HIST LSFM Project
This figure shows how at any given frame HIST images only at the intersection (red rectangle) of the beam waist (blue) and focal plane (black rectangle) by spatially filtering via using an ROI on the camera CCD.
The entire microscopy system, excluding the light engine.
An optical diagram produced by group (see Superfine et al.) detailing the specifics of the versatile optics system.
Currently, I am studying light sheet fluorescence microscopy (LSFM), a technique which uses a light source, typically a laser, focused by a cylindrical lens in one axis so that it appears as a light sheet (LS). In our group’s Versatile Optics System we use Steering Mirrors (SMs) and electronically tunable lenses (ETLs). SMs are capable of rapid and precise voice-coil driven motions, and with them we are able to control the position of the LS as well as its angle of attack. ETL dioptric power may also be rapidly and precisely controlled, which enables the swift and non-mechanical adjustment of both excitation and imaging focal planes. Custom-built LabVIEW software allows us to control these and other functions of the Versatile Optics System, allowing for precise and rapid control of the lightsheet’s wavelength, intensity, structure, orientation, and position..
Using this system, we will implement automated Highly Inclined Swept Tile LSFM, which is an advanced illumination scheme in which the light sheet forms a relative large angle with the focal plane. If we use the ETL to ensure that the focal plane of the light sheet intersects focal plane of the microscope, we obtain an ultra-high resolution focal intersection line across the sample where both illumination and optics are primed for optimal resolution. This can then be swept across the sample using the steering mirrors. By using a modern sCMOS camera in rolling shutter mode, we can image only the focal line by using the camera to sweep the region of interest across its CCD in sync with the focal line. This enables us to spatially filter out-of-focus light, drastically improving our signal-to-noise ratio, thus obtaining an incredibly sharp image at the focal plane. The reduction of background noise means that imaging requires less intense illumination, minimizing phototoxicity and photobleaching. After the focal line has scanned the entire plane, we may then adjust the ETLs to move both light sheet and camera focal planes upwards in the sample. By stitching together, the scans of various planes, we will form a three-dimensional volumetric scan. The novelty of this project is our ability to control so many aspects of the illumination scheme via our powerful optical train, which allows for the automation of advanced customization of the light sheet.
Creating this state of the art, single-molecule cellular volumetric scanning system with highly customizable, ultra high-speed control can then be implemented in our macrophage phagocytosis project, which is focused on the studying the physical process by which our immune cells mark and engulf foreign bodies, ultimately neutralizing them. Understanding this process is of great interest to many medical applications, including immunotherapy-based cancer treatments. Our research in this area focuses on both the biophysical interactions between the cell and the object being engulfed as well as the physical chemistry behind the properties of the proteins which enable this incredible process, especially actin. We also plan to use this system in conjunction with our physical chemistry research on the microfluidics of cilia. This project centers on constructing micropost arrays, and HIST LSFM would be used to conduct three-dimensional real-time tracking of fluorescent particles suspended in the fluid, enabling high definition spatiotemporal tracking of the fluid.
A more technical explanation of this project, adapted from a recent grant application, may be found below.