Ultra-rapid freezing and fixation method and apparatus for microscopic research specimens

Abstract

The present invention relates to a method for reducing the temperature of a sample, in particular a method for ultrarapid freezing and fixing a sample for time- and space-resolved microscopy measurements, and an apparatus for ultrarapid freezing and fixing a sample on a microscope at a specific time of interest. The method and apparatus are particularly useful for studying native molecular organization and (bio)chemical reactions within living cells with spatial and spectroscopic resolution beyond the fundamental limits imposed by molecular motion at positive Celsius temperatures.

Description

Detailed description of the invention

[0001] The present invention relates to a method for reducing the temperature of a sample, in particular to an ultra-rapid freeze-fixation method for a sample for time- and spatial-resolved microscopy measurements, and to an ultra-rapid freeze-fixation apparatus for a sample at a specific point in time of interest on a microscope. The method and apparatus are particularly useful for studying the innate molecular structure and (bio)chemical reactions within living cells with spatial and spectral resolution beyond the fundamental limitations caused by molecular motion at positive Celsius temperatures. [Background of the Invention] Fluorescence microscopy is a powerful tool for studying the structure and dynamics of molecular systems.

[0002] Recent advances in fluorescence microscopy have made it possible to track well-separated molecules with nanoscale and millisecond-level positioning accuracy (Balzarotti et al. 2017, Science, 355:606-12). Super-resolution techniques such as single-molecule localization microscopy (SMLM) (Betzig et al. 2006, Science, 313:1642-46), coordinate-targeted nanoscopy (e.g., stimulated emission suppression (STED) microscopy) (Hell and Wichmann 1994, Optics Letters, 19:780-82; Sahl, Hell, and Jakobs 2017, Nature reviews. Molecular cell biology, 18:685-701), or minimal photon flux (MINFLUX) nanoscopy (Balzarotti et al. 2017, Science, 355:606-12) enable resolution beyond the diffraction limit of light even in crowded environments. By adding spectral dimensions to photon readout in microscopy or nanoscopy, such as polarization (Runnels and Scarlata 1995, Biophys J, 69:1569-83; Varma and Mayor 1998, Nature, 394:798-801) and excited state lifetime (Gadella, Jovin, and Clegg 1993, Biophysical Chemistry, 48:221-39; Bastiaens and Squire 1999, Trends in Cell Biology, 9:48-52), it becomes possible to extract information about molecular reaction states within spatially resolvable volume elements.

[0003] However, spatial and spectral resolution are fundamentally limited by the number of photons collected due to the Poisson statistics of quantum interactions (Gratton et al. 2003, Journal of Biomedical Optics, 8; Balzarotti). (et al. 2017, Science, 355:606-12). The saturable cycle of the fluorescent dye molecule between the ground and excited states, determined by the molecular cross-section, fluorescence quantum yield, and excited state lifetime, sets a limit on the fluorescence photon flux. This results in the minimum achievable exposure time and motion blur due to Brownian motion during photon acquisition. Motion blur is inversely proportional to the size of the observed structure and fundamentally limits localization accuracy and resolution (Figure 1). This fundamental limitation on exposure time is further extended by technical constraints of the optical system, such as laser scanning in coordinate-targeted nanoscopy and switching of fluorescent dye molecules in coordinate-stochastic nanoscopy. Active movements, such as molecular motor-driven transport and cytoskeleton reconfiguration, further increase motion blur. Increasing the irradiation intensity to maximize the saturable flux of the fluorescent dye molecule increases the time the fluorescent dye molecule spends in the chemically reactive excited state, leading to strong bleaching. This not only limits the amount of useful photons that reach the detector, reducing resolution (Figure 1), but also increases phototoxicity and disrupts reactions within the sample.

[0004] A concrete example is fluorescence microscopy of living systems such as living cells. Here, cellular function is revealed on a micrometer scale, and emerges from the non-equilibrium pattern formation dynamics of nanometer-sized molecules (Bastiaens et al. 2006, Trends Cell). (BioL, 16:125-34; Battle et al. 2016, Science, 352:604-07; Koseska and Bastiaens 2017, The EMBO Journal, 36:568-82; Gnesotto et al. 2018, Reports on Progress in Physics, 81). Fluorescence microscopy and nanoscopy, in principle, enable imaging of molecular patterns within cells and measure the underlying reaction dynamics. However, the ultimate goal of relating the collective behavior of molecules to cellular function (Varma and Mayor 1998, Nature, 394:798-801; Bastiaens and Squire 1999, Trends in Cell Biology, 9:48-52; Wouters, Verveer, and Bastiaens 2001, Trends Cell Biol, 11:203-11; Sahl, Hell, and Jakobs 2017, Nature reviews. Molecular cell biology, 18:685-701; Balzarotti et al. 2017, Science, 355:606-12) is often unattainable due to motion blur and the fundamental problem of the photochemical reactivity of fluorescent probes.

