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OPERATION

Carl Zeiss

Checking and Adjusting the Laser Beam Divergence and the TIRF Angle

Laser TIRF 3

423683-7144-001

02/2014

4.5.3

Adjusting and Checking the TIRF

Angle Using the Adjustment Tool

Focus on the glass/medium interface of the

specimen (adherent beads) in epi-fluorescence.

Change to laser illumination and adjust 90°

angle of laser illumination in epi-fluorescence.

After having focused the specimen by means

of the TIRF objective, change to the adjustment
tool in order to adjust the angle.

Swivel the reflector module with the neutral-

density filter into the beam path.

Observe the live image of the camera.

Now, a white spot can be seen in the center of
the live image. This is the reflected laser beam.

Adjust the camera so that the resulting images
are not overexposed.

Change the TIRF angle of the Slider Laser TIRF:

−

The white spot moves upwards or
downwards, out of the center of the live

image.

−

To attain correct TIRF adjustment, the spot

must appear approx. 1 to 2 cm away from
the upper or lower edge of the live image

(Fig. 4-7).

−

Check the angle adjustment by means of
the bead specimen.

Swivel one of the line selection filters into the
beam path and observe the specimen.

In epi-fluorescence, the reflected laser beam is seen in

the center of the live image.

By changing the angle, the light beam travels upwards

(

) or downwards (

) from the center of the live

image. TIRF angle adjustment is attained in either
position (

Fig. 4-7

Presentation of the reflected laser
beam in the live image

Содержание TIRF 3

Страница 1: ...Laser TIRF 3 Imaging System Operating Manual...

Страница 2: ...ce Unless explicitly authorized dissemination or duplication of this document and commercial exploitation or communication of its contents are not permitted Persons in contravention of this copyright...

Страница 3: ...r safety Rev 2 31 2 6 Remote switch for laser mode 32 2 7 Laser Safety Concept 33 2 8 Configuration Overview of the Laser TIRF Imaging System 34 2 9 System Overview Laser TIRF Imaging System 35 2 9 1...

Страница 4: ...nce and the TIRF Angle 75 4 5 1 Delivery Scope of the TIRF Adjustment Tool Kit 75 4 5 2 Adjusting the Laser Beam Divergence Using the Adjustment Tool 77 4 5 3 Adjusting and Checking the TIRF Angle Usi...

Страница 5: ...afety Concept 93 6 2 Safety Kit for Customer Provided Laser Module 95 6 3 Remote switch for laser mode 96 6 4 Connecting the Remote Switch for Laser Mode 97 6 5 Basic kit laser safety Rev 2 98 6 6 Con...

Страница 6: ...g and information symbols explained below will help you recognize at a glance the type and purpose of the notes included in this operating manual CAUTION This symbol indicates a potential hazard to th...

Страница 7: ...scription blue edition Online version Operating manual for the installation of peripherals B 40 650 d Operating manual for the computer system Operating manual for HBO 100 B 46 0063 d reflected light...

Страница 8: ...ays keep the key for the laser key switch and where applicable the keys for any other laser starter switches in places which are not accessible to persons who are not authorized to handle the laser Do...

Страница 9: ...ning the device and replacing fuses Only use fuses for the given rated current which comply with the specifications on the fuse carriers and in this manual The use of makeshift fuses and short circuit...

Страница 10: ...he Laser TIRF imaging system is a high precision opto electronic instrument Improper handling of the instrument may easily affect its performance or even damage it and will void all warranty claims Mi...

Страница 11: ...r fall through the ventilation slots Health and safety protection regulations should be observed when handling the HXP 120 V UV radiation source Relevant national laws should also be complied with Fol...

Страница 12: ...rfaces Fig 1 1 Service interfaces between Laser Bench Tower and Slider Laser TIRF CAUTION The fiber mounts Fig 1 1 1 and 2 may be detached by Carl Zeiss Service staff only If one fiber end is not firm...

Страница 13: ...FRAP optional CAUTION The fiber mountings in the Laser Bench Rack Fig 1 2 1 on the laser manipulation slider Fig 1 2 2 and on the optional Laser TIRF Fig 1 2 3 and Cell Observer SD Fig 1 2 4 slider un...

