBeam for the Application Adjust
16. PALM Microlaser Technologies PALM MicroBeam User Manual PALM MicroBeam User Manual 0202 Z This document is delivered only to persons who are trained and authorized by P A L M Microlaser Technol ogies AG No part of this document may be reproduced or distributed in any form or by any means without the prior written consent of P A L M Microlaser Technologies AG The information in this document is subject to change without notice P A L M Microlaser Technologies AG assumes no responsibility for any errors that may appear Printed June 2002 Second Edition The following technologies have been patented by P A L M Microlaser Technologies Laser catapult technology Laser Pressure Catapulting LPCpat Patents US 5 998 129 EP 879408 B1 and others Three dimensional laser beam positioning system Patents US 5 689 109 EP 679325 B1 and others Additional patents pending P A L M Microlaser Technologies AG Am Neuland 9 12 82347 Bernried Germany Phone 49 0 8158 9971 0 Fax 49 0 8158 9971 249 E mail info palm microlaser com www palm microlaser com P A L M Microlaser Technologies AG 2001 All rights reserved 2 PALMO MicroBeam User Manual 0202 Z List of Contents Content Chapter Page 1 Important Basic Information 0 kk kk ka kk kk ka ak a a a a a a a 5 d l k al NObt Sres svt k ka ad A van M iets a a ha een ate Eee 5 1 2 Conventions for this User Manual
17. a new reference position The joystick serves to move the CapMover arm and consequently the cap in two dimensions The buttons have the following functions Left button If the button is pressed briefly the CapMover arm moves to the reference position that is currently saved By pressing the button a second time the arm moves back to the last position it was moved to If the arm has been moved from the reference position with the joystick then this position is accepted as the last targeted position If the button is pressed for a while then the cur rent position is saved as the new reference posi tion The LED will extinguish briefly as a confirmation signal Right button If the button is pressed briefly this will switch between the fast and slow speed ranges The LED will extinguish as a confirmation signal with each change of the speed range The faster speed range is suitable for examination at a low optical magnification factor whilst the slower speed range is useful in the case of high magnification Prolonged pressing of the button will cause the arm to move outside of the objective area for example in order to take up a new cap Speed change joystick and the functions of the left button are deactivated in this position Renewed activation of the right button will cause the arm to be retrieved and the cap to be moved to the saved reference position 5 8 Mechanical Manipulator Instead of the PALM
18. ane is not perfectly mounted Simultaneous stage movement will create a cut ting line within the sample This line will be fuzzy or fairly thick due to yet insufficient laser focus setting A star like three beam glass ablation around the laser focus indicates a good focussing 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 bigger pieces of glass will be blown out PALM MicroBeam User Manual 0202 Z Figure 8 and focus settings Find the correct focal plane in the following ways please refer to Part III PALM RoboSoftware Manual for using the graphic tools the automated laser functions and how to adjust energy and focus settings for the laser e use a plain membrane slide without tissue specimen e draw a zig zag line with the freehand graphic tool spanning at least the monitor screen e start automatic cutting with the Cut function e reduce the laser energy with the Page down key at the keyboard or with the slider for laser energy at 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 at the keyboard or the corresponding slider for laser focus at the mon itor screen to locate smallest cutting size e reduce the laser energy and repeat the above described procedure a second or third time until the smallest a
19. aution Do not move the stage manually Do not touch the moving stage Caution Avoid contaminating the guide of the stage with oil or chemicals Avoid any kind of mechanical stress e g putting heavy parts on it or leaning against as it is a precision instru ment Notice Do not clean the stage with alcohol 15 The PALM MicroBeam Survey PALMO CapMover 5 7 PALM CapMover The motorized PALM CapMover was developed for fast positioning with highest precision and quick exchange of the cap for sample collection The LPC LaserPressureCatapultingP t collection device can be mounted to the PALM CapMover This way the cap is positioned and moved via joy stick to check for the catapulted specimen 1 cap 2 cap mover 3 lever for cup height adjustment Figure 10 CapMover Manipulator 5 7 1 Control Apparatus At the front of the control apparatus there are two buttons a joystick and an LED The LED essentially indicates the operational readiness In the event of an error or a circuit overload this LED will extinguish In this case dis connect the apparatus from the power supply and ascertain the cause of the error You should not switch on the control apparatus until after having done this r Reference Slow Fast short El El long L_____ Set Ref Load Figure 11 Control Apparatus 16 Brief extinguishing of the LED also indicates a change of speed as well as the saving of
