Differential Interference Contrast Microscopy
Phase Contrast Microscopy
Microscopy in Combination with Epi-fl Attachment
Bright field Microscopy
System Condenser
Components for DIC Microscopy
Components for Ph Microscopy
Microscope Assembly
Mounting Epi-fl Attachment
System Condenser Assembly
Mounting the System Condenser
Mounting the DIC Sextuple Nosepiece 
Mounting the Objective DIC Prism
Mounting the Objective
Mounting the Analyzer Slider
Installing the Polarizer
Mounting the Lambda-plate
Troubleshooting Tables

Differential Interference Contrast Microscopy

Key Points of Microscopy

1. Use slide and cover glasses that are not deformed and that are free of dust or dirt.  Use a slide glass that is between 0.8 and 1.2 mm thick, and a cover glass that is 0.17 mm thick.
2. When using the system with an oil-type top lens mounted  on the system condenser, apply immersion oil between the top lens and the slide glass.  With the oil, the N.A. of the condenser lens become 1.4, making it possible to get optimum performance from the lens.   In addition, contrast is further improved in DIC microscopy.  
3. For the DIC method, the adjustments made in steps 6 and 7 below are especially important.  If these adjustments are not made properly, viewing will be poor.  
4. If the VFM epi-fluorescence attachment is also mounted on the microscope, see section "Microscopy in Combination With Epi-fl Attachment."

When using an oil-immersion type 10×DIC objective for DIC observation, there may be some unevenness in the view field.  

1. E800: Turn on the power switch of the power supply, set the sub-power switch of the microscope to the "DIA" to turn on the lamp that provides diascopic illumination.
   
   E600: Turn on the power switch of the microscope to turn on the lamp.
   
   
   
   
   
   
   
   
   
2. Insert the polarizer and the analyzer into the optical path. 
   
   
   
   
3. Rotate the condenser turret so that the "A" (empty position) indication is at the front.
   
   
   
   
   
   
   
   

4. Place the specimen on the stage, and focus on the specimen with the 10× objective.  (See the manual provided with the microscope).
   

5. Adjust the diopter and the interpupillary distance.  (See the manual provided with the microscope).

6. Center and focus the system condenser.  These adjustments are very important.  Do not skip this step.  (See "Focusing and Centering the System Condenser").
   
   
   
   
   
   
   
   
   
7. Adjust the orientation (direction of vibration) of the optical system.  This adjustment is very important.  Do not skip this step.
    - The analyzer slider of the  E600 does not have a rotating mechanism, so adjust only the polarizer. (See "Optical System Orientation Adjustment").
   
   
   
   
   
   
8. Move the stage and center the portion of the specimen to be viewed in the view field. 
   
9. Insert a DIC objective into the optical path.
   
   
   
   
   
   
   
   
   
   
   
   
   
10. Rotate the condenser turret so that the indication that matches the indication ([L], [M] or [H]) on the objective is at the front.  
    
    
    
11. If an oil-type lens is mounted on the system condenser, apply immersion oil between the specimen and the top lens. 
    
12. When using an oil immersion type objective, apply immersion oil between the specimen and the objective.  (See manual provided with the microscope).
    
13. Adjust the aperture diaphragm and the field diapragm.  

• Generally, stop down the aperture diaphragm to 70 to 80% of the N.A. of the objective. 
• Stop down the field diapragm so that it is just inside or just outside the view field.  (See the manual provided with the microscope).

14. Loosening the polarizer rotation knob and rotating the polarizer changes the image contrast. 
    

Color Contrast Microscopy

1. Perform steps 1 to14 of the DIC microscopy procedure. 
2. Insert an NCB filter into the optical path.
3. Remove the GIF (green interference) filter from the optical path to allow illumination by white light.
     The E600 does not have a GIF (green interference) filter, so this operation is omitted.
4. Insert the lambda-plate (sold separately) in the bottom of the system condenser.
5. The above procedure makes the background color of the view field sensitive, enabling observation with high contrast.  (If there are variations in the refractive index or thickness of the specimen, interference colors will appear according to the gradient of those variations).

