How To Focus A Slide Under The Microscope?

A microscope is an instrument that magnifies an object so that it may be seen by the observer. Because cells are usually too small to see with the naked eye, a microscope is an essential tool in the field of biology. In addition to magnification, microscopes also provide resolution, which is the ability to distinguish two nearby objects as separate. A combination of magnification and resolution is necessary to clearly view specimens under the microscope. The light microscope bends a beam of light at the specimen using a series of lenses to provide a clear image of the specimen to the observer. It is necessary to focus your microscope when viewing specimens to perform a proper analysis and give an accurate diagnosis.

Microscope Objective Lenses and Total Magnification

Your microscope has 4 objective lenses: scanning (4x), low (10x), high (40x), and oil immersion (100x). In addition to the objective lenses, the ocular lens (eyepiece) has a magnification. The total magnification is determined by multiplying the magnification of the ocular and objective lenses.

Scanning

Objective: 4x | Ocular lens: 10x | Total magnification: 40x

Low Power

Objective: 10x | Ocular lens: 10x | Total magnification: 100x

High Power

Objective: 40x | Ocular lens: 10x | Total magnification: 400x

Oil Immersion

Objective: 100x | Ocular lens: 10x | Total magnification: 1000x

The nosepiece is the rotating portion of the microscope above the stage. It will have three or four objective lenses attached to it. The objective lenses are the magnifiers that are attached to the nosepiece. They will be labeled with their magnification level, which is typically 4x, 10x, 40x, and 100x. There are generally two focus knobs on a standard microscope. The larger one is known as the coarse knob, as it zooms in/out by a large degree, while the smaller one is known as the fine knob, as it zooms in/out in smaller degrees.

Initial Setup Before Focusing

1. Plug in the microscope.
2. Turn on the microscope light. A light will appear from below the stage of the microscope. The stage is the flat, plate-like surface on the microscope that holds the slides you will be observing.
3. Place the stage as far away from the objective lenses as possible.
4. Rotate the revolving nosepiece until the lowest power objective lens clicks into place. As a general rule, always start and end with the lowest power objective on the microscope (usually 4x), as it is easiest to focus and center the sample at a lower power. Also, since it is the shortest objective lens, there is the least chance of scratching the lens when situating or removing the slide.
5. Find a flat, well-lit area to set up your microscope. A sturdy table or desk in a well-lit room will work perfectly.
6. If your microscope is monocular (it has only one eyepiece), close or cover your other eye for accuracy and comfort when viewing. If your microscope is binocular, adjust the interpupillary distance by either sliding or rotating the eyepieces until you can see only one circle of light with both eyes open.

Steps for Focusing on a Specimen

1. Looking at your stage from the side, place the specimen over the hole in the stage. Place the slide on the microscope stage with the specimen directly over the circle of light. Doing this will give you a 90% chance of finding the specimen as soon as you look through the eyepiece. Be sure that the slide is about ¼" away from the objective.
2. Still looking from the side, lower the lowest objective lens (4x) using the coarse adjustment knob until it is as close as possible to the stage.
3. Look through the eyepiece and slowly raise the objective lens using the coarse adjustment knob until your object comes into focus. Use the stage clips to hold the slide in place. Move the mechanical stage until your focused image is also centered.
4. To focus your object at the next highest power, look from the side of the stage and slowly rotate the objective lens (10x) into place and use the fine adjustment knob to bring your object into focus. Use the coarse knob to refocus and move the mechanical stage to re-center your image.
5. To focus at the highest power, rotate the nosepiece to the objective lens (40x) while looking at it from the side. If you do not look at it from the side you may crack your slide or the objective lens. At this point, only use the fine adjustment knob to focus specimens.
6. Remove the object from the stage and make sure the stage is clean.
7. Turn off the microscope light and unplug the microscope.

Using the Coarse and Fine Focus Knobs

Begin to focus on the object by adjusting the coarse focus knob. This will be the larger of the two knobs on the side of the microscope. Rotate the knob clockwise and counterclockwise until you see the best possible picture through the eyepiece. Adjusting the coarse focus knob may not give you optimum clarity. The coarse focus knob is meant to bring the object mostly into focus so that the fine focus knob can be effective.

After you have adjusted the coarse focus knob, you can fine-tune your focus by adjusting the fine focus knob. This will be the smaller of the two knobs on the side of the microscope. Turn this knob clockwise and counterclockwise until you see the best picture in the eyepiece.

When the 10x objective lens is in place, the distance between the lens and slide is much smaller than when the lower objective (usually 4x) is in place. This means the coarse focus knob must be used very carefully and slowly, not to push it into the slide, break the coverslip, or squash your specimen. If you have focused correctly using the lower power lens, when you move the 10x lens into place, the focus will be very close to where you want to be, so only a very small adjustment should be made using the coarse focus knob. The coarse focus knob should not be used with any higher powered lenses (40x, 100x) — use fine focus only with these.

Focusing Using the High Power Lens

To focus a microscope using the high power lens, first start with the low power lens (usually 10x) and focus on the specimen using the coarse adjustment knob. Once the specimen is in focus, switch to the high power lens (usually 40x or 100x) without moving the slide. Now, use the fine adjustment knob to bring the specimen into sharp focus. Do not use the coarse adjustment knob, as it may cause the high power lens to touch the slide and damage the lens or the slide. If needed, make minor adjustments to the stage position using the stage control knobs to center the specimen in the field of view.

Remember, if you can't focus under scanning and then low power, you won't be able to focus anything under high power. Do not skip objective lenses when increasing your magnification. Focus each objective lens before moving to the next magnification.

Adjusting Light and Diaphragm

Adjust the diaphragm as you look through the eyepiece. You will begin to notice that more detail is visible when you allow in less light. Too much light tends to give the specimen a washed-out appearance. If the specimen is too light or too dark, try adjusting the diaphragm. Adjust the diaphragm or light intensity to optimize the contrast and brightness of the image.

Troubleshooting Common Focusing Problems

• Image is too dark: Adjust the diaphragm and make sure your light is on.
• There's a spot in the viewing field that stays even when the slide moves: Your lenses are dirty. Use lens paper, and only lens paper, to carefully clean the objective and ocular lens. The ocular lens can be removed to clean the inside.
• Can't see anything under high power: Remember the steps — if you can't focus under scanning and then low power, you won't be able to focus anything under high power.
• Only half of the viewing field is lit (half-moon appearance): You probably don't have your objective fully clicked into place.
• Focus is just out of reach when switching to higher magnification: Some microscopes have a focus stop mechanism where you can set a max height for the stage. When set correctly, this prevents accidentally slamming your objective with a slide, but it can cause problems if it is set too low. Also check whether you are using a cover slip — higher magnification lenses usually have a shorter working distance and need thinner cover slips. Lower magnification objectives have greater working distance, so you may have picked something to focus on that is at the edge of the lower magnification objective's range, but out of working distance of the higher magnification objective.
• I see my eyelashes: You're too close to the objectives. Move your head back a little.
• Headache or eye strain: Relax. Try adjusting the ocular distance, check that the intensity of your light isn't too high or too low. Take breaks if needed.

Cleanup and Storage

Store the microscope with the scanning objective in place and the stage in its lowest position. Wrap cords around the microscope. Replace slides to the original slide tray. Regular cleaning ensures clear images. Use a soft brush or air blower to remove dust, and lens cleaning solution for stubborn stains. Do not touch the lens glass with your finger, as you will leave the lens smudged and hard to see through. Be patient and keep trying — using a microscope takes practice.