20. ctions for the single components of your PALMO MicroBeam especially for the micro scope the fluorescence attachment option the video camera and the laser P A L M Microlaser Technologies will not be responsible for mistakes and possible consequences caused by non obser vance of the users manual Please consider also the manuals provided for your periphery devices 1 1 Legal Notes P A L M Microlaser Technologies AG is not liable for damages resulting from the fact that this user manual was not or only partly observed The warranty extinguishes in case the customer opens the laser housing or uses the device for other than the specified purposes If used parts are replaced by similar parts which are however not identical to those recommended by the manufacturer in this manual P A L M Microlaser Technologies AG is only liable if the product supplied has already been faulty before the parts were replaced For details concerning the manufacturers war ranty please refer to the contractual conditions PALM MicroBeam User Manual 0202 Z 1 2 Conventions for this User Manual Special markings on the left margin point out information you should observe by all means Warning This sign warns you of possible most serious irreversible injuries to persons even going as far as to death Caution This sign signalizes possible moderate personal damage and possible damage of the device Notice This sign signalizes tha
21. e PALMO MicroBeam works with a pulsed UV laser wavelength 337nm of a high beam qual ity which is coupled via the epifluorescence path see Figure 7 Pos 6 into a research microscope and focused through the objective Depending on the applied objective the laser focus can reach less than one micrometer in diameter In theory the high beam quality of the laser allows to focus the laser beam to its diffraction limit However the cutting width is dependent on the laser beam itself the quality of the optics within the beam path the numerical aperture of the focusing objec tive and the absorption behavior of the specimen and on a correct laser focus setting Notice The correct laser focus setting is done by the user please see section 6 4 page 19 and is dependent on the focusing objective For cutting the stage is moved and not the laser itself PALM MicroBeam User Manual 0202 Z 5 4 Microscope Adapter The microscope adapter connects the laser to the microscope The laser beam is guided into the microscope and deflected into the objective 2 fluorescence illumination lamp option 3 Microscope adapter 1 filter wheel filter holder with shutter option Figure 6 Microscope adapter The especially designed beam splitter see Figure 7 Pos 2 allows to connect the fluorescence illu mination lamp and the filter wheel to the system and simultaneously use fluorescence illumination with laser ap
22. e dis mantled The device may only be operated if the entire protective equipment is perfectly working The application conditions and performance data defined in this user manual may not be al tered For Technical Data see section 10 page 26 PALMO MicroBeam User Manual 0202 Z 4 7 Pay Attention to Remaining Risks and Preventive Measures 4 7 1 Remaining Risks and Protective Measures Laser Remaining risks The laser works with invisible light When the casing is open the laser pertains to the laser protection class 3B The high power radiation leads to dimmed sight of the eye The casing must always kept closed and locked and may only be opened by the ser vice staff of P A L M Microlaser Technologies AG Unintentionally mirrored reflections must be avoided by a risk conscious behavior of the staff Protective measures taken by the manufacturer On the microscope lighting arm and the con denser if it can be swiveled out of the beam paths a safety switch is installed that shuts off the laser as soon as the lighting arm is swiv eled The travel paths of the laser beams as well as those of stage and CapMover are equipped with end switches to avoid damage 4 7 2 Remaining Risks Electricity Before cleaning current carrying parts make sure that the power has been switched off Even after switching off the electric energy supply life threatening voltage might occur in areas marked wi