[0005] To prevent molecular motion, such samples are typically chemically immobilized. However, immobilization by crosslinking or denaturation inherently alters the sample, making this method unsuitable for observing innate non-equilibrium states, particularly non-equilibrium molecular patterns in cells. The immobilization process itself is also imperfect, taking at least several minutes. Therefore, it cannot fix a defined state (Huebinger et al. 2018, Sci Rep, 8:17756). Post-processing to enhance photostability is another source of artifacts.

[0006] Motion blur can be mitigated by cooling the sample to a temperature where it becomes solid or reaches a very high viscosity, thereby reducing the photochemical reactivity of fluorescent dye molecules. However, this must be done very quickly to avoid changes in the sample due to phase transitions or loss of non-equilibrium states. For example, living cells need to be immobilized extremely rapidly below the glass transition temperature of around -136°C to avoid mechanical destruction of the cellular structure by ice crystals and denaturation of lipid membranes and proteins. In this way, water molecules in the sample are prevented from reaching thermodynamic equilibrium at this low temperature and are trapped in a liquid-like structure. Therefore, even macromolecular patterns that are out of equilibrium in cells can be preserved by ultra-rapid cooling.

[0007] However, when applying a liquid to a sample to cool it, a limiting factor is the limited wettability of the initially warm surface. Due to the Leidenfrost effect, an insulating vapor layer forms on the surface with significantly lower thermal conductivity compared to the liquid, thus limiting the cooling rate at the surface. Furthermore, there are technically possible flow rates and therefore cooling capacities. However, when using a gaseous cooling medium, its low density results in a volume-related heat capacity that is too low to achieve sufficient cooling capacity for practical investigations.

[0008] Direct, ultra-rapid cooling on a microscope is particularly desirable because it allows for accurate analysis in a state of no molecular motion, combined with dynamic measurements at positive temperatures, which are significantly reduced in photochemical reactivity of the same sample under freeze-stop conditions.

[0009] Methods for rapidly cooling samples, known as state-of-the-art techniques, include high-pressure freezing and propane jet freezing. High-pressure freezing requires a pressure of 2000 bar to lower the sample's melting point and rapidly pass through the temperature range where ice crystals can form. Encapsulation is necessary to pressurize the sample. Regarding propane jet freezing, the method and apparatus are unsuitable for microscopic observation of the sample before, during, and after the rapid cooling process, particularly with an inverted microscope, because the sample must be sprayed from both the top and bottom sides of the sample. In an inverted microscope, the sample is fed from above the microscope's objective lens.

[0010] European Patent EP2877828B1 discloses a method and control device for controlling the temperature of a sample. In this method, a supercritical medium is used to cool a sample placed on the front of a carrier substrate. The supercritical medium is supplied from below to the back of the carrier substrate, that is, against the direction of Earth's gravity. Although it is possible to cool the sample by the above method, the rate of temperature change is too small to continuously observe the sample without damaging the biological sample with ice crystals. Furthermore, even if the cooling medium is supplied against Earth's gravity, it is not possible to observe the sample using an inverted microscope.

[0011] An in-situ freeze-fixation method has been reported by Fuest et al (Journal of Microscopy, 2018, 272:87-95), in which the sample is placed on a heater in thermal contact with liquid nitrogen via an insulator. Despite the presence of a cryoprotectant, the achieved cooling rate was too low to prevent ice crystal formation (Fuest et al). (al. 2019, Scientific Reports, 9:19133). Therefore, this freeze-fixation method is only applicable to microorganisms that tolerate ice crystal formation, such as Caenorhabditis elegans and yeast.

[0012] Other samples, such as mammalian cells, have a high water content, very poor glass formation, and relatively low thermal conductivity (<0.6 Wm). -1 K -1 Therefore, stopping the process by freezing and fixing it is particularly difficult.

