Light sheet fluorescence microscopy uses parallelized excitation and a perpendicular geometry between excitation and detection to enable optically sectioned, high speed, volumetric imaging with massively reduced photo-damage and photo-bleaching compared to conventional imaging methods. Although this combination of benefits is enabling for biology, the spatial resolution of the light sheet microscopy has lagged behind other convention microscopy systems.
The main reason for the relatively poor resolution of light sheet microscopy is that the perpendicular geometry required necessitates the use of relatively low numerical aperture objectives. Acquiring images from multiple directions/views or using structured illumination to illuminate the sample in combination with image processing software does improve resolution, but these approaches fail to improve spatial resolution in light sheet microscopy applications to the extent possible in conventional microscopy, such as confocal microscopy. Furthermore, multi-view acquisition or structured illumination approaches have thus far required the sample to be exposed to more illumination than conventional, single-view light sheet fluorescence microscopy, thereby sacrificing some of the available signal/photon budget and mitigating the original advantages of light sheet fluorescence microscopy. In addition, such approaches require more images to be captured than a single-view light sheet fluorescence microscopy, thereby sacrificing the inherent speed.
As shown in FIG. 1, a conventional light sheet microscopy system, designated 10, is illustrated, in which a light sheet 18 generated from an illumination source (not shown), such as a laser source, is introduced into the sample (not shown) through a first objective lens 12. The illuminated sample emits fluorescence emissions 20 scattered at substantially a ninety degree angle relative to the light sheet 18 illuminating the sample and detected by a second objective lens 14. In particular, scanning the light sheet 18 through the first objective 12 while maintaining the focus in the second objective lens 14 generates an imaging volume of the sample. Although ninety degree detection ensures that the illumination plane defined through the sample is in focus, and that minimal out-of-focus light corrupts detection of the fluorescence emissions 20, it requires the use of relatively low NA optics. In addition, the majority of the fluorescence emissions excited by the light sheet 18 is scattered outside the aperture of the second objective 14 and wasted.
To collect a portion of the wasted fluorescence emissions 22, a third objective lens 16 may be positioned to capture some of the wasted fluorescence emissions 22 emitted by the sample. If the fluorescence emissions are imaged conventionally using only a tube lens and camera arrangement (e.g., conventional epifluorescence microscopy), relatively little of the fluorescence emissions 22 are used efficiently (i.e., “in focus”). This is because the depth of field of a high numerical objective is inversely proportional to the square of the numerical aperture.
Other techniques seek to capture more imaging planes at once and fusing them together to improve resolution relative to conventional light sheet fluorescence microscopy; however, such techniques suffer from out-of-focus light. As such, further improvements in the collection of fluorescence emissions in a light sheet microscopy system are desired.
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