• WARNING / CAUTION
• NOTES ON HANDLING THE SYSTEM
• NAMES OF COMPONENT PARTS AND OPERATIONAL PARTS
• MICROSCOPY
  • Ph Microscopy
  • Bright-field Microscopy
  • Dark-field Microscopy
  • Microscopy in Combination with Epi-fl Attachment
• OPERATION OF EACH PART
  • Ph Condenser
    • Condenser Centering and Focusing
    • Adjustment of Aperture Diaphragm
    • Ph Annular Diaphragm
  • Selecting Ph Objectives
  • Selecting Filters
• ASSEMBLY
• TROUBLE SHOOTING TABLES
• CARE AND MAINTENANCE

   WARNING

1. Intended product Use: The system should only be used for microscopic observation. Do not use the system for any other purpose.
   
2. Do Not Disassemble: Attempting to disassemble the microscope or the system could result in electric shock or damage to the equipment. Never attempt to disassemble any portion of the system unless the procedure is described in this manual. If you have any problems with the system, contact your nearest Nikon representative.
   
3. Read the Instruction Manuals Carefully: For your safety, carefully read this manual and the manuals provided with the other products used with the system. Make certain to heed the warnings and cautions at the beginning of each manual in particular. Furthermore, when using the system in conjunction with Epi-fl (Episcopic-fluorescence) attachment, be certain to carefully read the manual provided with the Epi-fl attachment. The mercury lamp used as the light source of the Epi-fl attachment requires careful handling.

Back to the top of this page

   CAUTION

1. Turn off the power when assembling the system, connecting or disconnection cables, or when replacing the lamp.
   In order to prevent electric shock and damage to the system, always turn off the power switch on the microscope (and the products used with the microscope) and unplug the power cord before assembling the equipment, connecting or disconnecting cables or replacing a lamp.
   
2. Do not spill liquid on the system.
   If the microscope or the power supply becomes wet, a short circuit may result and the system could be damaged or could become extremely hot. If you accidentally spill a liquid on the system, immediately turn the power switch off and unplug the power cord. Then use a dry cloth to wipe away the moisture. If any liquid gets inside of the system, do not attempt to use it; instead, contact your nearest Nikon representative.
   

3. Caution concerning assembly.
   Be careful not to pinch your hands or fingers when assembling the system.

Back to the top of this page

NOTES ON HANDLING THE SYSTEM

1. Handle the System Gently: The system is a precision optical instrument. Handle the system gently, avoiding the physical shocks.
   
2. Dirty Lenses: Do not get dust, fingerprints, etc., on the lenses. Dirt on the lenses, mirrors, etc., will adversely affect the image. If any of the lenses get dirty, clean them as described in "Care and Maintenance."
   
3. Installation Location: To avoid degraded performance and to prevent malfunctions, consider the following requirements when selecting an installation location:
   • Install the equipment in a location with little vibration.
   • Avoid installing the equipment in a location exposed to direct sunlight.
   • Avoid installing the equipment in a dusty location.
   • Avoid installing the equipment in a location subject to high temperatures (40º C or higher) or high humidity (60% or higher).  Such conditions could allow mold or condensation to form on the lenses and filters.

Back to the top of this page

NAMES OF COMPONENT PARTS AND OPERATIONAL PARTS

Attaching the C-C Ph turret condenser and Ph objective to the microscope makes Ph observation possible with your microscope.

• If the system is not yet assembles, see "Assembly" section first.
• For details on the assembly and handling of the microscope, see its respective manual.  

(Shown below are the Nikon ECLIPSE E600 microscope with the C-C Ph turret condenser and Ph objective mounted. Other components needed for the Ph observation are as shown. Some components may not be included in the set that you purchased.)

Back to the top of this page

MICROSCOPY

The general flow of the microscopy is described below.

For details on each step, see the corresponding section "Operation of Each Part." If the system is not yet assembled, see section "Assembly" first. For details on the assembly, handling and operation of the microscope and other products used with the system, see their respective manuals.