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Phase Contrast Microscopy

Key Points of Microscopy

The appearance of a Ph image depends on the phase difference and shape of the specimen, the characteristics of the objective, etc. Keep the following points in mind when preparing a specimen and when selecting the Ph objective.  If the DIC system or Epi-fl system is also mounted on the microscope, see section "Microscopy in combination with Epi-fl attachment."  Please note that with the combination of components described in this manual, you cannot perform Ph microscopy with the oil-type lens attached to the system condenser.

1. Select a specimen that will adversely affect the centering of the Ph annular diaphragm. 
   Specimens that scatter light or produce a prism or lens effect adversely affect the centering of the Ph annular diaphragm.  Especially when viewing a thick, live specimen, a large, coarse specimen or a specimen prepared with a microplate, care must be taken as the centering of the Ph ring diaphragm is shifted by a lens or prism effect, resulting in poor viewing.  
   
2. Select a specimen suited for the latitude and contrast of the objective.
   When using a dark contrast Ph objective, make sure that the phase difference of the specimen does not exceed the latitude (phase difference tolerance) of the objective.  If the phase difference of the specimen exceeds the latitude, the image will appear brighter than the background, making observation impossible.  When preparing a phase contrast specimen, the phase difference can be adjusted through the thickness of the specimen and the refractive index of the filling agent, the culture solution, etc. If the contrast of a specimen observed with a DLL objective is low, better results may be obtained with a DM objective. 
   
3. Stained specimens
   Specimens with a high contrast or stained too dark are not suitable for Ph microscopy.  Ph microscopy is suited for lightly stained specimens, de-colored specimens, or ultra-thin specimens for electron microscopes. 
   
4. Ph annular diaphragm centering
   In Ph microscopy, the exact alignment of the objective phase plate and the image of the Ph annular diaphragm in the system condenser is extremely important in order to maintain the phase contrast effect.  Re-check this alignment before starting microscopy.

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1. E800:  Turn on the power switch of the power supply, set the sub-power switch of the microscope to "DIA" to turn on the lamp that provides diascopic illumination.  
   
   E600:  Turn on the power switch of the microscope to turn on the lamp. 
   

2. Rotate the condenser turret so that the "A" (empty position) indication is at the front.
   
   
   
   
   

3. Place the specimen on the stage, and focus on the specimen with the 10× Ph objective (Ph1).  (See manual provided with the microscope.)

4. Adjust the diopter and the interpupillary distance.  (See the manual provided with the microscope).

5. Center and focus the system condenser.  These adjustments are very important.  Do not skip this step. (See "Focusing and Centering the System Condenser").

6. Rotate the condenser turret so that the "Ph1" indication is at the front. 
   
7. Open the condenser aperture diaphragm all of the way.  (Always leave the condenser aperture diaphragm fully open during Ph microscopy.)
   
8. Insert a GIF (green interference) filter in the optical path.
     The E600 does not have a GIF filter, so this operation is omitted.
   
   
   
   
   
   
   
   
   
9. Center the Ph annular diaphragm.  This adjustment is very important.  Do not skip this step.(See "Centering the Ph Annular Diaphragm").
   
   
10. Adjust the field diaphragm so that it is just inside or outside of the view field.  (See the manual provided with the microscope.)
    
11. If the objective has been switched, also switch the condenser turret in accordance with the Ph code of the objective.  After doing so, always center the Ph annular diaphragm.  (See "Centering the Ph Annular Diaphragm").    Also re-adjust the size of the field diaphragm.
    
12. When using an oil immersion type objective, apply immersion oil between the specimen and the objective.  (See the manual provided with the microscope.)

