Illumination of the sample with a high NA cone implies that incident rays, carrying the length of the ruler (the wavelength), are not all parallel to the optical axis. As a consequence an empirical correction factor has to be applied to height measurement [Mykura 1963, Creath 1989, Schultz 1991]. Such correction factor is not needed with DHM®.

In the 21st century, DHM® by Lyncée Tec refer to wavelengths for height measurements. By using ultrastable interferometric filters for selecting a precise wavelength band of a relatively broad spectrum laser source, DHM® operating wavelengths are precisely controlled and perfectly stable. The height values do not depend on any calibration,  precise positioning, or repeatability of interferometric piezo-controller, neither on any motorized displacement, or confocal detection system. DHM® provides thus precise measurements with very high repeatability.

In the Select Lenses for Calibration window, verify that the lenses are listed in descending order, from highest to lowest power.

Follow the onscreen prompt to switch to the next-highest power objective and select the corresponding software lens (some microscopes may select the software lens automatically)

When camera resolution is lower than computer-monitor resolution, the entire video is displayed in the top left of the main Neurolucida window, with black regions bordering the bottom and right of the video. If enabled, Fit Video in Window will stretch the live-camera feed to fill the software window; uncheck the box to prevent this stretching.

The wavelength filters used in the DHM® have a minimal dependence on temperature. For instance, changes on the transmission wavelength due to a change of temperature of 10°C are negligible for most applications.

Disable the option to Fit Video in Window when your video-camera resolution is lower than your computer-monitor resolution.

Do not check the box to Enable parfocal because parfocal correction is controlled by the microscope (not the software). Refer to your microscope manual for more information on adjusting parfocality. On a Zeiss microscope, use the touch screen to access Home > Configuration > Focus > Parfocality Adjustment.

Parcentric and parfocal calibration compensate for the deviations from parfocality (focal plane) and parcentricity (collimation) that are normally encountered between different microscope objective lenses. They are both critical for maintaining position when changing magnification.

Laser metrologyequipment

Measurement of standardized samples, as well as direct comparison of measurement performed with other metrological tools have demonstrated the high accuracy and repeatability of DHM® [Colomb 2010].

When camera resolution is higher than computer-monitor resolution, only the top left of the live-camera image will be visible in the main Neurolucida window. Select Fit video in Window to reduce the size of the live-camera image so that all of it is visible onscreen.

An alert is displayed to remind you that, for each lens, you need to click the exact same point on the grid, regardless of where it appears on the screen.

Lyncée Tec provides optionally a certificate issued by the Swiss National Metrology Center METAS for the transmission spectrum of the interferometric filter used to control the central wavelength and spectrum width. The operating wavelengths of the DHM® and thus heights measurements are fully controlled since no other critical distance or displacement are involved in the height measurement.

Laser metrologycourse

James Clerk Maxwell (1831–1879) has been one of the first to suggest using the wavelength as a natural gauge for length. In 1950, the standard meter in the new International System of Units (SI) is referred to the wavelength corresponding to the krypton-86 (605,780 nm).  The use of a precise wavelength as a reference for the measurement of  lengths is well established as the ideal measurement method.

Enable the option to Fit Video in Window when your video-camera resolution is higher than your computer-monitor resolution.

Once you've calibrated the lenses and verified calibration accuracy, you're ready to perform the parcentric/parfocal alignment.