Basic principles

Why Light sheet microscopy?

In wide-field and confocal microscopy, the sample is exposed to light also above and below the focal plane. The same is true for two-photon microscopy but here longer wavelengths are used and fluorescence excitation is mostly limited to the focal plane. In total internal reflection and light sheet microscopy, the specimen is only exposed to light in the focal plane. Selective illumination is especially beneficial for specimens that are much thicker than the focal plane.

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.

(A) The illumination and the detection objectives are oriented orthogonally. The sample is placed at the intersection of their focal planes. A single slice of the sample is illuminated with a thin sheet of laser light. (B) Viewed from top, the light sheet has a waist in the center of the field of view and overlaps perfectly with the focal plane of the detection objective.

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.

Comparison of photobleaching during volumetric imaging using light sheet and spinning disk confocal microscopy. LLC-PK1 mEmerald-EB3 cells. Imaged on two different microscopes with settings adjusted for similar initial signal-to-noise ratio. Light Sheet (green), Confocal (magenta). Intervals around mean indicate standard deviation. Image gallery shows maximum intensity projections of a 30×80µm region of interest. Scale bars: 20µm.

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.

Fixed Drosophila embryo with Sytox Green nuclear marker, mounted in 1.5% agarose and imaged with epi-fluorescence and light sheet illumination. Epi-fluorescence illumination: 470 nm LED excitation, 488 nm LP dichroic mirror. Light sheet illumination: 491 nm excitation. BP525/50 emission filter, 50 ms exposure time. Z-stacks w/ 1 μm z-spacing. Sample by Jeehae Park, Harvard Medical School. Scale bar = 50 μm.

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?

Two strategies to record z-stacks in a light sheet microscope. (a) Translating the sample. The specimen is moved through the static focal plane of the detection objective OLd, which is continuously illuminated with a light sheet. (b) Translating illumination and detection. The focal plane is moved through the static specimen, for example, by translating OLd. Simultaneously, a scan mirror maintains a light sheet in the focal plane of OLd.

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?

Obstacles in the field of view potentially result in stripes along the propagation direction of the light sheet when using single-sided illumination.

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.

Correction of sample-induced effects on the light sheet using double-sided illumination.

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.

By using a light sheet that pivots around the center of the field of view within one exposure, the propagation direction is constantly altered. Thereby, obstacles are homogeneously illuminated from a range of angles, and the sum of the excited fluorescence results in an image with a minimum number of visible stripes.

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).

Multiview acquisition using stepwise rotation of the sample. A specimen is rotated around its center in steps of, for example, 90°, and z-stacks are acquired from each angle (multiview acquisition). Single-sided illumination and detection result in about 25% coverage of the sample. Multiview registration and fusion combines the well-covered areas of each recording into a homogeneous dataset.

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.

Literature