Visualising cell behaviour has been the goal of many techniques developed in the 20th and 21st century. Cells were initially visualized after removing from the organism after death.
However, real-time visualisation of cells through fluorescent labelling still proved difficult due to the degree of transparency required to see the fluorescence. Similarly, imaging techniques have had problems with light scattering. Selective plane illumination microscopy (SPIM) or light-sheet microscopy differs offers high resolution imaging at various cell depths in live tissues.
SPIM techniques can differ slightly, but follow the same general principle: the light excitation originates at right angle to the optical lens. Because of this, the illumination path and the detecting path (the lens) are independent of each other and can be manipulated separately. This differs from conventional microscopy approaches, which typically have the same illumination and detecting pathway source. The object of interest is placed at the focal plane of the detecting lens where the illumination and detection path meet. The sample is excited by the illumination source and then detected by the detection optics and imaged using a camera. For a 3D image, the sample is moved slightly several times to acquire several images to visualize the depth.
Breaking the diffraction limit
The illumination source forms an important aspect of what type of image you get and how detailed. The perfect illumination would be a constant and thin light source across the entire illumination path. However, the light sheet is limited by diffraction. Instead, a cylindrical light source is used which varies in thickness across the path of illumination by converging towards the centre. The thickness of the light sheet is adjusted to keep the light uniform across the field of view, ranging from 1 µm for small samples (<100 µm) to 10 µm for larger samples (5 mm).
Imaging thick samples
The illumination source also determines the depth of the sample , which is usually around 10 µm or less. When SPIM was used on a fish, it reached an imaging depth of up to 500 µm. It has applications in the study of whole organisms, as the SPIM imaging apparatus can be built up around an organism’s chamber so that the animal is kept alive and in optimal conditions.
Microscopy: Super-Resolution: Structured Illumination Microscopy (SIM) (David Agard)
Increased axial resolution in developmental biology
In developmental biology, the detection apparatus needs to have long working distance lens (distance from lens to cover slip) and low numerical aperture (ability of optical system to gather or emit light). Such lenses have poor axial resolution, but in SPIM the amount that is illuminated is defined by the light-sheet which increases axial resolution. When SPIM was originally developed, authors cited axial point spread function (PSF) of 6 µm compared to the above 20 µm when the light sheet was removed.
Imaging single and multicellular structures
SPIM has applications in both single cells and multicellular structures. Cells can in other microscopy techniques be exposed to bleaching, where fluorescent molecules are destroyed by light. In SPIM, bleaching is low, which is particularly beneficial for single cell study. Low bleaching allows continuous acquisition of images of dynamic structures. With the advent of growing 3D cultures, SPIM is also proving useful to understand their development inside the 3D chamber.
Structure and dynamics of microtubules
Intracellularly, the biophysics and dynamics of microtubules have been better understood using SPIM. Low bleaching allowed for continuous acquisition for 15 minutes in 3D. Previous studies done on 2D surfaces have revealed the structural arrangements of various sections of the microtubules, but it was unclear how the centrioles move in 3D space. The data collected using SPIM can be used to refine models of how microtubules behave in a realistic, 3D space.
Imaging in vivo It is known that many biological processes, such as growth and apoptosis, are regulated or triggered by complex interactions around them. Thus, increasing research is focussed on how to investigate these phenomena in more natural settings, such as 3D cultures and organoids. SPIM investigates cells when the lines between in vivo and in vitro are blurred. Techniques for this are currently being developed, with the hope that this will further accelerate our understanding of fields already benefitting from 3D cell cultures, such as immunology and cancer research.