Nikon Alphaphot-2K
Compound Light Microscope with Köhler Illumination

Introduction

The Nikon Alphaphot-2 Compound Light Microscope is a very expensive piece of equipment that must be cared for properly. This includes its transport, use, and storage. This document was prepared for students and faculty to ensure that these microscopes will have a long life span. FOLLOW ALL INSTRUCTIONS CAREFULLY!

Parts of the Microscope

Please take a few moments to familiarize yourself with the parts of the microscope using the illustrations provided in Figure 1. Once you have reviewed this material, you may proceed through the rest of this document so that you can identify the parts on the actual Nikon microscope.

Figure 1.  Parts of the Nikon Alphaphot-2 Microscope with Köhler Illumination.

Preparing to Use Your Microscope

The Nikon Alphaphot-2 microscope is a very delicate and powerful instrument. In order to fully appreciate the specimens that you will be viewing, you MUST properly set up the microscope for YOU! By tailoring the instrument to your vision, it will make it much easier to see the details that you want to observe. At first, these steps may seem long and time consuming, but with practice, it should become "second nature" to you.

Setting Up the Microscope

1. If there is a DUST COVER, remove it and FOLD IT! Flatten the dust cover along its seams. Fold it neatly and place it in the middle of the bench, so that it is out of the way.
   
2. The POWER CORD is neatly wrapped around the arm of the microscope near its base. Carefully unravel the cord, straighten it out and plug the microscope in.
   
   NEVER PUSH OR DRAG THE MICROSCOPE ON THE BENCHTOP!!! ALWAYS LIFT IT TO MOVE IT!!! Pushing or dragging the microscope on the benchtop causes it to BOUNCE because of the little rubber feet. This jars the optical systems of the microscope, which will damage them over time!

Illumination System

The brightness control knob also acts as the ON/OFF switch. To turn on the microscope, rotate the brightness control knob toward you and select the desired brightness (Figure 2). Start out with a moderate setting.

Initial Setup

1. Ensure that the 10x objective is in place. If not, rotate the 10x objective using the revolving turret, ensuring that the objective clicks into place. DO NOT ROTATE OBJECTIVES BY GRABBING ONTO THE OBJECTIVES THEMSELVES!!! THIS DAMAGES THEM!!! ALWAYS use the revolving turret as shown in Figure 3.
   

2. Use the course focus knob to lower the stage as far down as it will go (Figure 4).   Turning the knob TOWARD you should lower the stage.  On the left side of the microscope, push the lever to open the slide clamp and place your specimen in the holder on the stage, as shown in Figure 5. ENSURE THAT THE SIDE WITH THE COVER GLASS IS UP! Release the lever on the clamp, and the slide will be held in place.
   
   
   

3. Use the condenser focus knob to move the condenser as high as it can go underneath the stage (Figure 6A).
   
4. Look through the microscope oculars. The circle of light that you see is the field of view. Adjust the interpupillary distance by grabbing the BASEPLATE OF THE OCULARS until you see only one circle, or a single field of view, through the oculars (Figure 7).

Ocular Diopter Adjustment.

1. With the 10x objective in place, look to the side of the microscope so that you can see both the slide and objective. Slowly turn the course focus knob to move the stage upwards until it is stopped. It should stop BEFORE the slide reaches the objective.  Turning the knob AWAY FROM you should raise the stage.
   
   You are looking at the side to ensure that the SLIDE AND COVER SLIP DO NOT RAM INTO THE OBJECTIVE!!! If the slide rams into the objective, the objective will be damaged, and the cover slip cracked, making the slide useless.
   
2. Looking through the oculars, focus the specimen using the course focus knob. The stage should move AWAY from the objective.
   
3. Continue looking through the oculars and use the fine focus knob to make any minor adjustments (Figure 8). At this point, it does not matter if the image is perfect, you only need it to be in relatively good focus.
   
4. Rotate the 40x objective into place using the revolving turret (Figure 3).
   
5. While looking down the oculars, close your RIGHT eye. The left eye should now be looking down the left ocular. Using the diopter adjustment ring on the left ocular, focus the specimen (Figure 7).
   
6. Repeat the above step, but this time with the other eye. While looking down the oculars, close your LEFT eye. The left eye should now be looking down the right ocular. Using the diopter adjustment ring on the right ocular, focus the specimen.
   
7. Rotate the 10x objective back into place, and look down the oculars to see if the specimen is in focus. If not, use the diopter adjustment rings as indicated in Steps 5 and 6.
   
8. Rotate the 40x objective into place and repeat Steps 5 and 6.
   
   You are repeating the ocular diopter adjustment to compensate for differences in your right and left eyes, and to also correct for the body tube length of the microscope. This will allow you to take full advantage of the high-quality optics and the parfocality of the lenses.

Aligning the Microscope for Köhler Illumination

Adjusting the Condenser and Field Iris Diaphragm

1. Rotate the 10x objective into place and focus the specimen.
   
2. While looking through the oculars, close the field iris diaphragm (Figure 10B). You should see a hexagon. Adjust the field iris diaphragm until about 2/3 of the original field of view is blocked off, and you can still see the hexagon, as illustrated in Figure 11.
   
3. While still looking through the oculars, focus the field iris diaphragm by moving the condenser focus knob. The field iris diaphragm is in focus when the edges of the hexagon are as SHARP as they can be.
   
4. After it is focused, open the field iris diaphragm, making sure that the edges reach the field of view at the same time. This ensures that the field iris diaphragm is centered. If not centered, use the field iris centering screws to center the field iris diaphragm in the field of view (Figure 12).
   