Страница 14: ...e sideport of the microscope CAUTION The screws Fig 1 3 1 on the retaining ring Fig 1 3 2 for the camera adapter should only be unscrewed by Carl Zeiss service technicians Non observance involves the...

Страница 15: ...a adapter camera protective cover customer interface The camera adapter Fig 1 4 1 is equipped with an interlock connection two reflected light barriers which automatically closes the laser shutter as...

Страница 16: ...120 optical waveguide HXP 120 illumination unit CAUTION The screws Fig 1 5 8 of the safety clamp Fig 1 5 9 for the optical waveguide on the coupling adapter and the same safety clamp on the illuminat...

Страница 17: ...N The clamp screw Fig 1 6 1 is a safety screw and may only be unscrewed by Carl Zeiss Service technicians If the lamp housing is removed laser class 3B radiation may emerge Non observance involves the...

Страница 18: ...ning and information labels affixed to the Laser TIRF imaging system and Axio Observer microscope Please check that all labels shown below are actually in place Contact Carl Zeiss Microscopy GmbH if a...

Страница 19: ...Carl Zeiss 02 2014 423683 7144 001 19 Fig 1 8 Warning and information labels on the Laser TIRF imaging system with laser safety cover for labels on the microscope stand see Fig 1 7 Fig 1 9 Warning an...

Страница 20: ...DEVICE SAFETY Carl Zeiss Safety Instructions Laser TIRF 3 20 423683 7144 001 02 2014 Fig 1 10 Warning and information labels on the Laser Bench Tower...

Страница 21: ...VICE SAFETY Laser TIRF 3 Safety Instructions Carl Zeiss 02 2014 423683 7144 001 21 Fig 1 11 Warning and information labels on the Laser Bench Rack 3F Fig 1 12 Warning and information labels on the SVB...

Страница 22: ...vided for defects caused by natural wear wearing parts in particular and improper use The instrument manufacturer shall not be liable for damage caused by faulty operation negligence or any other tamp...

Страница 23: ...means of an electromagnetic evanescent field generated by total reflection Typically this field is generated at the transition from the cover slip to the medium and decays exponentially with the dista...

Страница 24: ...escent field is typically 200 nm thick The TIRF method is based on the behavior of light when making the transition between media with different refractive indices for example from glass to water When...

Страница 25: ...ackground fluorescence known from epi fluorescence is clearly reduced and image contrast considerably enhanced This results in a correspondingly high Z resolution Therefore the Laser TIRF imaging syst...

Страница 26: ...n ZEN software blue edition Vibration damped table or anti vibration plate for Axio Observer The Laser TIRF imaging system is optimized for polarization retaining monomode fibers When using fibers fro...

Страница 27: ...ngle adjusting screw Fig 2 2 Structure of Slider Laser TIRF man mot The laser light is introduced via an optical fiber The Laser TIRF imaging system includes a polarization retaining element for defle...

Страница 28: ...nes and the corresponding electronics are housed either in the Laser Bench Tower or in the Laser Bench Rack depending on the equipment version selected The Laser Bench Tower and Rack are connected to...

Страница 29: ...are connected to the signal distribution box see operating manual SVB 1 Fig 2 5 Signal distribution box 2 3 4 TIRF Controller The motorized Slider Laser TIRF is controlled via the TIRF controller The...

Страница 30: ...The laser safety cover for reflected light provided for mechanical and scanning stages may be used in combination with different stages To check compatibility please see the table in Section 2 9 1 on...

Страница 31: ...distribution box Fig 2 8 3 and 4 are used to connect the following components Type A connection Slider Laser TIRF remote switch for laser mode laser manipulation slider Type B connection Laser safety...

Страница 32: ...activated button not depressed LED Active not lit If the Laser Operation mode is activated the ocular shutter is closed The Safe Fig 2 9 2 LED indicates the status of the interlock connections The Saf...

Страница 33: ...t work position Slider Laser TIRF Transmitted light illuminator mount Eyepiece shutter Reflector turret Laser safety cover for reflected light Camera input on sideport Reflected light illumination con...

Страница 34: ...ast one option must be selected from each group 1 10 The components in frames are mandatory for each equipment A corresponding number of laser lines can be selected for the capacity of a laser module...

