Applications of SPIM

SPIM is commonly used for studying development of intact, living embryos. Embryos are usually in size of a few millimetres which is a relatively large size in optical microscopy.

When imaging this large specimen, the light scatters and is absorbed into the tissue which lowers the resolution and the quality of images. However, if the sample is rotated during the imaging, the desired resolution can be achieved. For example, Huisken et al.

(2004) imaged live embryos of teleost fish Medaka and fruit fly Drosphila melanogaster with conventional SPIM-setup and green fluorescent protein (GFP). They succeeded in imaging the whole Medaka embryo in 3D with resolution higher than 6 μm in depth of 500 μm. In the study, they rotated the sample into four different orientations in order to achieve 3D reconstruction with high resolution. Besides the whole embryo, they imaged the heart of the Medaka embryo and were able to reconstruct the internal structure of it, including ventricle and atrium, and the heartbeat as a video. The sample rotation resulted in four image stacks that were usable even without further processing.

In addition to the large size of the specimen, the opacity of the specimen also affects the image quality commonly in optical microscopy. To show the performance of SPIM, Huisken et al. (2004) also imaged a fruit fly embryo (Drosophila melanogaster) which appears to be more opaque than other embryos, and they examined that SPIM was capable of reconstructing the embryo even without imaging it from many directions. The

embryo also developed normally despite the high amount of fluorophore excitations dur-ing the study which indicates the lower phototoxicity of SPIM compared to other fluores-cence methods. In addition, in a study by Krzic et al. (2012), an embryo of Drosophila melanogaster was imaged with a bit different setup of SPIM, in which there were two illumination and two detection lenses which imaged the specimen in two axes perpen-dicular to each other. This is called multiview SPIM which does not require rotation of the specimen, resulting in less phototoxicity, higher resolution and faster imaging since rotation can cause artefacts to the images because of the sample movement. The imag-ing time was very short, only 20 seconds for the whole embryo. As they also studied the effects of slow specimen rotation in order to improve the quality further, scanning of the sample from eight directions was achieved in time four times shorter than with conven-tional SPIM due to the imaging from multiple directions at time. On the whole, SPIM is an excellent method for developmental biology since it can achieve resolution below cel-lular level, it is usable in real time and an embryo can be scanned on the whole while it still stays alive during the process. The same principles might be usable also for other specimen in the scale up to a few millimetres.

The second application of SPIM is imaging the central nervous system which has been studied my multiple authors. For instance, Panier et al. (2013) studied the use of SPIM in imaging activity of a Zebrafish brain and achieved promising results. They succeeded imaging several regions of the brain in three dimensions by moving the sample into five different heights relative to the light sheet and recording the calcium activity of the neu-rons. However, during a single recording, they imaged 25 000 neurons which is not enough for formulating a model of the neural network. The sample movement turned out to have some disadvantages, such as the undesired stimulation of the neural system and extension of the imaging time since the sample has to be stationary in order to achieve high quality. In addition, the system did only provide single-cell resolution in the proximal parts of the brain because of the light sheet shape. However, Panier et al.

(2013) state that creation of a brain atlas will be possible with SPIM in the future.

Another example of examining the neural tissue is a study by Funane et al. (2018) where a combination of SPIM and FLIM was produced for lifetime imaging of a cleared mouse brain by injecting GFP carrying viruses into the brain before the removal process. Alt-hough they managed to collect 3D lifetime data with axial resolution of 9,9 μm and lateral resolution of 7 μm, the imaging time appeared to be quite long, approximately 100 minutes for single plane. SPIM-FLIM is an informative method when different neurologi-cal diseases are studied. Funane et al. also state that their imaging system can be ap-plied to other cleared organs and tissues that are in centimetre size. In similar way, Nie

et al. (2019) imaged a mouse brain in whole with multiangle-resolved subvoxel SPIM (Mars-SPIM) which combines more straightforward imaging than multiview SPIM and more complicated computational reconstruction. As mentioned above, scattering of the light appears to be a problem also when other tissue types are imaged with SPIM-based methods. Nie et al. (2019) were able to reconstruct the whole brain with better resolution and contrast than basic SPIM has achieved. They could also identify different regions of the brain and determine the density of the neurons in each region. According to Nie et al.(2019), the Mars-SPIM -system could also be applied to other organs and it has a high potential to work as a significant method when whole-brain atlases are created. In addi-tion, it could be useful for other histological applications, such as histopathological ex-aminations in support of diagnosis.

SPIM can be modified into open-top surface microscopy which has already been applied to breast tissue imaging during a surgical operation. Glaser et al. (2017) developed a light-sheet microscope with conventional perpendicular design that images the sample from underneath at 45 degrees angle. They imaged the surface of a stained breast tissue sample that was in scale of several centimetres. The imaging time was relatively short, less than 1 minute which would be a suitable amount of time if the system was used during a surgical operation as a diagnostic method. They were able to identify both be-nign tissue areas and malignant tumour areas from the surface-images. When compared with conventional histology samples that were fixed with formalin, embedded in paraffin and stained with H&E, the quality appeared to be very similar. In addition to the breast tissue surface, they performed a volume reconstruction to a core-needle biopsy of a prostate with their light-sheet microscope. They claimed that the system would be a prac-ticable method, for example, in margin status identification of breast tissue samples as a surface microscopy method and in visualizing complicated tissue structures in three dimensions. However, the 3D reconstructed sample was very thin in volume (1 millime-tre) and had to undergo a time-consuming clearing procedure before the imaging, where-upon it cannot be directly assumed that the method could succeed in imaging samples with greater volumes. On the other hand, as the group successfully reconstructed a core needle biopsy of a prostate and it is also commonly used in breast cancer diagnosis, SPIM would be also useful in such purpose.

The usefulness of SPIM has been proven with other additional applications, such as imaging the follicles of a pig’s ovarian with higher resolution and speed than conventional ultrasound microscopy (Lin et al. 2016). In addition, the use of SPIM for studying tumour genesis and formation of metastases has been implemented in several different studies.

For instance, Lloyd-Lewis et al. (2016) used SPIM to image mammary gland epithelia

cells of a mouse with different clearing methods, and resulted in 3D reconstructions us-able for further understanding of both normal and tumour tissue development of mam-mary gland. In similar way, Alladin et al. (2020) studied formation of a breast cancer tumour by tracking cell-cell interactions for 4 days within 3D cell culture of mammary gland cells. The metastasis formation was studied by Asokan et al. (2020) by performing SPIM imaging to track the movement and interactions of breast cancer and leukemic cells in circulation of a zebrafish embryo for 30 hours. Figure 4 presents breast cancer cells that are labelled with GFP and therefore are seen in green. On the left, the cancer cells are present as single cells, in the middle they are loosely together and on the right they have formed a cell cluster.

Figure 4. Breast cancer cells in circulation of a zebrafish embryo, imaged by SPIM.

The cancer cells appear green, as the structure of the embryo appears ma-genta. (Asokan et al. 2020)

All in all, the use of SPIM can lead to deeper understanding of cancer formation, for instance, in breast cancer research since samples of appropriate size can be imaged on the whole and tracking of cells can be performed with high resolution and continuous scanning which is a great advantage when compared with existing methods, such as MRI.