23. es AG 6 1 2 Switch Off the Device e Exit WINDOWS e Switch off the ControlUnit e Switch off the multiple socket 6 2 CCD Video Camera Adapter Caution Do not touch the camera If camera position is changed new calibration is necessary please refer to the PALMO RoboSoftware Manual how to calibrate your system Notice Depending of the Camera Adapter the com puter monitor only shows a section of the area visible in the ocular 18 6 3 Check of the Laser in the Microscope The laser is coupled into the microscope via the epifluorescence beam path Prior to the check of the laser make sure that the aperture of the iris diaphragm in the epifluorescence beam path is completely open that there is no filter within the beam path of the laser that a possibly existing filter slide is set to free passage that the filter block or slider in front of the objective is switched into the correct position called laser FL on adjustment failed that the FL shutter in the microscope is open FL appears in the display adjustment aid is in left position Caution Only use the objectives labelled with a PALM logo for the laser operation Objectives designed for oil immersion or water immersion must be used with the respective immersion medium otherwise the objectives might be damaged Caution Do not turn the objective revolver during simultaneous laser operation Never use the laser without an
24. es Ate wane ee ed sw i 12 52 Laser Interface 440a a ca glade kk de AA j aa e ene ae jn TA eee we A er ris it 12 Bios U SO ak Sa dae Wata er a G l ade Gu ne dia do A kr a sun Waran AD id Susan Da do 13 5 4 Microscope Adapters cc vues als a dead ha l A ae e Wa ki ay Be a E a eae EE a 13 5 5 Microscope Zeiss AxiOvert 2003 sonic sx ka sar a s l So ries an ay sana Di e ande e eR deb aA ad de 14 5 5 1 Fluorescence attachment ss lt s4 k k a hal ae xd n ka dha a kla e ar a kal dik 15 56 PALMS ROBOS tages i yay ans Wla ls Gy es tek wid al o ye Su Waal a ad 15 5 77 TPALMS Ca MORE Sta a an key nA A kek E een aaa A ka a pears Eya 16 5 7 1 Control APparatus ia a s aka dank a a lk J na ea dee a A Sar ak ee Ve n 16 5 8 Mechanical Manipulator 0 3 a a a al ras la ala le we a db k ya ee we al A wd L 16 5 9 CCD Video Camera sani sets A al ai lb al ee le t ln a dya V l Tea ee SS Wl aly ad 17 SO O0mp l F54 555 a 3 x ro EKA Xak q rade ba Ba d r AL a ere PL ae Ten ae NA A NE Ser KK Y ae a 17 6 How to Prepare the PALM MicroBeam for the Application 00000ee0eeees 18 6 1 Switch On OfFthe Devices s xal aaa Skea lana dek din A a A ns Dae id Wa C 18 6 1 1 Switch On the Device 2 axe t e ww Ay Gy n kl a da Y id eae n J 18 6 1 2 Switch Off the Device
25. f the art computer The computer itself is provided with a CD burner and with an image handling software for easy image selection and editing as well as archiving With the integrated network card you have the possibility to connect your computer with your local network If you want to connect it please contact your local area network administra tor Notice It is not allowed to change or modify soft or hardware components on the computer P A L M Microlaser Technologies AG is not responsible for any damage resulting from dis obeying these points Any modification of the computer hard or software underlies to the prior written consent of P A L M In any case P A L M is not liable for any loss due to any kind of computer viruses 17 How to Prepare the PALMO MicroBeam for the Application Switch On Off the Device 6 How to Prepare the PALMO MicroBeam for the Application 6 1 Switch On Off the Device 6 1 1 Switch On the Device e Set the key switch of the ControlUnit to ON and switch on the computer for details see PALMO ControlUnit on page 12 The PALMO RoboSoftware will be loaded auto matically If not please see Users Manual Part III PALMO RoboSoftware how to start it Notice If the red system LED of the control unit doesn t turn off automatically please witch off the PALM ControlUnit and after 10 seconds switch it on again Should the LED not turn off please contact P A L M Microlaser Technolo gi
26. l education is only allowed to work with the device under permanent survey of an experienced person 4 6 Points that must strictly be ob served Caution Service Works on the laser may only be per formed by P A L M Microlaser Technologies AG or its representative When service is needed please contact the P A L M Microlaser Technologies AG Service Protective equipment may not be circum vented removed or made ineffective at any rate 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 The device may not be operated or maintained without wearing the prescribed personal pro tective equipment Please find the respective instructions in the relevant chapters in this manual Persons under the influence of alcohol other drugs or substances that affect the ability to react are not allowed to operate the device The device may only be used in a technically faultless condition In case of troubles or prob lems with the power supply and or damage of the electric equipment the device has to be shut off immediately Any malfunctions or damage indicated by the system or detected otherwise must immedi ately be notified and removed The device may not be modified Protective equipment e g protective filters or covers of laser beam outlets may not b
