User:Kenji Doering/Notebook/Flow Cell Set Up/2011/06/16

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Kohler's Illumination and Preparing the Microscope for Gliding Motility Assay Observation

Setting up the Microscope and Kohler’s Illumination Technique

  • Note: This procedure is specific to our microscope, but the general concepts remain the same.

Turning on the microscope

● Turn one p the Mercury Lamp under the microscope table(I means on O means off)

○ The number on the Mercury lamp indicates hours of use on the bulb, when the number reaches 200, replace the bulb.

● Turn on the power supply on the right of the microscope, the button on the back of the power supply turns thower supply on , while the switch on the front supplies to power to the actual microscope.

● Towards the bottom of the microscope there is a green button with a light picture on it, this turns the light for the microscope on.

Setting up the microscope objective, slide and stage

● Take the glass slide sample and place it upside down on the stage such that the sample you wish to observe is on the side that will be closest to the objective.

● Making sure the slide is completely level, tape it down with basic scotch tape.

● Before placing the stage above the objective make sure to put a small drop of oil on the lens of the objective as to view the sample, to get a clear image, a medium is required between the lens and the sample.

Focusing the image, and the condenser.

● The handle like knobs farther from the front of the microscope move the stage left, right, front, and back. (The upper knob moves the stage front to back, the lower knob moves it left to right)

● The knob on the right and left of the machine closer to the front of the microscope do the small thing, The larger part of the knob moves the objective up and down with large difference, while the smaller part of the knob, moves the objective up and down with sensitive differences.

● The top dial on the front of the microscope adjusts the intensity of the light on the sample.

● Now carefully try to line up the objective with and edge of the actual sample you’re trying to observe.

● Gradually move the objective up towards the sample until the oil on the lens makes contact with the sample.

● Use the smaller knob that moves the objective up and down to make the image sharper.

Focusing the Condenser Diaphragm -The final goal of this process is to have the field diaphragm closed to its max, with each edge of the diaphragm visible and focused in the lens.

● Take the field diaphragm to half, and move the condenser diaphragm to its highest height.

● To move the condenser diaphragm up and down, use the large knobs near the back of the microscope, if the knobs you use aren’t moving the condenser diaphragm, you probably picked the wrong knob.

● The screw-like knobs on the condenser diaphragm adjust where the field of vision is located. How each knob moves the field of vision can be described as a string pulley system, where turning either knob right would move the field of vision towards that knob, but to truly understand how they affect the field of vision, you really just have to practice with it.

● Using the right(or left, personally I like using the right one because it takes me to the top left edge of the field diaphragm) knob gradually turn the knob clockwise until you reach a point where there is a boundary between light and dark

○ At this point in the focusing the line between these two regions will be pretty blurred, this line is the edge of the aperture on the field diaphragm.

● You can focus this line by moving the condenser diaphragm downward, however you can expect the line to gradually move out of your field of vision, try to keep the line within your field by using the smaller condenser diaphragm knobs. ● After you get to edge of the field diaphragm aperture focused, you can gradually start closing your field diaphragm until it is closed to it’s maxed ○ To not lose sight of edge of field diaphragm you can follow it by gradually shutting the field diaphragm with one hand, and turning the small condenser knob with the other hand( if you had used the right knob earlier, you can simply turn the knob in opposite direction, than you had in the initial directions, which is why I like using only one knob for this part.)

● Eventually you will reach a point where the edges of the field diaphragm begin to meet in your field of vision. After reaching maximum closure, simply use both of the smaller condenser diaphragm knobs in combination to make sure every edge of the field diaphragm is within the microscope field of visions. (This is why you should practice using the knobs together, to understand how each knob, and a combination of the knobs will move the view) It should look like a hexagon.

Congrats, you’re done.

A (very) short note on how Kohler’s Illumination works: Kohler’s Illumination is made up of several parts: Collector Lens, Field diaphragm, Condenser diaphragm, and the Condenser Lens. The Collecter Lens collects and focus the light into the Condenser Diaphragm, then the condenser lens projects an unfocused light through the sample into the objective. The objective then refocuses through the eyepiece back to the Microscope user.

Nadiezda Fernandez-OropezaGreat job Kenji! This notes are really detailed and easy to follow. I would be great just to attach a picture of the microscope so the readers would have a better idea of what you are describing, specially about the knobs.