Imaging developmental processes often requires time-lapse 3D-image acquisitions (4D imaging). The imaging speed of a microscope can be defined by its pixel (or voxel) rate, i.e. the number of pixels per unit time that can be obtained with sufficient signal and contrast. A high pixel rate permits capturing with adequate time resolution fast processes such as heart development (50–130 frames per second (fps) in [14
]), cilia beating (900 fps in [17
]) or fluid flow in developing embryos (44 fps in [18
]). A high pixel rate is also required to study slower large-scale processes such as collective cell migration or cell division patterns with a large number of pixels per image to reach the appropriate spatial resolution: for instance, in toto
imaging of early development [16
] typically requires acquiring ~100 million voxels per 3D-image stack in less than a minute.
In this context, point-scanning confocal or multiphoton approaches are usually too slow, as the image is recoreded one pixel at a time (). Indeed, in these approaches signal level prescribes pixel accumulation times of typically 1–10 μs, corresponding to pixel rates of only 105 to 106 pixels.s−1.
Strategies for improving acquisition speed in current fluorescence microscopy techniques
Several approaches have been explored during the last 15 years to improve the imaging speed of multiphoton microscopy up to ~107
, including fast point-scanning and multifocal approaches (, and , and [21
] for a review). However, besides hardware limitations (i.e. scanning speed, readout time, data transfer or storage) the pixel rate of any microscope is fundamentally limited by the signal level that can be obtained within the pixel accumulation time without causing fluorophore saturation or photodamage (including phototoxicity to the biological sample and photobleaching of the fluorophores). Hence, even though fast point-scanning can be implemented using resonant scanners, polygonal mirrors or acousto-optic deflectors [21
], the useful pixel rate is still limited by fluorophore photophysics of the single-point excitation approach (third column in ). The main strategy to circumvent this limitation is to parallelize the sample illumination and the signal detection. Using multifocal excitation (), overall pixel rate can be increased while maintaining the same illumination time per pixel (). However with this approach, an increase in imaging speed requires a proportional increase in laser average power ( and ), similar to linear microscopy (Supp. Table 1
). Available laser power therefore limits the achievable speed gain. Moreover, increasing the laser average power may eventually lead to linear absorption and photodamage, as it is the case in linear microscopy.
Strategies for fast acquisition speed in multiphoton microscopy: theoretical scaling of illumination parameters
Strategies for fast acquisition speed in multiphoton microscopy: example of experimental illumination parameters
Among the strategies for improving the imaging speed of multiphoton microscopy, the recent implementation of scanned light-sheet microscopy using two-photon excitation (2p-SPIM in [16
] and light-sheet 2p-microscopy in this review) introduces a new paradigm. In this technique, a sheet of light is generated by scanning a weakly focused Gaussian beam faster than the image acquisition time to illuminate an entire plane of the sample, which is then imaged with a camera oriented orthogonally to the sheet. Compared to a static light-sheet generated with a cylindrical lens [22
], this scanned light-sheet approach generates typically 100 times stronger 2PEF signal [16
], which is critical for live imaging. Light-sheet 2p-microscopy is the only technique improving overall pixel rate over point-scanning 2p-microscopy with longer pixel accumulation time and lower peak intensity ( and ). This fundamental property results from the orthogonal geometry of the illumination and detection pathways (which are collinear in conventional microscopy), allowing the use of a low numerical aperture (NA) illumination focusing (resulting in a large illumination volume) without degrading the axial resolution and the overall signal rate [16
]. The use of low-NA illumination has three important advantages for multiphoton live imaging. First, it results in lower peak intensity, and therefore less higher-order nonlinear photodamage to the tissue [23
]. Second, parallelization of the illumination is done along the light propagation direction, reusing the same excitation energy, thus requiring less laser power than in multifocal approaches, in turn limiting linear absorption and photodamage. Finally, the weakly focused excitation beam is less sensitive to sample-induced optical aberrations and resolution loss with depth than in the case of high-NA focusing [16
]. In addition, in the conditions presented in Table 3 [16
], the laser is scanned ~15 times during the image acquisition with 1 ms between two passes. This temporal excitation pattern potentially results in lower photobleaching, as time is given for fluorophore dark state relaxation [24
Overall, compared to other fast multiphoton techniques, light-sheet 2p-microscopy provides fast acquisition while reducing photodamage and requiring minimal increase in laser power as demonstrated in live embryos [16
] (). To date, it is the fastest implementation of multiphoton microscopy with up to ~1.1 107
pix/s (). We note however that light-sheet microscopy relies on widefield (camera-based) detection, which leads to compromises in terms of imaging depth, as discussed in the next section.
Selected applications of advanced multiphoton microscopy in developmental biology