Страница 35: ...INSTRUMENT DESCRIPTION Laser TIRF 3 System Overview Laser TIRF Imaging System Carl Zeiss 02 2014 423683 7144 001 35 2 9 System Overview Laser TIRF Imaging System Fig 2 11 System overview page 1...

Страница 36: ...INSTRUMENT DESCRIPTION Carl Zeiss System Overview Laser TIRF Imaging System Laser TIRF 3 36 423683 7144 001 02 2014 Fig 2 12 System overview page 2...

Страница 37: ...INSTRUMENT DESCRIPTION Laser TIRF 3 System Overview Laser TIRF Imaging System Carl Zeiss 02 2014 423683 7144 001 37 Fig 2 13 System overview page 3...

Страница 38: ...INSTRUMENT DESCRIPTION Carl Zeiss System Overview Laser TIRF Imaging System Laser TIRF 3 38 423683 7144 001 02 2014 Fig 2 14 System overview page 4...

Страница 39: ...00 Specimen stage 250x230 432017 9901 000 Mechanical stage 130x85 R L long coaxial drive 432016 9901 000 Mechanical stage 130x85 R L short coaxial drive 432047 9901 000 Scanning stage 130x85 PIEZO 432...

Страница 40: ...9420 000 XLmulti S1 DARK LS incubator x x Compatible with 424533 9001 000 424533 9011 000 Definite Focus x x 423638 9002 000 423638 9012 000 423638 9092 000 CSU X1M 1800 spinning disc unit CSU X1M 500...

Страница 41: ...F LS LS LS LS Laser units 405 nm 445 nm 488 nm 515 nm 561 nm LS LS LS LS 400600 9120 000 Laser 638 nm 75 mW LS LS 423635 9001 000 Laser manipulation slider DirectFRAP x x x x all versions ApoTome ApoT...

Страница 42: ...timally suited for combinations with the following objectives Thread size Contrast Objective lens M27 W0 8 TIRF H Ph DIC ACR Objective Plan Apochromat 63x 1 46 Oil Korr M27 420780 9970 000 DIC slider...

Страница 43: ...M TBP 468 537 604 489072 0000 000 CFP YFP mRFP dsRED O 73HE DBP Filter set 73 HE CFP YFP shift free EX DBP 410 39 509 18 BS DFT 443 523 EM DBP 470 20 554 50 489073 0000 000 CFP YFP O 76HE TBP Filter s...

Страница 44: ...E CFP YFP DsRed shift free EX TBP 406 489 561 BS TFT 427 503 578 EM TBP 459 525 608 489076 9901 000 CFP GFP DsRed O 77HE TBP Filter set 77 HE GFP mRFP Alexa 633 shift free EX TBP 483 564 642 BS TFT 50...

Страница 45: ...5 608 HE 489076 9901 000 CFP GFP DsRed O 77HE TBP Filter set 77 HE GFP mRFP Alexa 633 shift free TBP 483 564 642 HE TFT 506 582 659 HE TBP 526 601 688 489077 0000 000 GFP mRFP Alexa 633 640 50 SBP Fil...

Страница 46: ...ser 488 nm 100 400600 9060 000 488 400620 9030 000 FS 52 HE 489052 0000 000 Laser 515 nm 20 400600 9070 000 514 400620 9040 000 FS 53 488053 0000 000 505 nm 423052 9061 000 Laser 515 nm 50 400600 9080...

Страница 47: ...g module Laser Bench Tower 177 mm x 448 mm x 462 mm Weight Axio Observer Z1 without external power supply unit approx 15 kg Slider Laser TIRF approx 570 g Signal distribution box approx 2 kg TIRF Cont...

Страница 48: ...cy 50 60 Hz Power consumption max 300 VA Laser Bench Tower coupling module Environment for use Closed rooms Protection class I Ingress protection rating IP 20 Overvoltage category II Line voltage rang...

Страница 49: ...IP 20 Overvoltage category II Line voltage range 100 V AC to 240 V AC 10 Change of line voltage is not required Line frequency 50 60 Hz Power consumption max 60 VA Fuses 2x T 2 0 A H 250 V VP 232 2 e...

Страница 50: ...in compliance with DIN EN 61010 1 IEC 61010 1 including CSA and UL directives Overvoltage category II TIRF controller with desktop power supply Line voltage 100 V to 240 V 10 Change of line voltage is...

