Illuminating method of at least one biological sample, in which said at least one biological sample includes at least one or more fluorophores, at the focal point of at least one objective lens having a main optical axis (z-z), the method comprising the following operational steps: - Illuminating (step 10) said at least one biological sample with at least one excitation beam, which propagates between said at least one objective lens and said at least one biological sample along at least one first propagation axis (a-a); - Illuminating (step 20) said at least one biological sample with at least two depletion beams (DB, DB'), which propagate between said at least one objective lens and said at least one biological sample along the respective second propagation axes (b-b, b'-b'), said depletion beams being donut-shaped, each one in a plane orthogonal to the respective second propagation axis (b-b, b'-b'); whereby said at least one first propagation axis (a-a) and said at least second propagation axes (b-b, b'-b') are angularly inclined with each other, and said at least one first propagation axis (a-a) and said second propagation axes (b-b, b'-b') intersect on said at least one biological sample (s) only at the focal point of said at least one objective lens, so that an effective fluorescence volume is generated in said at least one biological sample which is limited both orthogonally and axially with respect to said main optical axis (z-z), characterized in that said step of Illuminating (step 20) said at least one biological sample with at least two depletion beams (BD, BD'), each along a respective second propagation axis (b-b, b'-b'), comprises: - deliver (step 21) at least one depletion radiation; - obtaining (step 22), from said at least one depletion radiation, at least two depletion beams (BD, BD'), that comprises: - obtaining at least one donut beam, from said at least one depletion radiation, - splitting said at least one donut beam into at least two donut beams (d1, d2), and - combine said two donut beams in respective depletion beams (DB, DB') which propagate substantially parallel to and spaced apart from each other, along a common path (d-d); and - supplying (step 23) said at least two depletion beams (BD, BD'), into a rear opening of said at least one objective lens, so that they enter into said rear opening substantially parallel to each other and to the main axis of said at least one objective lens (z-z), with the center of said rear opening of said at least one objective lens comprised between the centers (4, 4') of said two depletion beams (BD, BD'), optionally at the same distance therefrom, whereby said two depletion beams (BD, BD') exit from said at least one objective lens substantially inclined with each other. - Illuminating (step 10) said at least one biological sample with at least one excitation beam, which propagates between said at least one objective lens and said at least one biological sample along at least one first propagation axis (a-a); - Illuminating (step 20) said at least one biological sample with at least two depletion beams (DB, DB'), which propagate between said at least one objective lens and said at least one biological sample along the respective second propagation axes (b-b, b'-b'), said depletion beams being donut-shaped, each one in a plane orthogonal to the respective second propagation axis (b-b, b'-b'); - deliver (step 21) at least one depletion radiation; - obtaining (step 22), from said at least one depletion radiation, at least two depletion beams (BD, BD'), that comprises: - obtaining at least one donut beam, from said at least one depletion radiation, - splitting said at least one donut beam into at least two donut beams (d1, d2), and - combine said two donut beams in respective depletion beams (DB, DB') which propagate substantially parallel to and spaced apart from each other, along a common path (d-d); and - obtaining at least one donut beam, from said at least one depletion radiation, - splitting said at least one donut beam into at least two donut beams (d1, d2), and - combine said two donut beams in respective depletion beams (DB, DB') which propagate substantially parallel to and spaced apart from each other, along a common path (d-d); and - supplying (step 23) said at least two depletion beams (BD, BD'), into a rear opening of said at least one objective lens, so that they enter into said rear opening substantially parallel to each other and to the main axis of said at least one objective lens (z-z), with the center of said rear opening of said at least one objective lens comprised between the centers (4, 4') of said two depletion beams (BD, BD'), optionally at the same distance therefrom, whereby said two depletion beams (BD, BD') exit from said at least one objective lens substantially inclined with each other.