Method and device for ultra-rapid cryo-fixation of a sample for microscopic studies
Inventors
Bastiaens, Philippe I.H. • Hubinger, Jan • Grecco, Hernán Edgardo
Assignees
BUENOS AIRES, University of • National Scientific & Technical Research Council • Max-Planck-Gesellschaft zur Förderung der Wissenschaften eV
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Publication Number
US-12644806-B2
Publication Date
2026-06-02
Expiration Date
Abstract
The present invention relates to method for decreasing temperature of a sample, in particular a method for ultra-rapid cryofixation of a sample for time and spatially resolved microscopic measurements as well as a device for ultra-rapid cryofixation of a sample at any particular time point of interest on a microscope. Said method and device are particularly useful for studying native molecular organization as well as (bio)chemical reactions within living cells with spatial and spectroscopic resolution beyond the fundamental limits caused by molecular motion at positive Celsius temperatures.
Core Innovation
The invention provides a device for decreasing the temperature of a sample for cryo-arrest microscopy and inverse microscopy. The device includes a carrier substrate having a lower side for accommodating the sample and an upper side exposed for a supply of a liquid, and a pressure tank adapted for holding a fluid with an outlet valve located at the bottom. A supply line connects the outlet valve with the upper side of the carrier substrate and is adapted to pass the liquid with a pressure higher than atmospheric pressure through it.
The pressure tank is mounted to the upper side of the carrier substrate to keep the supply line as short as possible, and an expansion chamber surrounds the upper side of the carrier substrate. The sample is provided on the lower side of the carrier substrate at atmospheric pressure, and liquid nitrogen below its critical temperature is supplied from the pressure tank under pressure with helium at a pressure of at least 2.5 MPa by opening the outlet valve. The liquid nitrogen is brought into contact with the upper side of the carrier substrate, thereby decreasing the temperature of the sample.
The disclosed temperature-decrease device is integrated with optical microscopy to perform fluorescence microscopy on cryo-arrested samples, including fluorescence illumination and detection, stimulated emission depletion, and fluorescence-lifetime imaging microscopy using time-correlated single-photon counting. In stimulated emission depletion optical microscopy, the temperature decreased sample is illuminated with a first pulsed laser beam focused on at least one focal area and with a second torus-shaped laser beam comprising an intensity zero point in the focal area, and in fluorescence-lifetime imaging microscopy the fluorescence photons and photon arrival times relative to the laser pulse are detected to determine fluorescence-lifetime.
Claims Coverage
The consolidated independent claims cover five inventive features: inverse microscopy temperature decrease, a temperature-decrease device, fluorescence microscopy, stimulated emission depletion optical microscopy, and fluorescence-lifetime imaging microscopy with time-correlated single-photon counting.
Inverse microscopy temperature decrease via pressure-tank liquid nitrogen delivery under helium pressure
A method for decreasing temperature of a sample for inverse microscopy comprising providing the sample on the lower side of a carrier substrate at atmospheric pressure; providing in a pressure tank liquid nitrogen below its critical temperature and helium at a pressure of at least 2.5 MPa; and supplying the liquid nitrogen from the pressure tank to the upper side of the carrier substrate under pressure by opening an outlet valve located at the bottom of the pressure tank, wherein the liquid nitrogen is brought into contact with the upper side of the carrier substrate thereby decreasing the temperature of the sample.
Temperature-decrease device with short, pressurized liquid nitrogen supply and expansion chamber
A device for decreasing the temperature of a sample comprising a carrier substrate with a lower side for accommodating a sample and with an upper side exposed for a supply of a liquid; a pressure tank adapted for holding a fluid and comprising an outlet valve located at the bottom; a supply line connecting the outlet valve with the upper side of the carrier substrate wherein the supply line is adapted to pass the liquid with a pressure higher than atmospheric pressure through it; and an expansion chamber surrounding the upper side of the carrier substrate, wherein the pressure tank is mounted to the upper side of the carrier substrate, to keep the supply line as short as possible.