Страница 51: ...3B Laser power Laser 405 nm 20 mW Laser 405 nm 50 mW Laser 445 nm 25 mW Laser 445 nm 40 mW Laser 488 nm 30 mW Laser 488 nm 50 mW Laser 488 nm 100 mW Laser 515 nm 20 mW Laser 515 nm 50 mW Laser 561 nm...

Страница 52: ...tted and adjusted in all cases by the Carl Zeiss Microscopy GmbH Service department or persons authorized by the manufacturer If used as intended see Section 2 1 on page 23 the Laser TIRF imaging syst...

Страница 53: ...outlets at a maximum distance of 1 5 m from the Laser Bench Tower or Rack are required to connect the system Use the three multiple socket outlets delivered to connect the system components see Fig 3...

Страница 54: ...Installation Requirements Laser TIRF 3 54 423683 7144 001 02 2014 Fig 3 2 Terminal assignments of the multipoint connectors for the Slider Laser TIRF Cell Observer SD optional and DirectFRAP optional...

Страница 55: ...Z1 431007 9901 000 and higher stand the basic laser safety kit for the Axio Observer Z1 432927 9011 000 stand is needed for this The manufacturer accepts no liability if the product is not used as sp...

Страница 56: ...icroscope Settings Be sure to read the safety data sheet provided with Immersol 518 F immersion oil Select suitable fluorescence filter sets see Section 2 11 on page 43 Then insert the selected reflec...

Страница 57: ...Fig 2 7 page 30 shows the components of the laser safety cover Screw the supplied interlock magnet Fig 3 3 5 into the subframe Fig 3 3 7 Place the cover frame Fig 3 3 3 over the subframe Screw the cov...

Страница 58: ...ation with different stages To check compatibility please see the table in Section 2 9 1 on page 39 Only condensers of NA 0 55 can be used Please bear in mind that the laser safety incubator S TIRF S1...

Страница 59: ...safety system when the transmitted light illumination carrier is tilted frontward and the cover is positioned on the incubator The distance between the interlocks and the condenser must be adjusted i...

Страница 60: ...rrier sealing adjustable 4 Handle to open the right lamp carrier sealing 5 Right upper hinge door access to the microscope stage and condenser 6 Right lower sliding doors access to objectives and filt...

Страница 61: ...ng station for TFT display 432907 9901 000 must be used All condensers in the Axio Observer range can be used CAUTION The red incubator type may be used up to a wavelength of 561 nm only Non observanc...

Страница 62: ...lar blue bar on the right side Fig 3 7 6 through 90 Fig 3 11 2 Pull the left slide in base plate forwards and the right plate to the side to remove Mount all required components such as the fixed micr...

Страница 63: ...fully guide the XLmulti S1 RED LS incubator without slide in base plates Fig 3 7 8 and 9 from above over the microscope and position At the front the incubator must rest on the two pins Fig 3 12 betwe...

Страница 64: ...d and secure using the black M8 knurled screw Then plug the hose with the adapter onto the upper air outlet of the XL S1 heating unit To prevent any unwanted vibration ventilator the XL S1 heating uni...

Страница 65: ...rt Mxx S1 heating insert xx 06 12 24 or 96 The incubator is lightproof and offers protection against laser radiation in the 350 nm to 760 nm wavelength range If this incubator is used the laser TIRF i...

Страница 66: ...r on the heating insert Remove the film from the adhesive strips and attach both magnets to the corners of the rectangle as shown in the diagrams below 1 Temperature sensor inside incubator 2 Heated c...

Страница 67: ...of the incubator In this case ensure that the middle of each magnet Fig 3 17 2 is directly opposite the red point Fig 3 17 1 otherwise the contact will not trigger The position of the magnets is shown...

Страница 68: ...the big screw Fig 3 18 3 by means of the small M3x8 screw Fig 3 18 1 with the aid of the T10 wrench Do not overtighten otherwise the magnet could be broken Apply the contact plug for laser safety to t...

Страница 69: ...on page 39 Only condensers of NA 0 55 can be used The red type of this incubator can be used up to 561 nm The black type DARK is usable up to 640 nm CAUTION The red incubator type may be used up to a...

Страница 70: ...ld diaphragm slider from the stand Carefully push the Slider Laser TIRF into the receptacle of the luminous field diaphragm of the microscope until it clicks into place Fig 3 20 Fix the Slider Laser T...

