In every implementation of fluorescence microscopy, we want to illuminate a specimen with light of a specific wavelength to excite fluorophores, and then collect the emitted photons by eye or with a detector. Typically, illumination and detection share a part of the microscope’s beam path – at least the objective lens. Hence, illumination occurs from the same direction as detection, resulting in a large part of the specimen being illuminated. Fluorescence microscopy requires powerful illumination, which can have detrimental impacts on specimens, namely photo-bleaching and photo-toxicity. Thus, it is much more effective to limit illumination to the parts of the sample that are actually being imaged. Light sheet microscopy is an elegant implementation of this idea.
The principle of light sheet microscopy – also known as selective plane illumination microscopy (SPIM) – is to illuminate the sample from the side in the focal plane of the detection objective. The illumination and the detection path are distinct and perpendicular to each other. The sample is placed at the intersection of the illumination and the detection axes. The light sheet excites the sample in a thin volume around the focal plane and the emitted fluorescence is collected by the detection optics.
Compared to other illumination schemes in fluorescence microscopy, photo-bleaching is dramatically reduced in light sheet microscopy. Live specimen can be imaged over longer periods of time and/or with higher frequency, while being kept at a healthy state.
The selective illumination of the focal plane in light sheet microscopy not only results in healthier samples, but also provides optical sectioning. Without out-of-focus fluorescence signal, image contrast is significantly improved and the specimen can be reconstructed in 3D.
How is 3D image Data recorded?
Typically, a z-stack is recorded by moving the stage with the mounted sample at a constant velocity through the focal plane, while having the camera record at full speed. Scientific cameras record somewhere around 100 frames per second, with some slower (about 10 fps) and some much faster (>1000 fps). There needs to be enough time to collect fluorescence signal at each plane, but mostly the z-stack recording is limited by the speed of the camera. The linear stages used in the Flamingo are fast enough to keep up with those speeds, as we are are just looking at small volumes (~50-500 µm) and small distances between each plane (a few µm).
If you want to record fast 3D events, a linear stage will set a limit to the repetition rate, because it needs to reset/return after each z-stack. This is when faster solutions like a piezo are helpful, because you can constantly swipe back and forth through the sample. At this point the sample is often kept stationary, with the focal plane and the light sheet swiping through the sample instead.
Why are there two illumination arms?
An illumination arm generates a light sheet that projected into the focal plane of the detection objective from one side. The specimen itself scatters, refracts and absorbs light, so the illumination quality will suffer the deeper the light sheet penetrates the sample. As a result, only one half of a larger sample can be imaged well.
With a second illumination arm, the sample can be illuminated from the opposite side. The effects the sample imposes on the light sheet illumination are the same, but now you record the one half missing from the first image. The “good” parts of both images can be combined into one image covering both halves of the sample.
We integrated an additional technology in the Flamingo to increase the image quality even more. Constantly pivoting the light sheet at high speed drastically reduces stripes and shadows induced by the absorbing and scattering parts of the sample.
Both double-sided illumination and pivoting light sheet are summarized as multi-directional SPIM, or mSPIM.
Why rotating the sample?
The Flamingo features both linear stages as well as a rotational stage. Rotating the specimen can be useful in several scenarios. Often, you want to be able to image your sample from just the right angle (dorsal, ventral, lateral etc.) or want to image an organ that is only visible from certain angles (like the heart).
The other big application for sample rotation is so-called multi-view imaging, where you record z-stacks from multiple angles and then fuse the useful parts of each dataset to generate one 3D image that covers the entire sample. This is useful for larger samples, because both the illumination and the detection quality will suffer the deeper you image, and you cannot capture all the details from one angle.
- Icha, Weber, Waters & Norden, BioEssays 2017: Phototoxicity in live fluorescence microscopy, and how to avoid it
- Weber, Mickoleit & Huisken, Methods in Cell Biology 2014: Light sheet microscopy
- Shah, Weber & Huisken, Wiley 2017: Light sheet microscopy