See also Good Microscopy Practices (Video)
General Guidelines for Set-Up of Transmitted Light, Brightfield Microscopy Session
- Turn on microscope. Set light level to factory recommended position (usually indicated by an icon of a camera).
- Start with 10x lens, if possible. Find area of interest in the specimen slide.
- If light is too bright to your eyes, insert neutral density filters (generally along the side of the microscope); or, if there are no filters visible, decrease brightness of illumination.
- Set Koehler illumination. If uncertain, check with local expert, or for online instructions visit imagingandanalysis.com/koehler_illumination.html.
- Open camera software, if not already opened. Set camera exposure to automatic. Once satisfied with exposure, change exposure to Manual.Note: You may have to further adjust the brightness level in the manual mode by increasing / decreasing the Exposure (sometimes named Brightness or Shutter). Adjust camera exposure so that the white background is slightly less than saturated (pure white). Be careful not to over expose the background, because you will remove near-white features and this results in lost data.
After setting the microscope light intensity, do not change for the duration of your imaging session (unless you use LED or xenon light sources).
- Use stage controls to move specimen out of the field of view to show only non-specimen, white area. White balance the camera in one of the following ways in the camera software (or find out how your software white balances from a colleague):
- You may have to create a Region of Interest (ROI) on the image display where there is no specimen first, then press a White Balance button.
- A button labeled AWB (Auto White Balance) may need to be checked/unchecked.
- A 2nd button may be labeled Manual: be sure to check the Manual button to preserve the White Balance for the rest of the microscopy session.
After setting White Balance, do not change illumination levels and keep the same White Balance for entire session.
- When changing to other magnifications, and physically checking with your eyes on the microscope, the light may be too dim or too bright. Add or remove neutral density filters to dim or brighten, or use the camera software viewer to see your images and adjust exposure to dim or brighten.
- Take all the pictures you need at various magnifications. Save all images as TIFF files to preserve metadata (acquisition parameters from the acquisition software) and for consistent color rendition (as opposed to the JPEG format, which is inconsistent). Capturing images at a bit depth of 8-bits is also recommended (for a further explanation why, see Getting Fooled by Image Appearance in Software Applications).