Страница 71: ...power lasers Ensure that you are not exposed to the radiation of such lasers In particular never look into the laser beam 4 1 Control and Functional Elements 4 1 1 Manual Slider Laser TIRF mot 1 Focus...

Страница 72: ...tch for laser mode When laser operation mode is activated the eyepiece shutter is closed The Safe LED Fig 4 1 3 indicates the status of the interlock connections The Safe LED lights up only if all int...

Страница 73: ...RATION Laser TIRF 3 Control and Functional Elements Carl Zeiss 02 2014 423683 7144 001 73 4 1 3 Laser Bench Rack 1 Key switch Laser ON OFF Fig 4 3 Control and functional elements of the Laser Bench Ra...

Страница 74: ...Switch on the ON OFF switch Fig 4 5 4 of the TIRF controller The four pilot LEDs Fig 4 5 1 2 3 5 light up The red and the yellow pilot LEDs Fig 4 5 2 and 3 will go out after about 5 seconds If the re...

Страница 75: ...on 4 5 Checking and Adjusting the Laser Beam Divergence and the TIRF Angle TIRF divergence is adjusted using the Focus rotary button Fig 4 1 1 on the Slider Laser TIRF The focus of the laser beam can...

Страница 76: ...the cover slip See the table below for use of the adapter rings Name Character istics Thread size Order number Pupil position OAF mm Delta Z for laser focus mm Adapter for adjustment tool Order numbe...

Страница 77: ...images are not overexposed 8 Change the TIRF angle so that the laser point appears in the center of the live image 9 Now swivel the divergence adjustment tool into the beam path You see now an enlarge...

Страница 78: ...n in the center of the live image This is the reflected laser beam Adjust the camera so that the resulting images are not overexposed 7 Change the TIRF angle of the Slider Laser TIRF The white spot mo...

Страница 79: ...nce of total reflection This is particularly beneficial for multicolor TIRF too Therefore move the beads or the object to be examined into observation position Focus on the surface of the cover slip e...

Страница 80: ...uorescing movable beads can now observed Change the TIRF angle slowly until you can no longer see the camera image A sharp contrast interface which marks the transition to total reflection will be vis...

Страница 81: ...stem before actually using it The purpose of calibration is to render the illumination angle and the desired penetration depth reproducible This basically means that after the calibration process the...

Страница 82: ...cent field under TIRF conditions is determined using the following equation 1 sin sin 4 2 1 2 0 g n d Where 0 488 nm 1 n 1 518 2 n 1 33 g 61 2 The calculated average refractive index of normal cover s...

Страница 83: ...h analogue potential from 10 to 10 Volts At 0 Volt the laser beam should ideally appear at 0 above the sample i e vertically above it Every angle in the sample therefore corresponds to a voltage Toler...

Страница 84: ...e calibration routine Once the critical angle is reached an exponential fall of the mean grey scale value can be observed in the rear objective pupil cf the three example images to the left The invers...

Страница 85: ...rs the instruments Do not use any solvents Dry the cleaned surfaces with a lint free cloth Cleaning of glass surfaces To remove dirt or fingerprints from glass surfaces use a clean optics cleaning clo...

Страница 86: ...rrier tilted backwards Bring transmitted light illumination carrier to vertical position Reflector turret not clicked into position or pulled out Click reflector turret into position or push it in No...

Страница 87: ...off the instrument by means of the power switch Disconnect the power plug Remove the fuse carrier Fig 5 1 1 by pulling it towards you You may use a small screwdriver if necessary Remove the fuses Fig...

Страница 88: ...lace relevant fuses 2x T4 0 A H 250 and 1x T1 0 A H 250 V Fig 5 3 2 and 4 Insert fuse carrier Connect the power cable 5 5 Changing the HAL Halogen Lamp The laser must always be switched off before cha...

Страница 89: ...4 2 is mounted CAUTION The camera adapter Fig 5 4 1 is firmly fastened to the sideport by means of a retaining ring Fig 5 4 3 and must not be removed Non observance involves the risk of eye damage Fi...

Страница 90: ...from the microscope or from the coupling connector see Fig 1 5 on page 16 Fig 5 5 HXP connection Switch off HXP 120 Remove the protective cap Fig 5 5 1 from the sensor ring which is fastened to the i...