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Microscopy in Combination with Epi-fl Attachment

It is possible to combine DIC microscopy (or Ph microscopy) with Epi-fl microscopy by mounting both the components for the DIC microscopy (or the Ph microscopy) and Epi-fl attachment on the microscope.  For example, use the DIC method (or the Ph method) instead of the Epi-fl method (which causes colors to fade) to search for the target portion of the specimen.  It is also possible to use both methods simultaneously in order to compensate for their individual shortcomings.  For details on the microscopy procedure when using the Epi-fl method, see the manual provided with the Epi-fl attachment; for details on the microscopy procedure when using the DIC method or the Ph method.

1. Switching the methods
   Perform the following steps when switching between the different microscopy methods. 

When switching to DIC microscopy

• Move the shutter on the Epi-fl attachment into the optical path to block off the light source for epi-fl microscopy. 
• Move the diascopic filter block (DIA ILL) into the optical path.
• Insert the analyzer into the optical path.
• Swing the polarizer into the optical path.
• Bring a DIC objective into the optical path. 
• Insert the objective DIC prism into the optical path.
• Rotate the condenser turret so that the indication that is the same as the indication on the objective is at the front.  
• Adjust the size of the aperture diaphragm (normally, to about 70 to 80% of the N.A. of the objective). 

When switching to Ph microscopy

• Move the shutter on the Epi-fl attachment into the optical path to block off the light source for epi-fl microscopy. 
• Move the diascopic filter block (DIA ILL) into the optical path. 
• Remove the analyzer from the optical path. 
• Swing the polarizer out of the optical path. 
• Bring a Ph objective into the optical path. 
• Rotate the condenser turret so that the indication that is the same as the Ph code of the objective is at the front. 
• Open the aperture diaphragm completely.  

When switching to Epi-fl microscopy

• Turn off the microscope's diascopic illumination lamp.  (Viewing is difficult if the lamp is left on.)
• Remove the shutter on the Epi-fl attachment from the optical path.  
• Bring the desired filter block into the optical path.  
• Remove the analyzer from the optical path. 
• Remove the objective DIC prism from the optical path.  (Only when using a DIC objective.)
• Adjust the size of the aperture diaphragm (normally, to about 70 to 80% of the N.A. of the objective).

2. Simultaneously microscopy
   When using both the DIC method (or the Ph method) and the Epi-fl method simultaneously, follow the procedure described below. 

• Use the DIC method (or the Ph method) to find the portion of the specimen to be observed.  
• If there is a green interference (GIF) filter in the optical path for diascopic illumination, remove the filter from the optical path. 
•  Bring the desired excitation filter block into the optical path. 
• Open the shutter on the Epi-fl attachment and recheck the focus. 
• Use the ND filters of the Epi-fl attachment to adjust the brightness of the fluorescent image. 
• Use the microscope's ND filters to adjust the brightness of the DIC image (or the Ph image). 

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Bright field Microscopy

For bright field microscopy, perform the steps described below.  For details on the microscopy procedure, see the manual provided with the microscope. 

• Rotate the condenser turret so that the "A" (empty position) indication is at the front.
• If the polarizer, analyzer, or objective DIC prism is in the optical path, remove it.  Since removing any of these will increase the illumination, adjust the brightness by inserting ND filters into the optical path. 
• When using a 2× or 4× objective, swing out the top lens and fully open the field and aperture diaphragm during observation. 

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OPERATION OF EACH PART

System Condenser

1. When using a 2× or 4× objective
   When using a 2× or 4× objective, hold the top lens swing-out knob and quietly push the slider to the left, to swing out on the top lens.  (Do not move the swing-out slider quickly.  Fully open the filed and aperture diaphragm when the top lens is out of the optical path.)  Doing so expands the illuminated area so that the observation using a 2× or 4× objective is possible.  (Do not swing out an oil-type top lens, since doing so will not only spill the immersion oil but may damage the specimen by pushing it up from the bottom since the distance between the top lens and the specimen is very short.)
   
2. Focusing and centering the system condenser
   Focus and center the system condenser, referring to the section on focusing and centering the condenser in the manual provided with the microscope.  When doing so, keep the following points in mind. 