Ph 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 Epi-fl attachment is also mounted on the microscope, see section "Microscopy in Combination with Epi-fl Attachment."

1. Select a specimen that will not 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 on a microplate, are must be taken as the centering of the Ph annular diaphragm is shifted by a lens or prism effect, resulting in poor viewing.
   
2. Prepare a specimen that matches 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 specimen for Ph microscopy, adjust the phase difference through the thickness of the specimen, 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.

Characteristics of Ph Objectives

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 phase plate (inside the Ph objective) and the image of the Ph annular diaphragm in the condenser turret are extremely important to maintain the phase contrast effect. Recheck this alignment before starting microscopy.

Back to the top of this page

1. Turn on the microscope to turn on the diascopic illumination.
2. Rotate the condenser turret so that the "A (hollow)" indication faces the front. 

3. Place the specimen on the stage, and focus on the specimen with the 10x Ph objective (ph1). (See the 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 condenser. These adjustments are very important. Do not skip this step (See OPERATION OF EACH PART: Ph Condenser).

6. Rotate the turret so that the "Ph1" indication faces the front. (See OPERATION OF EACH PART: Ph Condenser)
7. Place the green interference filter (GIF) on the filter holder around the field lens.

8. Center the Ph annular diaphragm. This adjustment is very important. Do not skip this step (See Centering the Ph Annular diaphragm).
9. Adjust the filed diaphragm so that it is just inside or outside of the view field. (See the manual provided with the microscope)
10. If you switch the objective, also switch the condenser turret so that it matches the Ph code of the objective. At this time, check that the phase plate of the objective and the image of the Ph annular diaphragm are aligned. Also readjust the size of the field diaphragm.

11. When using an oil immersion type objective, apply immersion oil between the specimen and the objective. (See manual provided with the microscope)

NOTE: The C-C Ph turret condenser is designed for dry use only. Never place immersion oil between the tip of the condenser and the specimen.

Back to the top of this page

Bright-field Microscopy

Perform the following for bright-field microscopy. Refer to the manual provided with the microscope for details.

• Rotate the condenser turret so that the indication "A (hollow)" faces the front.
• Objectives with the magnification higher than 4x can be used for bright-field microscopy. Vignetting occurs on the view filed periphery during UW microscopy using a 4x objective.
• The condenser performs just like an Abbe condenser described in the manual provided with the microscope.

Back to the top of this page

Dark-field Microscopy

Perform the following for dark-field microscopy. Dark-field microscopy with the C-C Ph turret condenser can be used as a finder to search for the object to be viewed in a specimen.

• Rotate the condenser turret so that the indication "DF" faces the front.
• Insert an objective having a magnification of at least 10x and numerical aperture of no more than 0.7 into the optical path.
• Raise the condenser as far as it will go when viewing specimens with a thick slide glass. If the slide glass is too thick, the illumination may not reach the specimen surface. When this happens, prepare the specimen again using a thinner slide glass.

Use a condenser especially designed for dark-field microscopy when performing dark-field microscopy in the main.

Back to the top of this page

Microscopy in Combination with Epi-fl Attachment

It is possible to combine Ph microscopy with Epi-fl microscopy by mounting both the Ph and Epi-fl attachment on the microscope. For example, use the Ph microscopy instead of the Epi-fl microscopy (which causes colors to fade) to search for the target on the specimen. It is also possible to use both methods simultaneously in order to compensate for their individual shortcomings.

For details on Epi-fl microscopy, see the manual provided with the Epi-fl attachment.

1. Switching the methods
   Performing the following steps when switching the microscopy.

When switching to Ph microscopy

• Insert the shutter of the Epi-fl attachment into the optical path so as to block the excitation light of the Epi-fl attachment.
• Remove the excitation filter block from 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

• Rotate the condenser turret so that the indication "C (shutter)" faces the front.
• Insert an excitation filter block in the optical path.
• Remove the shutter of the Epi-fl attachment from the optical path.