27. lid The higher the magnification aperture and UV Transmission of the objective the smaller the focus of the laser and the higher the energy 19 How to Prepare the PALMO MicroBeam for the Application 6 4 1 Laser Energy Adjustment The nitrogen laser emits a fixed laser energy For precise laser micromanipulation a precise laser energy adjustment is required A neutral density filter is mounted into the laser beam path which allows to continuously adjust the laser energy to the individual requirements without beam displacement This gradually coated filter is turned by a high precision DC stepping motor which is conveniently regulated on the monitor screen by means of the of the PALMO RoboSoftware please see the PALMO RoboSoft ware Manual 6 4 2 Laser Focus Correction Precise laser focus alignment allows laser cutting with a nearly diffraction limited laser focus The coincidence of the laser focus with the optical focus of the microscope is mandatory for precise laser micromanipulation Every wavelength is diffracted differently When performing laser micromanipulation at a greater distance from the specimen slide or within a thicker or strongly refracting object the offset of the laser focus position has to be compensated The same applies when using different objectives To compensate for the so called chromatic aberra tion the laser focus position has to be changed independent from the microscope focus The
28. plication Notice Due to the high power laser for simultaneous fluorescence illumination especially designed beam splitters have to be used For more infor mation please contact P A L M Microlaser Technologies AG Caution Do not touch or remove the screws on the microscope adapter The alignment of the sys tem would be destroyed and if the mirror is removed invisible radiation of a laser class 3B might be guided unprotected to your eyes 13 The PALM MicroBeam Survey Microscope Zeiss Axiovert 200 5 5 Microscope Zeiss Axiovert 200 1 display 4 fluorescence attachment option 5 microscope adapter 6 epifluorescence channel 2 special coated beam splitter 3 button for fluorescence shutter Figure 7 Survey of the Microcope The high optical and mechanical quality Zeiss inverse microscope Axiovert 200 is part of the PALMO MicroBeam This microscope can also be used as a normal research microscope Please observe the Axiovert 200 user s manual The laser beam is guided through the microscope adapter see section 5 4 page 13 into the epiflu orescence channel of the microscope It is reflected by a special coated beam splitter in the filter revolver and focused by the objective It is necessary to observe the following instruc tions otherwise the laserbeam might be blocked and performing microdissection will not be possi ble e The electromagnetic fluorescence shutter in the microscope has to
29. r manual must be stored close to the device at a place accessible and known to the staff where it is available at any time This user manual must be read understood and observed in all points by the responsible and oper ating staff Please observe all safety instructions and other information given in the user manual and the attached manuals of the accessories Caution Any use other than the ones defined in this user manual might lead to personal injuries or material damage which P A L M Microlaser Technologies AG is not liable for 4 3 Laser Safety The PALMO MicroBeam is a laser device of Class 1 M with no special safety requirements if operated according to the instructions given in this user man ual Built in is a Laser of class 3B It bears a potential risk A direct or reflected beam must not strike the unprotected eye do not look directly into the beam In service case the device has to be operated by the service staff or an authorized representative of P A L M Microlaser Technologies AG even with the laser casing open The customer must take the following safety mea sures in order to prevent any direct looking into the beam or any uncontrolled mirrored reflections PALMO MicroBeam User Manual 0202 Z The laser may only be operated in a surveyed area In case there are other work places located in the operating room the device must be shielded by laser protective walls during service works Only
30. reen o k LED is orange or red please contact P A L M Microlaser Technologies AG 24 PALM MicroBeam User Manual 0202 Z 9 Maintenance of the PALM MicroBeam Caution Before starting any maintenance works dis connect all system components from the mains 9 1 Laser See Laser Manual 9 2 Microscope For detailed actions see Microscope Manual 9 2 1 Cleaning of the Optical Parts e Clean all easily accessible optical parts once a week e Carefully wipe the optical parts with a Kleenex tissue moistened with alcohol of 70 to 80 9 2 2 Cleaning When Actually Dirty Actual dirt e g caused by finger prints or immer sion oil must immediately be removed otherwise the optical functions will permanently be impaired Dirt on surfaces of lenses or filters burnt in by laser light can not be removed any more e Slightly moisten a Kleenex tissue with pure alcohol 100 or spirit and wipe the dirty parts very carefully e Clean dry objectives and oculars smoothly with a cotton bud or a tooth pick padded with cotton wool Only use surgical cotton from the phar macy e Remove cotton fluffs and similar by blowing with a small pair of bellows such as an enema syringe Caution Moisten cleaning tissue only slightly do not soak Excessive solvent might dissolve the cementing of the lenses Do not use acetone for cleaning at any rate 25 PALMO MicroBeam User Manual 0202 Z Technical Data Elect