• Keep the top lens in the optical path; do not swing it out of the optical path.  If the top lens is swung out of the optical path, the field diaphragm image will not be focused on the specimen surface.  
• Rotate the condenser turret so that the "A" (empty position) indication is at the front. 
• If the polarizer, analyzer, or objective DIC prism is in the optical path, remove it.  Since removing any of these will increase the illumination, adjust the brightness by inserting ND filters into the optical path. 

3. System condenser aperture diaphragm adjustment
   Adjust the size of the aperture diaphragm, referring to the section on the aperture diaphragm in the manual provided with the microscope.  Normally, the diaphragm should be closed to 70 to 80% of the N.A. of the objective.  When doing so, keep the following points in mind.

• Keep the top lens in the optical path; do not swing it out of the optical path.  If the top lens is swung out of the optical path, the aperture diaphragm will not function. 
• For Ph microscopy, open the aperture diaphragm all the way.  If the aperture diaphragm is closed at all, the Ph annular diaphragm may be blocked, making Ph microscopy impossible. 

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Components for DIC Microscopy

1. Polarizer

• To remove the polarizer from the optical path, swing the upper part of the polarizer out of the optical path.
• The polarizer can be rotated by loosening the polarizer rotation knob; the background color can be changed by rotating the polarizer.  The polarizer rotation knob can be used in either of the top tap holes on the front of the polarizer. 

2. Analyzer

• Insert the analyzer into the optical path by pushing it into the second click position.  To remove the analyzer from the optical path, pull it out. 
• The analyzer can be rotated by loosening the analyzer rotation clamp screw and moving the rotation knob; normally, the analyzer is clamped in position at the index. 
  -- The analyzer slider of the E600 does not have a rotation mechanism.  

3. Optical system orientation adjustment (adjustment of direction of vibration)
   Adjust the orientation of the polarizer and the analyzer so that they are perpendicular.  Perform this adjustment carefully, since it determines the basic performance of the DIC method. 

1. Focus the center of the system condenser (See "Focusing and Centering the System Condenser").
2. Focus on the specimen. (See the manual provided with the microscope.)
3. Insert a 40× objective into the optical path, and rotate the condenser turret so that the "A: (empty position) indication is at the front. 
4. Remove the objective DIC prism, located above the 40× from the optical path.  
5. Insert the analyzer into the optical path.  
6. Loosen the analyzer rotation clamp screw and move the rotation knob to align the analyzer with the index, and clamp it in place. 
   -- The analyzer slider of the E600 does not have a rotating mechanism, so this operation is omitted. 
7. Swing the polarizer into the optical path. 
8. Loosen the polarizer rotation knob, align the upper part of the polarizer with the index, and clamp it into place. 
9. Remove the specimen from the view field.
10. Adjust the angel at which the polarizer is mounted on the microscope.
    Method 1: Use a hexagonal screwdriver to loosen the polarizer clamp screw.  Next, while looking through the eyepieces, rotate the entire polarizer and then fix it in place at the position where the view field is darkest.
    Method 2:  Remove an eyepiece, and then use an adapter (sold separately) to mount a centering telescope (sold separately).  Close the aperture diaphragm to a minimal opening, and then turn the eyepiece of the centering telescope so that the aperture diaphragm is brought into focus.  Open the aperture diaphragm as far as it will go.  Loosen the polarizer clamp screw with a hexagonal screwdriver.  Next, rotate the entire polarizer and then fix it in place at the position at which the dark cross images become visible.
11. Install an objective DIC prism e for a 40× objective) in the revolving nosepiece. 

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Components for Ph Microscopy

1. Ph annular diaphragm
   Ph microscopy is performed by matching the system condenser Ph annular diaphragm with the objective phase plate. 

Ph Code
One of the phase codes (Ph1, Ph2, or Ph3) is displayed on a Ph objective, depending on the size of the phase plate.  (The Ph code has no bearing on the magnifying power of the objective.)  Rotate the system condenser so that the Ph annular diaphragm with the same code is in the optical path.  Ph microscopy is not possible if different codes are used in combination. 

