Method and device for ultra-rapid cryo-fixation of a sample for microscopic studies

A device for decreasing the temperature of a sample comprising:

• (a) a carrier substrate with a lower side for accommodating a sample and with an upper side, which is exposed for a supply of a liquid;
• (b) a pressure tank adapted for holding a fluid, the pressure tank comprising an outlet valve located at the bottom;
• (c) 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 the atmospheric pressure through it;
• and
• (d) 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.

A method of fluorescence microscopy comprising the following steps:

• (a) providing a sample containing fluorescent molecules on the lower side of a carrier substrate at atmospheric pressure;
• (b) providing in a pressure tank liquid nitrogen having a temperature below its critical temperature and helium having a pressure of at least 2.5 MPa;
• (c) 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, wherein the temperature of the sample is decreased, preferably rapidly decreased;
• (d) illuminating the sample;
• and
• (e) detecting the fluorescence produced by the sample.

A method for performing optical microscopy with stimulated emission depletion of a cryo-arrested sample, comprising

• (a) providing a sample containing fluorescent molecules on the lower side of a carrier substrate at atmospheric pressure;
• (b) providing in a pressure tank liquid nitrogen having a temperature below its critical temperature and helium having a pressure of at least 2.5 MPa;
• (c) 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, wherein the temperature of the sample is decreased, preferably rapidly decreased;
• (d1) illuminating the sample by a first pulsed laser beam to excite the fluorescent molecules for fluorescence in the sample, wherein the first pulsed laser beam is focused on at least one focal area;
• (d2) illuminating the sample by a second torus-shaped laser beam to de-excite the fluorescent molecules in the sample, wherein the second laser beam comprises an intensity zero point in the at least one focal area;
• (e) detecting the fluorescence produced by the sample;
• and
• (f) optionally repeating steps (d1), (d2) and (e) at different positions of the focal area of the first pulsed laser beam and at different positions of the intensity zero point of the second torus-shaped laser beam.

A method for performing fluorescence-lifetime imaging microscopy with time-correlated single-photon counting of a cryo-arrested sample, comprising

• (a) providing a sample containing fluorescent molecules on the lower side of a carrier substrate at atmospheric pressure;
• (b) providing in a pressure tank liquid nitrogen having a temperature below its critical temperature and helium having a pressure of at least 2.5 MPa;
• (c) 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, wherein the temperature of the sample is decreased, preferably rapidly decreased;
• (d) periodically exciting the fluorescent molecules to emit fluorescence photons by illuminating the sample with a pulsed laser beam;
• (e) detecting the fluorescence photons produced by the sample together with the photon arrival times relative to the laser pulse;
• and
• (f) determining the fluorescence-lifetime from the detected fluorescence photons and the photon arrival times.