A recently developed technique known as light-sheet fluorescence microscopy has led to many biological discoveries by allowing researchers to create 3D images of tissue, even live animal embryos, using fluorescent tags. Now, researchers report the ability to increase the imaging depth of light-sheet fluorescence microscopy with the use of an optical phenomenon known as three-photon absorption.
In The Optical Society (OSA) journal Optics Letters, the researchers report in detail how three-photon absorption can be used with light-sheet fluorescence microscopy to image deeper into tissues. As a demonstration, they used the combined technique to produce clear images throughout a ball of cultured cells, known as a spheroid, about 450 microns in diameter.
"This demonstration is very important as it addresses an unmet need of better imaging at depth, which could help scientists gain better data about biological processes," says research team leader Kishan Dholakia from the University of St. Andrews in the UK. "This approach could be especially useful for neuroscience and developmental biology studies and might find application in imaging multiple samples in an automated way for drug discovery."
The light needed to image fluorescent labels can be damaging and even deadly to delicate biological samples such as brain tissue or animal embryos used to study development and disease processes. Light-sheet fluorescence microscopy allows fast, high-resolution imaging with little optical damage because it illuminates a sample with just a thin sheet of light; other parts of the sample don't get any unnecessary light exposure.
"We expect three-photon light-sheet fluorescence microscopy to make a large impact on imaging the brain in rodents such as mice and rats, where it could be used to capture very wide-field images with subcellular resolution," says the paper's first author, Adrià Escobet-Montalbán.
The researchers wanted to compare three-photon light-sheet fluorescence microscopy to the previously used two-photon absorption. In multiphoton absorption, the fluorescent label gives off light after absorbing, or being excited by, two or three photons rather than the one photon used to produce traditional fluorescence.
Multiphoton absorption reduces out-of-focus light and minimizes light that could harm the sample because it uses longer wavelengths, which are scattered less by tissue, and by confining the excitation light to a small volume. When three photons are used to produce fluorescence rather than two, these benefits are amplified.
To demonstrate their new technique, the researchers used a standard optical setup for light-sheet fluorescence microscopy with a pulsed laser that is traditionally used for two-photon excitation. Although this laser was not the most appropriate for creating efficient three-photon excitation, it was ideal for comparing two-photon and three-photon excitation.
The research team imaged spheroids of human embryonic kidney cells using two-photon and three-photon excitation. Near the spheroid's surface, both imaging modalities performed similarly. However, at the far side of the spheroid, the image quality for the three-photon light-sheet fluorescence microscopy preserved image contrast while the quality of the two-photon image declined considerably.
Optimizing the technique
To further improve the depth imaging and field of view, the researchers experimented with changing the light intensity profile of the laser to a Bessel beam, which has a central bright core surrounded by concentric rings, rather than the traditional solid Gaussian laser beam like that of a laser pointer.
"Bessel beams can be used in two-photon light-sheet mode but may yield potential artifacts due to their concentric rings," says co-author Federico Gasparoli. "For the first time, we show numerically and experimentally that these problems are suppressed in three-photon light-sheet fluorescence microscopy and that the beam goes even deeper, making this approach very attractive."
Next, the researchers plan to improve the technique by using laser systems at longer wavelengths that are specifically designed for three-photon absorption. This should enable imaging at increased depth. In parallel, the researchers are working to develop ways to detect the light emitted from fluorescent labels deep inside samples.