Страница 91: ...performed only by the Carl Zeiss Microscopy GmbH Service or by explicitly authorized specialist staff To ensure optimum settings and trouble free functioning of your Laser TIRF imaging system over a...

Страница 92: ...id The manufacturer does not assume liability for any modifications to the Laser TIRF imaging system If the customer s own lasers are used the Laser Bench Rack 400615 9030 000 Laser Bench Tower 400615...

Страница 93: ...er in the correct work position Slider Laser TIRF Transmitted light illuminator mount Eyepiece shutter Reflector turret Laser safety cover for reflected light Camera input on sideport Reflected light...

Страница 94: ...USING THE SLIDER LASER TIRF WITH CUSTOMERS OWN LASERS Carl Zeiss Laser Safety Concept Laser TIRF 3 94 423683 7144 001 02 2014 Fig 6 2 Connection overview schematic...

Страница 95: ...r beam must be fully blocked by the shutter blade Fig 6 3 2 in the down currentless position Fig 6 3 Laser shutter converter board and connection cable included in the TIRF Safety Kit for customers ow...

Страница 96: ...t depressed Active LED not lit If laser operation mode is activated the eyepiece shutter is closed The Safe LED Fig 6 4 2 indicates the status of the interlock connections The Safe LED lights up only...

Страница 97: ...he Remote Switch for Laser Mode Connect one connector of the Y cable to the safety cable of the Slider Laser TIRF Fig 6 5 2 Connect the second connector of the Y cable to the remote switch for laser m...

Страница 98: ...r Fig 6 6 5 see Fig 6 7 on page 99 The four ports of the distribution box Fig 6 6 3 and 4 are used to connect the following components Type A connection Slider Laser TIRF remote switch for laser mode...

Страница 99: ...able of the door interlock switch to the plug and tighten the two screws Connect the distribution box for laser safety 1 Connection plug laser safety cover incubator 2 Connection plug laser shutter fo...

Страница 100: ...pin sub D ports on the distribution box Fig 6 7 6 as follows Laser safety cover or laser safety incubator Type B jack Fig 6 7 1 Laser shutter for customer s own laser module Type B connector Fig 6 7...

Страница 101: ...unit to the TIRF controller Fig 6 8 2 Fig 6 8 Connect Slider Laser TIRF mot 6 7 Coupling a Laser to the Laser TIRF Imaging System This step is necessary only if you intend to use your own laser modul...

Страница 102: ...using the threaded retaining ring Fig 6 9 5 Take care that the lug of the fiber connector is introduced horizontally into the Slider Laser TIRF Fig 6 9 C Fix the unscrew protection cap Fig 6 9 3 to t...

Страница 103: ...up does not light up Laser shutter closed Eyepiece shutter open Laser operation mode activated lights up lights up Laser shutter open Eyepiece shutter closed Open laser safety cover laser safety incu...

Страница 104: ...a or protective cover removed from the camera adapter lights up does not light up Laser shutter closed Eyepiece shutter open Reflected light lamp e g HXP 120 or the protective cover are not on the ref...

Страница 105: ...ystem components see Fig 5 10 The multiple socket outlets must not be connected in series Fig 6 10 Terminal assignments of the multipoint connectors The Laser TIRF imaging system must be connected to...

Страница 106: ...RS OWN LASERS Carl Zeiss Power Requirements Laser TIRF 3 106 423683 7144 001 02 2014 Fig 6 11 Terminal assignments of the multipoint connectors for the Slider Laser TIRF Cell Observer SD optional and...

Страница 107: ...for fluorescence HXP Mercury short arc lamp IEC International Electrotechnical Commission IP Ingress Protection protection rating for housing ISO International Standardization Organization LS Laser s...

Страница 108: ...lements 72 Laser Bench Rack 74 Laser Bench Tower 73 Customer interfaces 12 D Device safety 6 Dimensions 48 F Fluorescence filter set 44 Fuses Replacement 88 I Incubator 61 70 Installation instructions...

Страница 109: ...3 Operating principle 24 Operation 48 72 Optical fiber 47 R Remote control switch for laser operation 32 97 Connection 98 S Safety check 104 Sample compartment 30 Service interfaces 12 Service request...

Страница 110: ......

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