Centering the Ph annular diaphragm

1. If the aperture diaphragm is closed at all, open it fully. 
2. Remove an eyepiece, and then use an adapter (sold separately) to mount a centering telescope (sold separately).
3. Turn the eyepiece of the centering telescope so that the images of the objective phase plate and the system condenser Ph annular diaphragm image are brought into focus.  
4. If the images of the phase plate and the Ph annular diaphragm image are brought into focus.  If the images of the phase plate and the Ph annular diaphragm are not in alignment, use two hexagonal screwdrivers to adjust the Ph ring diaphragm centering screws on the condenser turret so that the centers of the two images are aligned.  (Insert the screwdrivers in the screw holes at the rear side of the stage.) 
   Note that if the image of the Ph annular diaphragm extends beyond the phase plate, the phase image contrast deteriorates.  If the stage handle is in the way, loosen the stage rotation clamp screw and rotate the stage slightly. 

Ph objective
The Plan Fluor Ph objective can be used for bright field microscopy, DIC microscopy, and Epi-fl microscopy.  The Plan Apochromat Ph objective can be used for bright field microscopy, DIC microscopy, and Epi-fl microscopy, excluding UV excitation.  However, because both have a phase plate inside, the "view" may differ from an objective intended specifically for the microscopy method in question.  
For the absolute best results, use an objective intended specifically for the microscopy method in question. 

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ASSEMBLY

Microscope assembly
Assemble the microscope as described in the microscope manual.  However, do not mount the revolving nosepiece, the objectives or condenser.  (If not performing DIC microscopy, mount the revolving nosepiece).

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Mounting the Epi-fl attachment
If Epi-fl attachment is also to be used, mount the Epi-fl attachment on the microscope as described in the manual provided with the Epi-fl attachment.  However, do not mount the shielding plate until after mounting the components for DIC microscopy (or the Ph microscopy).  In addition, do not mount the shielding tube, since it is not used. 

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System Condenser Assembly

When performing DIC microscopy, install the condenser DIC prisms inside the system condenser, when performing Ph microscopy, install the Ph annular diaphragm inside the system condenser.  Three of each type can be installed. 

1. Select the condenser DIC prisms and Ph annular diaphragms to be installed in the system condenser.  
   
   Selecting a condenser DIC prism
   Which DIC prisms should be installed is determined according to the top lens type and the N.A. (numerical aperture) of the objective. 
   

(Note 1) The DIC prism with the indication [SS] has smaller shearing amount than the prism with the indication [H].  [DIC SS] condenser prism should be used together with the [SS] objective DIC prism and [H] objective.

Selecting a Ph annular diaphragm
Install Ph annular diaphragm labeled with the same Ph codes instead on the Ph objectives. 

2. Loosen the screw located in the center of the back of the system condenser, and then remove the turret from the system condenser.  There are a total of seven holes in the turret.  Leave the smallest hole vacant.  (That hole is used for system condenser centering and bright field observation.)
   
3. Set a condenser DIC prism (with the label indication facing up) in the hole with the pin.  Sufficiently loosen the DIC prism locking screw.  Remove the top lens swing-out knob, which also works as DIC prism insertion/removal tool, from the system condenser slider and screw it into the threaded hole.  Set the prism in the hole so that the notch on the prism is aligned with the position of the pin in the hole.  Using a hexagonal screwdriver, tighten the DIC prism locking screw to lock the prism in place.  Remove the top lens swing-out knob, and replace it in its original position on the slider. 
   
4. When performing Ph microscopy, set a Ph annular diaphragm (with the Ph code indication facing up) in the hole with the spring.  Using a hexagonal screwdriver, sufficiently loosen the Ph annular diaphragm centering screws.  Use the side of the diaphragm to push the spring and set the diaphragm in place.  (Tighten both screws an equivalent amount.)
   

5. Apply labels to the side of the turret in accordance with the DIC prisms and Ph annular diaphragms that were installed.  Apply each label in the position that appears in the front when the system condenser is mounted on the microscope and the DIC prism or Ph annular diaphragm in question is inserted into the optical path.  (The label will be on the diagonal from the DIC prism or Ph annular diaphragm in question).  Place the label "A: on spaces for vacant holes.

