TIRF Vesicle Fusion
Dr. Shyam Krishnakumar
Department of Neurology, Yale School of Medicine, CT, USA
Background
Dr. Shyam Krishnakumar, Assistant Professor in the Department of Neurology at Yale University, leads a research program focused on uncovering the molecular mechanisms that govern neuronal communication. “Nerve cells communicate by releasing chemical compounds called neurotransmitters - often within just a millisecond,” he explains. “We aim to understand the protein components involved, how they assemble and function together to support this ultrafast fusion process, and how their activity can be modulated to meet physiological demands.”
Traditionally, studies of neurotransmitter release have relied on animal models or neuronal cultures. While powerful, these systems often involve multiple protein isoforms and compensatory mechanisms, making it difficult to extract precise mechanistic insights. To overcome these limitations, Dr. Krishnakumar’s lab has developed a cell-free system that reconstitutes neurotransmitter release in vitro using fully defined, minimal components. “We construct a suspended lipid bilayer - mimicking the presynaptic membrane - on a holey silicon chip and track the docking and fusion of individual artificial vesicles that simulate synaptic vesicles,” he says. “This setup gives us precise experimental control and allows us to generate direct mechanistic insights. We've published extensively on this system, primarily using fluorescence microscopy to image fusion events on timescales of tens to hundreds of milliseconds.”
Figure 1: Measurement of Calcium-Evoked Fusion Events. Calcium-triggered rapid fusion of vesicles docked to a suspended lipid bilayer was measured using a content-release assay with Sulforhodamine-B loaded vesicles. Sulforhodamine-B is highly self-quenched when concentrated inside vesicles. Upon fusion, the dye is diluted, leading to a measurable increase in fluorescence. This reconstituted assay recapitulates vesicle fusion driven by synaptic SNARE proteins, while key regulatory factors—including Synaptotagmin, Complexin, Munc18, and Munc13—are incorporated to precisely control the probability and kinetics of fusion. Fluorescence signals were captured using a Teledyne Photometrics Kinetix camera at 500 frames per second, providing a temporal resolution of approximately 3 milliseconds. Graph shows a fluorescence intensity trace of SRB fusion kinetics at 500 fps.
Challenge
The main challenge for the lab was imaging speed. “To study fusion at physiological time scales—on the order of 1 to 5 milliseconds—we needed to significantly enhance our temporal resolution,” Dr. Krishnakumar explains. “At the same time, we required a large field of view to track multiple vesicles, since the synchronicity of fusion events is an important aspect of our research.”
The [Kinetix] camera enables high-speed recordings in our reconstituted system, allowing us to isolate and study individual vesicle fusion events and analyze the synchronicity of release in response to calcium signals - all at near-physiological timescales.
Dr. Shyam Krishnakumar
Solution
To meet these imaging challenges, Dr. Krishnakumar adopted the Kinetix CMOS camera, which combines high-speed imaging with excellent sensitivity at low light levels and a large field of view - ideal for high-magnification studies. “Our collaborator, Professor Kirill Volynski at the UCL Institute of Neurology - who has used a similar setup for years - recommended the Kinetix to me,” says Dr. Krishnakumar. “Integration to our TIRF microscopy setup was seamless, and operationally, it’s been incredibly straightforward. We haven’t had any issues.”
“The Kinetix camera has been transformative for our work. We’re now able to perform the recordings we need with high temporal precision,” Dr. Krishnakumar adds.
