| abstract |
The invention is a method of observing a fluorescent sample (10), extending in a plane (P 10 ), said sample plane, the sample comprising a fluorescent agent (10f) able to emit a wave fluorescence (Δ f ), in a fluorescence spectral band (Δλ f ), when it is illuminated by an excitation light wave (Δ e ), in an excitation spectral band (Δλ e ), the process comprising the following steps: a) illuminating the sample (10) with a first light source (11), according to a first illumination spectral band (Δλ 1 ), according to the excitation spectral band (Δλ e ) and acquiring a first image of the sample (I 1 ), in the fluorescence spectral band, using an image sensor (30); b) illuminating the sample (10) with a second light source (12), in a second spectral band (Δλ 2 ), outside the fluorescence spectral band, and acquiring a second image of the sample (I 2 ), in the second spectral band, using the image sensor; the image sensor (30) being coupled to an optical system (20) such that: in the fluorescence spectral band (Δλ f ), the object focal plane of the optical system (P 1 ) coincides with the plane of the sample (P 10 ); in the second spectral band, the object focal plane of the optical system (P 2 ) is offset relative to the plane of the sample (P 10 ), the offset being greater than 20 μm. |