6. Mount the turret on the system condenser.  Insert the turret into the system condenser so that the round cutout at the rear of the turret comes just under the top lens.  You can feel a slight click spring at this moment.  Lightly press in the turret and tighten the screw at the rear of the turret. 
   
7. Mount the top lens on the system condenser.  

• When performing DIC microscopy, mount a top lens that is suited to the types of DIC prisms that were installed.  
• When performing Ph microscopy, mount a dry-type top lens.  (This system does not permit Ph microscopy when using an oil-type top lens).

8. Adjust the position of the swing-out knob according to the top lens type.  

• When a dry-type top lens is mounted, screw in the swing-out knob to the hole at the end of the swing-out slider. 
• When an oil-type top lens is mounted, screw in the swing-out knob to the hole a the center of the swing-out slider. 

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Mounting the System Condenser

Using the condenser focus knob, lower the condenser carrier (the bottom portion of the substage) as far as it will go.  (If mounting another condenser, loosen the condenser clamp screw and remove the condenser).   Slide the system condenser in horizontally so that the limit pin on the circular dovetail of the system condenser fits in the notch on the condenser carrier.  Tighten the condenser clamp screw to lock the condenser in place.  (Tighten it so that it does not loosen even when the turret is rotated).  

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Mounting the DIC Sextuple Nosepiece (only when performing DIC microscopy)

Use a hexagonal screwdriver adequately loosen the microscope's revolving nosepiece clamp screw.  Align the revolving nosepiece with the notch on the revolving nosepiece mount on the microscope and slide the revolving nosepiece from beneath, pushing it toward the rear as far as it will go.  Tighten the revolving nosepiece clamp screw to fix the revolving nosepiece in place.  

Note when removing the revolving nosepiece: Lower the stage and remove any objective in the nosepiece.  Be sure to hold the revolving nosepiece while removing it so that it does not fall. 

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Mounting the Objectives

Lower the stage.  Screw the objectives into the revolving nosepiece so that the objectives are in order of increasing power when the revolving nosepiece is rotated clockwise (when viewed from above).

Note when removing the objectives: Lower the stage, and if there is a specimen on the stage, remove it.  Use both hands when removing the objectives so that they do not fall. 

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Mounting the Objective DIC Prism (only when performing DIC microscopy)

Remove the dummy slider located directly above the DIC objective.  Replace the dummy slider with a DIC prism slider that corresponds with the DIC objective.  

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Mounting the Analyzer Slider (only when performing DIC microscopy)

Remove the cover on the right side of the microscope arm and insert the analyzer slider.  (The analyzer enters the optical path at the second click position.  Pulling the slider out removes it from the optical path).
For the E600, remove the dummy slider from the top of the front side of the DIC sextuple nosepiece, and insert the E600 analyzer slider in its place.  (The analyzer enters the optical path at the second click position.  Pulling the slider out removes it from the optical path).

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Installing the Polarizer (only when performing DIC microscopy)

Loosen the polarizer clamp screw, rotate the polarizer so that the index lines match, and then lock it in place.  Place the polarizer over the field lens in the microscope base, and then tighten the clamp screw to lock the polarizer in position where the index line is in front.  (Be sure to make adjustments of the directions of vibration before performing microscopy).  

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Mounting the Lambda-plate (only when performing DIC microscopy)

With the lambda symbol facing up, insert the lambda-plate in the bottom portion of the system condenser.  

- ASSEMBLY IS NOW COMPLETE -

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TROUBLESHOOTING TABLES

Improper use of the microscope may adversely affect performance even if the microscope does not suffer damage.  If any of the problems listed in the table below arise, take the countermeasures indicated.  If a problem not covered in the tables below arises, or if the countermeasures indicated in the tables below do not resolve the problem, see the manual provided with the microscope.  

DIC Microscopy

Ph Microscopy

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