Abstract: A method of investigating a sample using a wide-field multiphoton microscope with tunable excitation wavelength (?) is disclosed. The sample is illuminated at some excitation wavelength. Images of the sample are acquired at wavelengths different from the excitation wavelength, e.g., at half the excitation wavelength for SHG microscopy, using an image sensor. Based on the obtained images, an autoalignment procedure is carried out for optimizing the position of the sample relative to the illumination and/or collection beam paths. An image or spectrum that has been obtained for the optimal relative position is stored. The procedure is repeated for multiple excitation wavelengths.