2. Simultaneous microscopy
   When using both the Ph and Epi-fl microscopy simultaneously, follow the procedure described below:

• Use the Ph method to focus on 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.
• Remove the shutter on the Epi-fl attachment from the optical path 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 Ph image.

Back to the top of this page

OPERATION OF EACH PART

Ph Condenser

1. Condenser centering and focusing
   Refer to the section entitled "Condenser focusing and centering" in the manual provided with the microscope.

• Turn the turret so that the indication "A (hollow)" faces the front.
• Fully open the aperture diaphragm.

2. Adjustment of aperture diaphragm
   In the case of the bright-field microscopy (turret indication: A), the aperture diaphragm should normally be closed to 70 to 80% of the numerical aperture of the objective.

   Refer to the section entitled "Aperture diaphragm" in the manual provided with the microscope. [The aperture diaphragm is removed from the optical path when not performing the bright-field microscopy (turret indication: A).]
   

3. Ph annular diaphragm
   Aligning the Ph annular diaphragm of the condenser and the phase plate of the objective allows the microscope to be used for phase contrast microscopy.

Ph codes:

• A Ph code (Ph1, Ph2, or Ph3) indicated on the Ph objective shows the size of the phase plate inside the objective. (The Ph code is irrelevant to the magnifying power of the objective).
• Rotate the condenser turret so that the Ph annular diaphragm of the same code is inserted into the optical path. 
• Phase contrast effects cannot be obtained if different codes are used in combination.

Centering the Ph annular diaphragm

• Switch the objective to Ph1 (10x) and rotate the condenser turret to "Ph1".
• Turn the stage motion control knobs to bring under the optical path, the part of the specimen where there are no objects yet is covered with a cover glass.
• Remove the eyepiece from the eyepiece tube and insert a centering telescope in its place.
• Turn the eyepiece part of the centering telescope while holding onto the flange and focus on the phase plate of the objective.

• If the phase plate of the objective and the annular diaphragm of the image of the condenser are not in alignment, turn the two centering knobs on the condenser after loosening their clamp screws. Move the entire turret with the centering knobs to align the annular diaphragm image with the phase plate. Tighten the clamp screws when finished.  It is necessary that the phase plate and annular diaphragm image are aligned since it is not, proper Ph image cannot be obtained.

• NOTE: The annular diaphragms on the turret of the C-C Ph turret condenser are pre-adjusted based on the Ph1 annular diaphragm. In general, once Ph1 has been centered, centering of other annual diaphragms is not required. However, phase contrast images change slightly by the quality of alignment of the annular diaphragm on the condenser and the phase plate on the objective. In case of the precision observations or photomicrography, it is best to check that the annular diaphragm and phase plates are concentric at each magnification. When the annular diaphragm and phase plate are slightly off-centered, three-dimensional image can be  obtained through the shadowing effect, which may be effective for some specimens.

Back to the top of this page

Selecting Ph Objectives

• Ph objectives are in-dispensible to Ph microscopy.
• Ph objectives consist of Achromat, Plan Achromat, Plan Fluor and Plan Apochromat objectives according to chromatic aberration and the degree of correction of image surface curvature.  
• According to the properties of the phase plate within the Ph objective, there are "DM " objectives suitable for the specimens with low phase difference, "DLL" objectives for high phase difference, and "DL" objectives for intermediate phase difference.
• It will not be possible to obtain good results if the properties of the phase plate do not match the amount of phase difference of the specimen.

Back to the top of this page

Selecting Filters

NCB11 (Color Balancing Filter)

• This filter is normally used during microscopy. It is essential for obtaining good color reproduction in color photomicrography.
• This filter is contained in the base of the E600. It should be placed in a filter holder around the field lens in the E400.

GIF (Green Interference Filter)

• Used to improve contrast during Ph microscopy.
• This filter should be placed in a filter holder around the field lens in both the E600 and E400.

Infrared Cutoff Filter

• This filter cuts out infrared rays contained in the illumination. Used when living specimens, etc. may be damaged by heat.
• This filter is installed in the lamphouse in E600. A separately sold filter (Ø45) should be placed in a filter holder around the field lens in the E400.

