Traditional wide-field optical fluorescence microscopes are proven invaluable tools that accomplish the most diverse imaging tasks at the cellular and sub-cellular level. Nevertheless, when the systems (organisms or tissues) containing the fluorescent structures grow in size and complexity, traditional microscopy methods become limited or unusable. The main reason for this is that wide-field microscopes detect both the desired in-focus and undesired out-of-focus light. In a thick sample the high-resolution information from the focal plane can become “buried” in the blurred light from the surrounding tissue. The problem is more evident when the task involves following fast dynamical processes over time, with a limited amount of photons. That is why having an alternative technique that would allow the observation of fast events with high spatial resolutions over a large field of view (FOV) is extremely important. To overcome the problem of out-of-focus light, techniques referred as laser point scanning microscopy (LSM), such as confocal and multi-photon microscopies, have been introduced [1
]. LSM techniques generate images only from in-focus light providing intrinsic optical sectioning
. Then, by digitally combining a stack of these images a three dimensional representation of the fluorescent sample can be obtained. In addition to out-of-focus light, another important issue to take into consideration when imaging biological samples is the photodamage (photobleaching and phototoxicity). In LSM techniques, as excitation and collection occurs along the same axis, the entire sample is repeatedly irradiated when taking an image stack. As a consequence, cumulative photodamage is induced within the sample [2
To overcome such problems, selective plane illumination microscopy (SPIM) was proposed [3
]. In SPIM, a static sheet of excitation light is produced onto the sample plane using a cylindrical lens. Then, the fluorescence light emerging from this plane is collected through a microscope objective (MO) placed along the axis orthogonal to the excitation sheet. This uncoupling between the excitation and collection branches provides SPIM with: i) 2D optical sectioning capability in large fields of view that does not require point-scanning, and ii) decoupled resolution in the transversal and axial directions, determined by the collection numerical aperture (NA) and light-sheet thickness, respectively [3
]. Perhaps the most valuable benefit of this technique is the reduction of the photodamage to the sample, due to the restriction of the irradiation to the plane under observation [4
]. Since it also can provide rapid acquisition speed, SPIM has emerged as a powerful tool for in vivo
time lapse studies, from single cells to whole organisms and tissues [5
]. Even though SPIM has proven to be a good alternative to conventional fluorescence microscopy methods, it still holds some drawbacks: i) a broadening of the light sheet deep inside the sample caused by scattering and aberrations, ii) the formation of stripe artifacts induced by absorption and scattering along the illumination axis, and iii) inhomogeneity of the sheet due to diffraction generated by the limiting diaphragm. Moreover, for having large FOVs, the depth of field in the cylindrical lens should be increased. This is achieved by using low NA lenses. However, this also reduces the optical sectioning capability of SPIM as the thickness (waist) of the generated cylindrical beam is increased. The balancing between both parameters has to be chosen carefully for the specimen of interest.
Recently two-photon excited fluorescence single plane illumination microscopy (2p-SPIM) was demonstrated for imaging the pharynx of cameleon labeled Caenorhabditis elegans
]. The use of two-photon excitation allows better out-of-focus light rejection, improving the quality of the optical sections and reducing the photodamage. These improvements rely on: i) the use of NIR excitation wavelength matching the optical window of biological samples and therefore allowing less sensitivity to scattering, better penetration depth, and reduced linear absorption; and ii) the nonlinear nature of the absorption in TPEF virtually eliminates the conversion of the scattered excitation into fluorescence [7
]. However, compared to two-photon LSM, in 2p-SPIM the total intensity of the nonlinear excitation beam is reduced as the beam is distributed over a plane as opposed to a single point. This drastically reduces the efficiency of fluorescence excitation.
Another interesting alternative implementation of SPIM (in which the beam is static) relies on the generation of the light sheet by scanning in one direction a focused Gaussian beam. This is termed digital scanned (laser) light sheet microscopy (DSLM) [8
]. There are several advantages to this implementation over widefield SPIM: i) The full power of the excitation light is concentrated into the single scanned line providing better illumination efficiency and lower exposure times, ii) each line in the specimen is illuminated with the same intensity generating a homogenous light-sheet, where the height can be easily controlled with the amplitude of the scanning. Nevertheless the degrading effects of excitation scattering present in SPIM are inherited by DSLM. Further improvements were reported (Keller et al
.) by combining DLSM (and SPIM) with structured illumination (SI), with the aim to mitigate the blurring effects of the out-of-focus scattered light [10
]. In this approach the sheet is modulated to create sinusoidal patterns over the sample. Digital post-processing of the obtained images allows for the rejection of fluorescence generated by scattered excitation light, resulting in an enhanced optical sectioning and increased contrast. Multidirectional selective plane illumination microscopy (mSPIM) [11
] has also been proposed to reduce absorption and scattering artifacts. In mSPIM the light sheet is i) rapidly tilted about the detection axes, and ii) sequentially directed onto the sample from two opposing directions, providing an evenly illuminated focal plane. The two images obtained are further combined by digital image fusion techniques [12
]. Notwithstanding, for large and highly scattering samples, and due to the short excitation wavelength, some of the aforementioned problems remain: undesired intensity modulations, loss of resolution and limited penetration depth.
Recently, Truong et al.
] reported on the use of a scanned light sheet microscope using TPEF (2p-DSLM) for live imaging of fruit fly and zebra fish embryos. They show the advantages of using 2p-DSLM for imaging large highly scattering samples over the conventional 2p-LSM and 1p-DSLM. Basically, the use of TPEF increases the penetration depth, improves background rejection and reduces phototoxic effects. In addition, the line scanning configuration improves the excitation efficiency and increase the tolerance to aberrations. These advantages allow deep, fast, non-phototoxic imaging of living organisms. Another improvement that has been implemented in order to alleviate the deleterious effect of scattering on scanned sheet microscopy is the use of Bessel beams (BB) [14
]. Self-healing properties of these beams allowed imaging 50% deeper inside human skin when compared with Gaussian beams. However, as side lobes of the BB normally introduce a certain amount of background signal to the images acquired, the use of confocal-line detection should be implemented. Another alternative is the use of high NA objective lenses to combine BB with both TPEF and SI. This technique was reported in terms of achieving enhanced isotropic 3D resolutions and was compared to other super-resolution techniques for imaging intracellular features in single cells in a small field of view [15
In this paper we will show how 2p-DSLM combined with advanced spatial shaping of the beam, by using BB, can be used to improve the optical sectioning, the resolution and the intensity distribution uniformity of the light sheet in large fields of view and for moderately large specimens. This is compared with Gaussian beams in the nonlinear regime and with both Gaussian and BB in the linear regime. We present results on the system characterization and on imaging living C. elegans
. To the best of our knowledge, this is the first time that 2p-DSLM has been combined with BB excitation to image multi-cellular organisms. The results are put in to context (and for reference purposes only), by producing an image stack using a well demonstrated and optimized SPIM imaging system [16
] (in this case working at the excitation wavelength of 488 nm and having a standard GFP band-pass filter (GFP: 526/39) in the collection path).