Super temporal resolution microscopy allows imaging of fluorescent molecules 20 times faster than traditional lab cameras normally allow. A general method has been developed to let a microscope capture 3D spatial information along with the fourth dimension: molecular movement over time. This will help scientists who study dynamic processes view where molecules of interest are located and how fast they move; for example, within living cells.
The method to expand the capabilities of existing wide-field fluorescence microscopes is based on creation of custom phase masks — transparent, spinning disks that manipulate light’s phase to change the shape of the image captured by the microscope’s camera. The shape contains information about a molecule’s 3D position in space and how it behaves over time within the camera’s field of view. A phase mask turns the blurry blob in a microscope image into an asset. This blob — called a point spread function — is used to get details about objects below the diffraction limit that are smaller than all visible light microscopes are able to see.
Algorithms were developed to make it practical to design custom phase masks that modify the shape of the point spread function. The new phase mask design — called a stretching lobe phase mask — decouples space and time. When the targets are at different depths, the lobes stretch farther apart or come closer, and the time information is now encoded just in the rotation.
The method manipulates light at the spinning phase mask to optimize the pattern for different depths by the refractive pattern programmed into the mask by the algorithm. Each layer is optimized in the algorithm for different detection depths; all three spatial dimensions and fast time behavior can be seen simultaneously.