Spatially modulated illumination allows for light sheet fluorescence microscopy with an incoherent source and compressive sensing

Abstract

Light sheet fluorescence microscopy has become one of the most widely used techniques for three-dimensional imaging due to its high speed and low phototoxicity. Further improvements in 3D microscopy require limiting the light exposure of the sample and increasing the volumetric acquisition rate. We hereby present an imaging technique that allows volumetric reconstruction of the fluorescent sample using spatial modulation on a selective illumination volume. We demonstrate that this can be implemented using an incoherent LED source, avoiding shadowing artifacts, typical of light sheet microscopy. Furthermore, we show that spatial modulation allows the use of Compressive Sensing, reducing the number of modulation patterns to be acquired. We present results on zebrafish embryos which prove that selective spatial modulation can be used to reconstruct relatively large volumes without any mechanical movement. The technique yields an accurate reconstruction of the sample anatomy even at significant compression ratios, achieving higher volumetric acquisition rate and reducing photodamage biological samples.

Enlarged Field of View in Spatially Modulated Selective Volume Illumination Microscopy

“…The three-dimensional vascular network is recovered at a lateral resolution slightly higher than the axial. The theoretical depth of field of the detection objective is 52 μ m, but the reconstruction in 3D shows an acceptable blurring for at least twice the DOF (Calisesi et al, 2019). This motivates the choice of illuminating a thickness Δ z , larger than the depth of field.…”

“…Furthermore, reconstructions of thicker volumes might also be obtained by enlarging the modulated area at the DMD and, consequently, moving the illumination subsystem. This approach fits compressed acquisition routines which would lower the total acquisition time by an order of magnitude (Calisesi et al, 2019). In this paper, however, we investigate the reconstruction quality obtainable with a static sample by proposing a deconvolution method tailored to smSVIM.…”

“…We recently proposed an alternative approach, namely spatially modulated Selective Volume Illumination Microscopy (smSVIM) (Calisesi et al, 2019). Similarly to SPIM, in this technique, the detection axis is perpendicular to the illumination direction, but without relying on single plane illumination.…”

“…This method can be implemented with incoherent light sources, such as Light Emitting Diodes (LED), making the system attractive for low-cost, shadowing free, volumetric microscopy applications. Furthermore, smSVIM can be combined with compressed sensing strategies (Candes & Wakin, 2008; Calisesi et al, 2021), reducing the number of acquisitions needed for a 3D reconstruction (Woringer et al, 2017; Calisesi et al, 2019). This can be beneficial for high-speed imaging and to lower the photodamage of the sample during the measurement.…”

Instantaneous non-diffracting light-sheet generation by controlling spatial coherence

“…It is promising to use a laser with tunable spatial coherence [23] such as degenerate cavity lasers [24]. Our simple approach has potential for advancing LSFM systems [25], including generation of multiple light-sheets in 3D [26,27].…”