The Vitrobot is an automated laboratory instrument used in modern biological research. It allows scientists to visualize biological structures like proteins, viruses, and cellular components. This device functions like a molecular flash-freezer, preserving delicate biological specimens for detailed structural analysis. Its precision and automation have transformed microscopic research.
The Goal of Vitrification
When water freezes slowly, its molecules form sharp ice crystals. These crystals can severely damage delicate biological structures like cell membranes and proteins, distorting their natural shape and making accurate scientific study impossible.
Vitrification freezes a sample so rapidly that water molecules do not form destructive crystalline structures. Instead, water instantly locks into an amorphous, glass-like solid state, preserving biological material in its near-native condition. This rapid solidification prevents damaging ice, ensuring the sample’s molecular integrity.
How the Vitrobot Works
The Vitrobot maintains a controlled environment within its chamber, regulating temperature and relative humidity. This prevents the liquid sample from evaporating or drying out prematurely, ensuring it remains stable and hydrated during initial preparation.
A drop of the biological sample, typically 2-5 microliters, is applied onto a porous metal mesh called an electron microscopy (EM) grid. This grid, often made of copper, gold, or nickel, supports the sample during freezing and imaging. The sample can be applied manually or automatically, with precise control over application and wait times before blotting.
Robotic filter paper arms then blot away excess liquid from both sides of the grid. This precisely timed action, often 3-4 seconds, creates an ultra-thin sample layer, typically less than 100 nanometers thick. This thinness is essential for successful vitrification, enabling the rapid cooling needed to prevent ice crystal formation. Blotting parameters like force and duration are adjustable, and multiple blotting actions are possible.
Following blotting, the robotic arm rapidly plunge-freezes the grid into liquid ethane. This ethane is pre-cooled to approximately -180 degrees Celsius by liquid nitrogen. The combination of the ultra-thin sample layer and the fast plunge ensures water molecules vitrify instantly, preserving biological structures in their native, hydrated state.
Preparing Samples for Cryo-Electron Microscopy
The Vitrobot is the initial step in the workflow for Cryo-Electron Microscopy (Cryo-EM). Cryo-EM is a powerful imaging technology that determines the three-dimensional structures of biological molecules at near-atomic resolution. It captures images of proteins, nucleic acids, and other macromolecules in their natural, hydrated state, without crystallization. The Vitrobot prepares these delicate samples for effective imaging.
The Vitrobot’s output—a vitrified sample embedded in an amorphous ice layer on an EM grid—is directly compatible with Cryo-EM instruments. This grid, with its frozen-hydrated specimen, is then transferred under cryogenic conditions into the electron microscope. The quality and uniformity of the vitreous ice layer, along with the even distribution of sample particles, are important for high-resolution images. Without the Vitrobot’s meticulous sample preparation, the electron microscope’s capabilities would be underutilized.
The Impact of Automated Sample Preparation
Before automated systems like the Vitrobot, preparing samples for cryo-electron microscopy was a manual process. This approach was difficult to control, often leading to inconsistencies and sample failure due to variations in blotting and plunging. Consistently achieving the precise, ultra-thin vitreous ice layer was a significant challenge.
The Vitrobot’s automation brought two advancements to structural biology. First, it introduced reproducibility, ensuring samples are prepared under nearly identical conditions, including temperature, humidity, blotting force, and freezing velocity. This consistency is invaluable for reliable experimental results. Second, automation greatly increased throughput, allowing scientists to prepare many high-quality samples quickly. This acceleration has made Cryo-EM more accessible and routine.