Science, Maths & Technology

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Introduction to microscopy

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# 1.2 Using a light microscope

This section describes the basic operation of a light microscope and is intended to be used as a short practical guide. Since the layout and functions of light microscopes vary, the details vary between instruments - we strongly recommend that you receive training and orientation on the specific instrument that you are using.

Identify the main components of the microscope, including the following:

• a.The light source, and field iris (diaphragm).
• b.The on/off switch for the light, and slider/knob to vary the voltage.
• c.The condenser, the focusing knob and centring controls for the condenser.
• d.The stage to hold the sections and clips to hold the sections in place.
• e.The controls for moving the stage up/down and left/right.
• f.The objectives and the objective turret (nosepiece).
• g.The coarse and fine focusing controls.
• h.The eyepiece(s) and any independent focus for individual eyepieces.
• i.Any controls that switch in filters, phase-contrast or polarisation filters.
• j.A control that switches the light output between the eyepiece and the camera.

Most microscopes have most of these controls. More advanced microscopes have many of their functions driven by motors, controlled by a computer and/or a joystick, particularly stage movement (e), switching of objectives (f) and focusing (g). Advanced microscopes often have digital cameras attached and project the image directly to a computer screen. In general, more senior staff have access to more sophisticated microscopes.

1. Turn on the light source, starting with the voltage low, so as not to put a power surge through the bulb. Adjust the voltage and filters to a suitable illumination level.
2. Focus and centre the condenser and adjust the field iris to illuminate the area under the section and the objective.
3. Select a low power objective, set the slide on the stage and carefully turn the focus so the objective approaches the section, observing from the side to ensure that the objective does not actually touch the section. Then, while observing though the eyepiece, focus back to the correct position using coarse and fine focus controls, i.e. the objective is moving away from the slide while focusing.
4. If the microscope has independent eyepieces, focus the fixed eyepiece using the focusing controls, then focus the independent eyepiece - this is only useful if your two eyes have different focusing strength.
5. Scan the section, first at low power and then switch to a higher power objective, having located the tissue on the slide.
6. When closing down a microscope, the steps above should be reversed so that the instrument is left with the lowest power objective in position and the light voltage set at a low value.