Fluorescence microscopy with cooled fluorescent sample using pressure-tank cryo-arrest
A method of fluorescence microscopy comprising providing a sample containing fluorescent molecules on the lower side of a carrier substrate at atmospheric pressure; providing in a pressure tank liquid nitrogen below its critical temperature and helium having a pressure of at least 2.5 MPa; supplying the liquid nitrogen from the pressure tank to the upper side of the carrier substrate under pressure by opening an outlet valve located at the bottom of the pressure tank, wherein the liquid nitrogen is brought into contact with the upper side of the carrier substrate, thereby decreasing the temperature of the sample; illuminating the temperature decreased sample; and detecting the fluorescence produced by the illuminated sample.
Stimulated emission depletion optical microscopy with cryo-arrested sample and torus-shaped beam
A method for performing optical microscopy with stimulated emission depletion of a cryo-arrested sample comprising providing a sample containing fluorescent molecules on the lower side of a carrier substrate at atmospheric pressure; providing in a pressure tank liquid nitrogen below its critical temperature and helium having a pressure of at least 2.5 MPa; supplying the liquid nitrogen from the pressure tank to the upper side of the carrier substrate under pressure by opening an outlet valve located at the bottom of the pressure tank, wherein the liquid nitrogen is brought into contact with the upper side of the carrier substrate, thereby decreasing the temperature of the sample; illuminating the temperature decreased sample by a first pulsed laser beam to excite the fluorescent molecules for fluorescence with the first pulsed laser beam focused on at least one focal area; illuminating the temperature decreased sample by a second torus-shaped laser beam to de-excite the fluorescent molecules with the second laser beam comprising an intensity zero point in the at least one focal area; detecting the fluorescence produced by the illuminated sample; and optionally repeating steps (d1), (d2) and (e) at different positions of the focal area and at different positions of the intensity zero point of the second torus-shaped laser beam.
Fluorescence-lifetime imaging microscopy with time-correlated single-photon counting of cryo-arrested sample
A method for performing fluorescence-lifetime imaging microscopy with time-correlated single-photon counting of a cryo-arrested sample comprising providing a sample containing fluorescent molecules on the lower side of a carrier substrate at atmospheric pressure; providing in a pressure tank liquid nitrogen below its critical temperature and helium having a pressure of at least 2.5 MPa; supplying the liquid nitrogen from the pressure tank to the upper side of the carrier substrate under pressure by opening an outlet valve located at the bottom of the pressure tank, wherein the liquid nitrogen is brought into contact with the upper side of the carrier substrate, thereby decreasing the temperature of the sample; periodically exciting the fluorescent molecules in the temperature decreased sample to emit fluorescence photons by illuminating the sample with a pulsed laser beam; detecting the fluorescence photons produced by the sample together with the photon arrival times relative to the laser pulse; and determining the fluorescence-lifetime from the detected fluorescence photons and the photon arrival times.
Across the independent claims, the core inventive scope is the cryo-arrest temperature decrease for inverse microscopy and subsequent fluorescence readout, using liquid nitrogen delivered under pressure from a pressure tank through a bottom outlet valve to the upper side of a carrier substrate, with helium at a pressure of at least 2.5 MPa.
Stated Advantages
Enables fluorescence microscopy modalities on cryo-arrested samples, including stimulated emission depletion (STED) and fluorescence-lifetime imaging microscopy (FLIM) with time-correlated single-photon counting (TCSPC).
Preserves fluorescence imaging while avoiding ice crystal formation, including avoiding detectable ice crystals and ice crystals larger than a size threshold.
Documented Applications
Fluorescence microscopy of cryo-arrested samples with fluorescent molecules, including temperature decreased sample illumination and fluorescence detection.
Optical microscopy with stimulated emission depletion (STED) of a cryo-arrested sample, using a torus-shaped laser beam with an intensity zero point.
Fluorescence-lifetime imaging microscopy (FLIM) of a cryo-arrested sample using time-correlated single-photon counting (TCSPC), including photon arrival times and determination of fluorescence lifetime.
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