[0013] Recent approaches to avoiding ice crystal formation in living cells have involved replacing water with cryoprotectants prior to cooling (Fuest et al. 2019, Scientific Reports, 9:19133). However, since this method alters the sample (Huebinger 2018, PLOS ONE, 13:e0205520), it may affect the study of biological samples. Therefore, rapid cooling methods that avoid the use of cryoprotectants are strongly desired.

[0014] EP3418710A1 relates to a method and apparatus for preparing a sample for inverted microscopy research, wherein the sample is irradiated with light to a first region at time t1, irradiated with light to a second region of the sample at time t2 after time t1, and fixed at time t3 after time t2. The sample can be fixed by freezing (such as in liquid nitrogen) or chemical fixation using a glutaraldehyde solution and a photocurable resin. WO2013 / 152239A1 relates to a freezing preparation system for almost instantaneously vitrifying biological samples. The system comprises a capsule structure configured to hold the sample in a recess while the sample is subjected to ultra-rapid freezing by being exposed to a cryogenic cooling jet. Pulsed microwave energy is applied during rapid cooling to avoid ice crystal formation within the sample. These methods do not disclose how to generate a cooling jet that effectively avoids the formation of an insulating gas layer to cool the sample sufficiently quickly. EP3128267A1 discloses a food freezing tunnel apparatus for cooling food to cryogenic temperatures. A pulsed flow of liquid refrigerant is applied from a pressurized tank. The pulsed refrigerant flow is generated by alternately opening and closing two valves. However, the pressure is limited to 1.379 MPa by the pump, which limits the cooling rate.

[0015] A general drawback of conventional methods arises from the special design for freezing samples. Other tempering processes, such as rapid sample thawing, cannot normally be implemented by conventional methods. However, in the investigation of dynamic systems, it is important that after capturing still images and completing fixation by heating, processes within the system can continue without being impeded as much as possible. For example, in the case of biological samples, what is important is that heating is performed in a manner such that ice crystals do not form during thawing and the sample is not overheated.

[0016] The object of the present invention is to avoid the drawbacks and limitations of the prior art, and provide an improved method and apparatus for performing rapid temperature adjustment and lowering the temperature of a sample for microscopic investigation. More precisely, the present invention includes a method for ultra-rapid cryofixation of samples for spatially-resolved microscopic measurement without using a cryoprotectant, and an apparatus for ultra-rapid cryofixation of samples on a microscope. In particular, the present invention aims, compared with the prior art, to enable temperature change of a sample at a higher temperature adjustment rate, to achieve temperature increase or decrease, to set the final temperature after temperature change and / or to monitor the sample. Rapid temperature adjustment needs to be performed at any timing, particularly during observation of the sample. Specifically, the present invention aims to provide an improved technique for vitrifying biological samples and controlling the heating thereof such that ice crystal formation and overheating do not occur, thereby enabling microscopic measurement of the sample before, during and after vitrification (reversibly) without changing the sample. In other words, the present invention provides a technique for fixing ("freeze-stopping") short-lived states of a sample in a targeted, non-invasive ("native") manner, and aims to enable investigation of these states using fluorescence microscopy, for which recording speeds would otherwise be too slow to study these states. A further object of the present invention is to provide an apparatus and a method for cooling a sample on an inverted microscope sufficiently fast to enable continuous observation of the sample before and after cryofixation.

[0017] The object of the present invention is resolved by teaching the independent claims. Further advantageous features, aspects and details of the present invention are evident from the dependent claims, specification, drawings and examples of this application. [Brief description of the invention] This objective was addressed by a method for lowering the temperature of a sample (1) for inverted microscopy, which includes the following steps.

[0018] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) supplying at least one liquid (2) having a temperature below the critical temperature and a gas (500) having a pressure higher than atmospheric pressure into a pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), at least one liquid (2) is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the at least one liquid (2) into contact with the upper side (11) of the carrier substrate (10), thereby lowering the temperature of the sample (1), preferably rapidly. At least one liquid (2) is nitrogen, and the gas (500) is helium at a pressure of at least 2.5 MPa.

[0019] In other words, the present invention relates to a method for lowering the temperature of a sample (1) for inverted microscopy, comprising the following steps.

[0020] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly.

