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; <EOS>