31. ricity 10 Technical Data 10 1 Electricity Input voltage Voltage fluctuations Frequency Frequency fluctuations 10 2 Nitrogen Laser 26 Wavelength Pulse energy Pulse duration Pulse frequency Beam divergence Laser control Pulse frequency of laser control 115 230 V AC 5 50 60 Hz 2 Hz 337 nm gt 270 y 3 nsec 1 30 pulses per second 0 3 mrad Laser control can be changed by impulse packages external by earth free foot stepper or via RS232 interface automatic permanent impulses Impulse width 0 1 ms Adjustable via computer PALMO MicroBeam User Manual 0202 Z
32. t information is given demanding your special attention Info This indicates tips on correct use and offers useful notes for getting the best use of the soft ware 1 3 How You Can Contact Us Business hours 9h 15 00 h Mo Fr Phone 49 0 81 58 99 71 100 Fax 49 0 81 58 99 71 249 E mail Info palm microlaser com support palm microlaser com Service Address P A L M Microlaser Technolo gies AG Am Neuland 9 12 82347 Bernried Germany Homepage www palm microlaser com Installation 2 Installation Your PALM MicroBeam is only to be installed by Palm service engineers or their representatives In case you want to move an already installed unit please contact P A L M Microlaser Technologies for assistance The PALMO Laser Systems basically are mainte nance free Necessary service actions are the exchange of the laser cartridge which has a garan teed lifetime of two years or 20 10 pulses In case of any malfunction of the device please con tact the PALM Service see section 1 3 page 5 3 Operating Room In order to guarantee a correct function and com fortable work with your new PALMO MicroBeam System the following requirements has to be paid attention The system should be installed in a room which is adequately large about 6 square meters and well aired There should be a free space available about 90cm above the table and all shelves wall cup boards etc should be removed SN
33. th an electric flash as well as in the fol lowing supply systems dangerous areas Process computer DC power unit Laser fan Stepping motor control Network feed lines 4 7 3 Remaining Risks Infectious Material Danger of virus particles due to virus contami nated tissue Works on virus contaminated tissue must be car ried out under a sterile protective cover When working with infectious material the respectively applicable safety regulations must be observed Clean the working area of the microscope after use if required The cleaning agent should neither be abrasive nor strongly basic or acid as this would attack the protective coating 4 7 4 Remaining Risks EMV Regarding interference resistance the device meets the protection requirements according to DIN EN 61000 6 2 Regarding interference emission the device meets the protection requirements according to DIN EN 55011 Do not operate any devices emitting high fre quency radiation in the same room where the PALM MicroBeam is placed 4 7 5 Remaining Risks Fire Extinction To extinguish fire use sand carbon dioxide or fire extinguishing powder Never ever use water because of the electric dangers 4 8 How to Ensure a Safe and Ecological Disposal Dispose the following materials in an environmen tally friendly way Microscope lamps with incorporated bulb or individual mercury vapor short arc lamps as hazardous waste Laser as
34. vailable focal spot is found The perfect laser focus setting is achieved when with a minimum of laser energy a fine cutting line is visible which immediately disappears as soon as the laser focus is slightly shifted up or down The cutting edges have to show identical symme try when the object is moved into x or into y direction With increasing energy the membrane margins should still appear symmetrically Notice For each type of sample the laser focus adjust ment has to be performed individually The individually determined values can be stored in a separate file 21 PALM MicroBeam with Fluorescence Illumination Cutting speed 6 6 Cutting speed The microdissection of big areas usually should be performed with a low magnification objective at comparatively high energy values with a high cut ting speed These parameters automatically yield a broad cutting line To reach a fine cutting line it is necessary to opti mize the laser energy with regard to the cutting speed as the laser pulse repetition rate is fixed at 30 pulses per second 22 7 PALM MicroBeamwithFlu orescence Illumination 7 1 Operatethe Microscope with Fluores cence Illumination The epifluorescence equipment is attached with clamping screws to the nozzle of the illumination insert at the back of the microscope Notice Make sure that there is no filter within the beam path of the laser that could block the laser transmission
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