Back to the top of this page

ASSEMBLY

   WARNING: Before assembling the system, be sure to read the WARNING and CAUTION section is at the beginning of this manual, and also the section entitled, "Notes on Handling the System." Be certain to heed all the warnings and cautions. Also be sure to read the manuals for any other products that you are using with the system (the microscope, Epi-fl attachment, etc.), and heed all the warnings and cautions in those manuals. In order to prevent accidents, burns and injuries caused by electric shock, fire, or ultraviolet light, turn off the power switches for the microscope and other products used with the system during assembly.

Refer to the illustrations while assembling the equipment.

For details on the assembly, handling, and operation of the microscope, Epi-fl attachment, etc. see their respective instruction manuals.

Scratches or fingerprints on the lenses and prisms will adversely affect the image. Handle these components carefully during assembly in order to keep them free from scratches and fingerprints.

1. Microscope Assembly
   Assemble the microscope as described in the microscope manual. However, do not mount the objectives and the condenser.
   
2. Mounting the C-C Ph Turret Condenser

• Using the condenser focus knob, lower the condenser carrier (the bottom portion of the sub-stage) as far as it will go. (If another condenser is mounted, loosen the condenser clamp screw to lock the condenser.)
• Slide in the C-C Ph turret condenser horizontally so that the limit pin on the circular dovetail of the 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.)

3. Mounting the Objective

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

4. Filters

• Be careful so that the fingerprints and other dirt do not get on the filters and field lens. Place the filters in the filter holder located around the field lens.

Back to the top of this page

TROUBLESHOOTING TABLES

Ph Microscopy

Dark-field Microscopy

Back to the top of this page

CARE AND MAINTENANCE

Filter and Lens Cleaning

Do not get dust, fingerprints, etc., on the lenses or filters. Dirt on the lenses, filters, etc., will adversely affect the image. If any of the lenses or filters get dirty, clean them as described below.

• Use an air blower to blow away dust. If that does not suffice, brush away the dust with a soft brush, or else wipe it away gently with gauze.
• Only if there are fingerprints or grease on a lens or filter, dampen a piece of soft, clean cotton cloth, lens tissue, or gauze with absolute alcohol (ethyl alcohol or methyl alcohol) and wipe. However, do not use the same area of the cloth, etc., to wipe more than once.
• Use petroleum benzine to clean off immersion oil. Wiping with absolute alcohol (ethyl alcohol or methyl alcohol) after the oil has been removed finishes the clean up process. If you cannot obtain petroleum benzine, use methyl alcohol. However, because methyl alcohol does not clean as well as petroleum benzine, it will be necessary to wipe the surfaces repeatedly. (Usually, three or four times are sufficient to clean lenses or filters.)
• Use petroleum benzine only to remove immersion oil from objectives; do not use petroleum benzine for cleaning the entrance lens on the eyepiece tube, filters, etc.
• Because absolute alcohol and petroleum benzine are both highly flammable, be careful when handling, when around open flames, when turning the power switches on and off, etc.
• Use absolute alcohol and petroleum benzine according to the instructions given by their manufacturers.

Cleaning of Painted Components

• Do not use organic solvents (such as alcohol, ether or paint thinner) on painted components, plastic components, or printed components. Doing so could result in discoloration or in the peeling of printed characters. For persistent dirt, dampen a piece of gauze with diluted detergent and wipe lightly.

Storage

• Store the equipment under conditions of low humidity where mold is not likely to form.
• Store the objectives, eyepieces, filter blocks, etc. in a desiccator or similar container with a drying agent.
• Put the vinyl cover over the equipment to protect it from dust.
• Before putting on the vinyl cover, turn off the power switches for the microscope and the Epi-fl attachment light source, and wait until the lamphouse is cool.

Regular Inspections

Regular inspections of this equipment are recommended in order to maintain peak performance. Contact your nearest Nikon representative for details about regular inspections.

Back to the top of this page