First TIRF Live cell Study of Internal Cell Structures Uses Andor Camera

US scientists have performed an advanced study of structures inside live cells using total internal reflection fluorescence microscopy (TIRF), thanks to a high-speed camera and software from Andor Technology.

The experiment, which was carried out by two postgraduates from Yale University, captured the first time-lapse pictures of the APPL1 endosome, a type of vesicle that carries signals inside a cell from its surface. By tracking the vesicle using time-lapse imaging, they found APPL1 helps cells communicate with each other.

Previously, scientists had believed most intercellular communication occurred at a cell’s surface. The new research shows signalling between cells is much more complex than originally thought.

Furthermore, it gives an insight into diseases associated with APPL1 like Dent disease, a rare kidney disease, and Lowe syndrome, a rare eye, kidney and brain condition. The APPL1 vesicle could also have a role in cancer and metabolic diseases such as diabetes.

“TIRF is designed to image things close to the surface of the cell with unmatched definition and signal-to-noise,” says Dr Roberto Zoncu, one of the postgraduates who performed the experiment. “But it wasn’t thought to be ideal for endosomes.”

The researchers predicted that some vesicles would station close to the cell surface after they entered the cell, meaning they could be observed using TIRF. They captured time-lapse images of the fluorescent-labelled vesicles as they moved through the cell using a highly-sensitive Andor iXon 897 EMCCD camera.

“We have proved we can image things moving from the surface of the cell inside using TIRF,” says Dr Zoncu. “And that’s an advantage because it’s the most sensitive technique of all.”

The team also attributes their success to the Andor camera and software. “Because the vesicles are very small, the light sensitivity of the camera has to be as high as possible. This camera is the most sensitive available in our experience. It’s better than any competitor around,” says Dr Zoncu.

The fluorescent dots indicating each vesicle were tracked from image to image using Andor’s iQ image analysis software. According to Dr Zoncu: “The software comes with the camera, but it has many advantages. It is very user-friendly during image acquisition and it’s flexible. It also allows us to do some image analysis, which is very important in our work.’

The time-lapse results suggest APPL1 transforms into an early endosome – a compartment that acts like a ‘sorting office’, dispatching signals to different parts of the cell. The team also discovered that a phospholipid (fat) molecule called phosphatidylinositol-3-phosphate (PtdIns(3)P) is the molecular switch that turns the APPL1 vesicle into an endosome.

Dr Zoncu has high hopes for future research on APPL1. “Thanks to this technology, we can focus further studies on diseases where this endosome is likely to be involved,” he says.

The research was published in Cell last year.