All too often we encounter a scientist who believes their sample is “too small”, or the detail they need to resolve “too fine”, for a lightsheet microscope. Or worse yet, we never encounter them because they never even consider lightsheet microscopy as an option for their imaging because of their preconceived notions on what lightsheet microscopy is good for – larger samples, embryos, cleared tissue and the like. This, however, couldn’t be further from the truth. The benefits of lightsheet imaging (low phototoxicity, minimal photobleaching) can now be applied to nearly ANY sample you can image on an inverted microscope, using the Tilt lightsheet imaging system.
The idea behind lightsheet microscopy or SPIM (selective plane illumination microscopy) has been around for some time. The setup traditionally makes use of two objectives oriented at 90 degrees from each other. One objective creates the sheet and introduces it into the sample and a second, orthogonal objective is used to capture the image. In contrast to widefield or confocal imaging, in which the entire sample is illuminated at any given time, the lightsheet excites only a single focal plane, offering modest optical sectioning combined with unrivaled phototoxicity and photobleaching properties.
While the first generation of lightsheet instruments have been great for large samples that can be suspended and rotated between the objectives, mounting procedures typically precluded the use of these microscopes on flat samples, such as cellular monolayers or single cells. Additionally, because of physical constraints of focusing a light sheet at the focal length of a second objective – and the requirement to “fit” the sample between the objectives – imaging in this way has been limited to lower resolution applications using long working distance, low magnification, low numerical aperture (NA) objectives.
This is where the technology behind the Tilt offers several advantages. By using an interference pattern to improve the characteristics of the lightsheet “waist” – and by tilting the optics at a slight angle – a narrow sheet of light can be placed in focus above any objective. This includes the high resolution, high magnification, and high NA objectives previously incompatible with lightsheet microscopy. To add to this, sample prep on the Tilt requires nothing more than a coverslip bottom and a clear, flat side to the sample chamber. Anything you can image with a standard inverted setup (widefield, point scanners, spinning disc, TIRF, etc.) – you can image with the Tilt with the lowest possible photobleaching and phototoxicity available
We want to continue to change perceptions about what is possible with lightsheet imaging. Bring your fish, worms, yeast, embryos, oocytes, tissue culture, cell extracts, whatever – and imagine the information you can get with the healthier cells, longer experiments, and higher time resolution that lightsheet makes possible.