5. Once centered, open the field iris diaphragm until the edges of the hexagon just disappear from the field of view, as in Figure 13.

Adjusting the Substage Diaphragm

1. With the specimen still focused, carefully remove the right ocular.
   
2. With your right eye, look down the open ocular tube that held the right ocular. You should see a circle of light. You are looking directly at the back focal plane of the microscope.
   
3. While looking at the back focal plane of the microscope, adjust the substage diaphragm using the substage diaphragm lever as illustrated in Figure 14. You should see a hexagon increasing and decreasing in diameter as you move the lever.
   
4. Open the substage diaphragm until the edges of the hexagon are just barely visible in the back focal plane.   Figure 15 shows the substage diaphragm being open 70-80%.   You want 95-99%.
   
5. Carefully put the right ocular back into the ocular tube. The microscope has now been aligned for Köhler illumination.

NOTE: All of the steps outlined in this section on Aligning the Microscope for Köhler Illumination MUST BE COMPLETED EACH TIME YOU SWITCH OBJECTIVES OR SLIDES!!! Each objective has slightly different optical characteristics, such as reduced fields of view, so these adjustments must be made each time you switch objectives. Slides with cover slips have variable thicknesses, which can also affect Köhler illumination.

The field iris diaphragm controls the amount of light traveling through the specimen. If opened larger then necessary, extraneous light will travel through the specimen and enter the field of view, reducing image quality and contrast. Use the brightness control knob to control specimen brightness, NOT the field iris diaphragm.

The substage diaphragm also helps to set the contrast of the image. If the diameter is wide, the image will be "washed out," while if the diameter is too small, the image will have too much contrast, and specimen details will be lost.

Köhler illumination was developed by Professor August Köhler to strike the best balance between contrast and resolution in specimens viewed with the compound light microscope.

Oil Immersion

Using the Oil Immersion Lens

The 100x objective is the ONLY objective that can be immersed in oil.

To use the oil immersion objective:

1. Focus your specimen using the 40x objective and align the microscope for Köhler illumination.
   
2. Rotate the revolving turret to the 4x objective or to a position BETWEEN the 40x objective and the 100x oil immersion lens.
   
3. Place a small drop of immersion oil over the cover glass in the circle of light that can be seen passing through the specimen. Ensure that there are no air bubbles in the oil. Air bubbles will produce a poor image. To get rid of air bubbles, add more oil, or rotate the 100x objective through the oil droplet several times.
   
4. Rotate the revolving turret so that the 100x objective is in place. Focus the specimen and align the microscope for Köhler illumination.
   
5. When you are done, ENSURE THAT YOU DO NOT ROTATE THE 40X OBJECTIVE THROUGH THE OIL!!!

Cleaning the Oil Immersion Lens (100x Objective)

The oil immersion lens does not have to be cleaned UNTIL THE END OF THE LABORATORY SESSION! When you are ready to put away your microscope, you must clean the 100x objective as follows:

1. TURN OFF THE MICROSCOPE!!!
   
2. Use LENS PAPER to wipe off any excess oil from the 100x objective and slides.
   
3. Dip a cotton swab in the cleaning solution provided. WARNING! THE CLEANING SOLUTION IS HIGHLY FLAMMABLE! DO NOT BREATHE IN THE FUMES!!!
   
4. Use the end of the cotton swab to clean the 100x objective lens, removing any excess oil.

IMPORTANT ADDITIONAL INFORMATION

There is another ring between the arm and the coarse focus knob as illustrated in Figure 16. DO NOT TURN THIS RING UNDER ANY CIRCUMSTANCES!!! This is the coarse focus knob tension adjustment ring.

Putting the Microscope Away (Storage)

Before putting away your microscope, please follow ALL of these steps!

1. TURN OFF THE MICROSCOPE!!!
   
2. ROTATE THE 4X OBJECTIVE INTO PLACE USING THE REVOLVING TURRET.
   
3. Turn the coarse focus knob until the microscope stage as LOW as it can go.  You must do this to prevent the power cord from scratching the glass components.

1. If you have an ODD numbered microscope, move the mechanical stage all the way to the BACK of the stage.
   
2. If you have an EVEN numbered microscope, move the mechanical stage all the way to the FRONT of the stage.

4. On the right side of the microscope, ensure that the horizontal (X) bar of the mechanical stage is flush with the stage.
   
5. Turn the condenser focus knob so that the condenser is as close to the stage as possible (highest position).
   
6. Unplug your microscope and wrap the cord around the base of the microscope, underneath the stage and condenser and around the field iris diaphragm.
   
7. Unfold the dust cover if the microscope had one. Cover the microscope. The word NIKON should be on the right side of the microscope.
   
8. USING TWO HANDS, return the microscope to the cabinets. ENSURE THAT THE MICROSCOPE ARM FACES OUTWARDS!
   
   ENSURE THAT THE MICROSCOPE IS RETURNED TO ITS PROPER NUMBERED POSITION IN THE CABINET!!!
   
   When placing the microscopes in the cabinets, DO NOT LET THE OCULARS HIT ANY PART OF THE CABINET! The clearance should just be enough to push the microscope straight in by the ARM.
   
   NOTE: PARTS OF THE MECHANICAL STAGE HANG OVER EACH SIDE OF THE MICROSCOPE!!! BE CAREFUL NOT TO DAMAGE THE MECHANICAL STAGES OR THE MICROSCOPES ON EITHER SIDE!!! The space on either side of each microscope in the cabinet is EXTREMELY LIMITED! BE EXTREMELY CAREFUL!!!