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US-20260126358-A1 - Rapid vitrification of biological samples

US20260126358A1US 20260126358 A1US20260126358 A1US 20260126358A1US-20260126358-A1

Abstract

A method of sample preparation in association with an electron microscope. In one embodiment, a carrier supporting a biological sample is received. A high-pressure freezing (HPF) protocol is then applied to the carrier. The protocol comprises a first phase, and a second phase that follows the first phase. The first phase comprising (i) comprising: a pressurizing operation applying a pressure to the carrier, and (ii) a rapid cooling operation using a coolant that avoids a Leidenfrost effect at a carrier-coolant interface by maintaining nucleate boiling or substantially suppressing film boiling upon contact with the carrier. The pressurizing and rapid cooling operations freeze the biological sample while preserving its structural integrity. The second phase involves raising a temperature of the carrier and the frozen biological sample to a given temperature for one of: storage, and use.

Inventors

  • Alex DE MARCO

Assignees

  • NEW YORK STRUCTURAL BIOLOGY CENTER

Dates

Publication Date
20260507
Application Date
20251106

Claims (12)

  1. 1 . A method of sample preparation in association with an electron microscope, comprising: receiving a carrier supporting a biological sample; and applying to the carrier a high-pressure freezing (HPF) protocol comprising a first phase, and a second phase that follows the first phase; the first phase comprising (i) a pressurizing operation applying a pressure to the carrier; and (ii) a rapid cooling operation using a coolant that avoids a Leidenfrost effect at a carrier-coolant interface by maintaining nucleate boiling or substantially suppressing film boiling upon contact with the carrier, wherein the pressurizing and rapid cooling operations freeze the biological sample while preserving its structural integrity; the second phase raising a temperature of the carrier and the frozen biological sample to a given temperature for one of: storage, and use.
  2. 2 . The method as described in claim 1 , wherein the coolant is one of: liquid helium, liquid neon, slush nitrogen, liquid propane, and combinations thereof.
  3. 3 . The method as described in claim 1 , wherein the temperature is the temperature of liquid nitrogen.
  4. 4 . The method as described in claim 1 , wherein the pressure is in the range of 1800 – 2400 bar.
  5. 5 . The method as described in claim 4 , wherein the pressure is 2100 bar.
  6. 6 . The method as described in claim 1 , wherein the biological sample has a thickness up to 200μm thick.
  7. 7 . The method as described in claim 1 , wherein the biological sample has a thickness up to 300μm thick.
  8. 8 . The method as described in claim 1 , wherein the carrier is formed of silicon carbide (SiC).
  9. 9 . The method as described in claim 1 , wherein the carrier is formed of synthetic diamond.
  10. 10 . The method as described in claim 1 , wherein the pressurizing operation occurs before or concurrently with the rapid cooling operation.
  11. 11 . The method as described in claim 1 , further including performing one of: a structural study, a functional study, and a combination thereof, on the frozen biological sample.
  12. 12 . The method as described in claim 1 , wherien the coolant has a temperature at or below 120 K and a specific heat capacity greater than that of liquid nitrogen.

Description

BACKGROUND The subject matter herein relates generally to the field of biological sample preparation and preservation techniques. More particularly, the subject matter concerns methods and apparatus for high-pressure freezing (HPF) of biological specimens for subsequent microscopic examination and analysis. Single particle cryo-Electron Microscopy (cryo-EM) revolutionized structural biology, enabling resolution of the structure of molecules in a purified near-native condition, as well as resolution of dynamics in large complexes that were, up until a few years ago, impossible to analyze. Cryo-electron tomography (cryo-ET) is a technique akin to a single particle that recently emerged as an enabler for performing structural analyses directly in the cell, where biology occurs without purification. Cryo-ET provides snapshots of the cellular landscape, revealing the spatial relationships between macromolecules, organelles, and other cellular structures in their native state at molecular resolution. It bridges this gap between isolated molecular structures and cellular context. The preservation of biological samples (or more generally aqueous samples) in their native state is crucial for structural and functional studies at the cellular and molecular level using cryo-EM or -ET systems. Traditional chemical fixation methods often introduce artifacts and fail to capture the true ultrastructure of biological specimens. This limitation has driven the development of physical preservation methods, particularly those employing rapid freezing techniques. High-pressure freezing (HPF) was developed for aqueous sample preservation in the 1960s. The technique involves the application of high pressure (approximately 2100 bar) during the freezing process, which prevents the formation of crystalline ice and instead promotes the formation of vitreous ice. This vitrification process is essential for maintaining the structural integrity of biological samples. Conventional freezing methods at atmospheric pressure are limited by the formation of crystalline ice structures. Even if a sample is plunged in liquid ethane (T˜80K) the water will transition from liquid to crystalline if the thickness is higher than 10um. The phase diagram of water reveals that at typical ambient conditions on Earth (approximately 0.1 MPa), cooling results in the formation of hexagonal ice (Ice Ih). This crystallization process causes mechanical stress through volume expansion and chemical alterations within the sample, leading to structural artifacts and compromised analysis. While cryoprotectants can modify this process, traditional freezing methods cannot completely prevent crystalline ice formation in samples thicker than a few micrometers. Moreover, the phase diagram demonstrates that different crystalline ice forms emerge in distinct regions of pressure-temperature space: at atmospheric pressure and temperatures below 0°C, Ice Ih is the stable phase, which is particularly problematic for biological preservation due to its hexagonal crystal structure. The application of high pressure during freezing addresses these limitations by exploiting water's complex phase behavior to promote vitrification rather than crystallization. The water phase diagram reveals multiple triple points where three phases can coexist, with the liquid-Ice Ih-Ice III triple point occurring at 209.9 MPa and -22°C (251.15K), and the liquid-Ice V-Ice VI triple point at 632.4 MPa and 0.16°C. High-pressure freezing typically operates at pressures around 2100 bar (210 MPa), strategically chosen near the liquid-Ice Ih-Ice III triple point. At these conditions, rapid cooling can bypass the formation of any crystalline ice phases (including Ice III and Ice V) through a pressure-induced vitrification process. This process is governed by two primary mechanisms: first, the increased pressure alters water's phase transitions, as evidenced by the phase diagram's liquid water region extending to lower temperatures under pressure. Second, the combination of pressure and rapid cooling rates (>10,000°C/s) promotes the formation of amorphous ice, where water molecules maintain a disordered, liquid-like arrangement. The transition between low-density amorphous (LDA) and high-density amorphous (HDA) forms of ice occurs in this pressure regime, with evidence suggesting a second critical point in the phase diagram associated with this transition. However, this remains subject to experimental verification. The phase diagram reveals that at pressures above 209.9 MPa, water can transition through multiple crystalline phases (Ice III, V, and VI) depending on the specific pressure-temperature path. High-pressure freezing protocols must therefore be carefully designed to avoid these crystalline phases, as their formation would compromise sample preservation. This is achieved by selecting pressure-temperature conditions and cooling rates that favor direct vitrification while avoiding the thermodynamically st