Method for observing a fluorescent sample, lying in a plane ( P 10) referred to as the sample plane, the sample comprising a fluorescent agent (10 f) able to emit a fluorescence light wave (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 method comprising the following steps: a) illuminating the sample using a first light source, in a first illumination spectral band ( Δλ 1), the first illumination spectral band lying in the excitation spectral band (Δλ e), and acquiring a first image ( I 1) of the sample, in the fluorescence spectral band, using an image sensor; b) illuminating the sample using a point second light source, in a second spectral band (Δλ 2), outside of the fluorescence spectral band, the sample lying between the second light source and the image sensor, and acquiring a second image ( I 2) of the sample, in the second spectral band, using the image sensor; the image sensor defining a detection plane ( P 30), the image sensor being coupled to an optical system placed between the image sensor and the sample, the optical system having chromatic aberrations such that: - in the fluorescence spectral band (Δλ f), the object focal plane ( P 1) of the optical system is coincident with the sample plane ( P 10), the image plane of the optical system being coincident with the detection plane ( P 30); - in the second spectral band (Δλ 2), the object focal plane ( P 2) of the optical system is offset with respect to the sample plane ( P 10), or the image focal plane of the optical system is offset with respect to the detection plane ( P 30), the offset being larger than 20 μm; such that: - the first image ( I 1) is a focused image of the sample, which image is representative of a fluorescence of the sample; - the second image ( I 2) is a defocused image of the sample, which image is representative of a structure of the sample; the method being such that the image sensor, the optical system and the sample remain stationary between the acquisition of the first image ( I 1) and the acquisition of the second image ( I 2), the method comprising a step c) of applying a holographic reconstruction operator ( h) to the second image ( I 2), so as to obtain a reconstructed image ( A z,M z,φ z) of the sample in a reconstruction plane ( P z). a) illuminating the sample using a first light source, in a first illumination spectral band ( Δλ 1), the first illumination spectral band lying in the excitation spectral band (Δλ e), and acquiring a first image ( I 1) of the sample, in the fluorescence spectral band, using an image sensor; b) illuminating the sample using a point second light source, in a second spectral band (Δλ 2), outside of the fluorescence spectral band, the sample lying between the second light source and the image sensor, and acquiring a second image ( I 2) of the sample, in the second spectral band, using the image sensor; - in the fluorescence spectral band (Δλ f), the object focal plane ( P 1) of the optical system is coincident with the sample plane ( P 10), the image plane of the optical system being coincident with the detection plane ( P 30); - in the second spectral band (Δλ 2), the object focal plane ( P 2) of the optical system is offset with respect to the sample plane ( P 10), or the image focal plane of the optical system is offset with respect to the detection plane ( P 30), the offset being larger than 20 μm; - the first image ( I 1) is a focused image of the sample, which image is representative of a fluorescence of the sample; - the second image ( I 2) is a defocused image of the sample, which image is representative of a structure of the sample;