[0021] In other words, a method for lowering the temperature of a sample (1) for inverted microscopy includes the following steps:

[0022] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Liquid nitrogen having a temperature below its critical temperature and a pressure of at least 2.5 MPa The helium is supplied into the pressure tank (400), (c) Applying liquid nitrogen to the upper side (11) of the carrier substrate (10) from the pressure tank (400) under pressurized conditions by opening the outlet valve (22) located at the bottom of the pressure tank (400), thereby bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly.

[0023] In other words, a method for lowering the temperature of a sample (1) for inverted microscopy includes the following steps:

[0024] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) Applying liquid nitrogen to the upper side (11) of the carrier substrate (10) from the pressure tank (400) under pressurized conditions by opening the outlet valve (22) located at the bottom of the pressure tank (400), thereby bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly.

[0025] In other words, a method for lowering the temperature of a sample (1) for inverted microscopy includes the following steps:

[0026] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) Opening the outlet valve (22) located at the bottom of the pressure tank (400) to allow liquid nitrogen to pass from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, thereby bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly.

[0027] Thus, rapid cooling of the sample (1) contained on the lower side (12) of the carrier substrate (10) is achieved by supplying liquid nitrogen having a temperature below the critical temperature from a pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions. The liquid nitrogen is pressurized by gas (500) (helium) which is contained at high pressure in the pressure tank (400).

[0028] The helium pressure is preferably at least 2.5 MPa, more preferably at least 3.0 MPa, more preferably at least 3.5 MPa, more preferably at least 4.0 MPa, more preferably at least 4.5 MPa, and most preferably at least 5.0 MPa.

[0029] Therefore, in a preferred embodiment, a method for lowering the temperature of a sample (1) for inverted microscopy includes the following steps:

[0030] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 5 MPa into a pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly.

[0031] In further embodiments, the helium pressure is preferably between 2.5 MPa and 100 MPa, more preferably between 3.0 MPa and 100 MPa, more preferably between 3.5 MPa and 100 MPa, more preferably between 4.0 MPa and 100 MPa, more preferably between 4.5 MPa and 100 MPa, and most preferably between 5.0 MPa and 100 MPa.

[0032] Therefore, in another preferred embodiment, a method for lowering the temperature of a sample (1) for inverted microscopy includes the following steps:

[0033] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen having a temperature below the critical temperature and helium having a pressure between 5 MPa and 100 MPa into a pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly.

[0034] Liquid nitrogen, which acts as a cooling medium or heat sink, is applied suddenly (or rapidly or instantaneously) by opening the outlet valve (22) of the pressure tank (400). Therefore, the outlet valve needs to be configured to release liquid nitrogen rapidly. In this regard, the outlet valve needs to be switchable; that is, it needs to open and close completely at very high speed. Also, the outlet valve needs to be designed to have the maximum diameter necessary for the rapid release of liquid nitrogen.

[0035] The carrier substrate (10) that separates the sample (1) from liquid nitrogen functions as a thermal conductor and is therefore made of any material having the desired thermal conductivity and sufficient strength under the pressure of the tempering solution. It is preferable that the carrier substrate (10) includes or consists of a diamond disk.

[0036] In one embodiment, liquid nitrogen is completely released from the pressure tank (400) in step (c) to maximize cooling of the sample by bringing the maximum amount of nitrogen into contact with the upper side of the carrier substrate. Thus, a method for lowering the temperature of a sample (1) for inverted microscopy includes the following steps:

[0037] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is completely supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly.

[0038] According to the present invention, a sudden, rapid, or explosive temperature drop is achieved. This means that the sample temperature decreases from the initial temperature (start temperature) to the final temperature (target temperature) at a rate (cooling rate, tempering rate) where the rate of molecular processes (such as ice crystal formation) within the sample is slower or negligible.

[0039] In other words, a method for lowering the temperature of a sample (1) for inverted microscopy includes the following steps:

[0040] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is completely supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and causing an explosive decrease in the temperature of the sample (1).

[0041] In another embodiment, in step (c), the entire contents of the pressure tank (400), i.e., first liquid nitrogen and second helium, are supplied to the upper side (11) of the carrier substrate (10). Cooling of the sample is mainly caused by passing the liquid over the upper side (11) of the carrier substrate (10), but additional cooling may be achieved by helium having the same temperature as the liquid nitrogen. Thus, in one embodiment, a method for lowering the temperature of a sample (1) for inverted microscopy includes the following steps:

[0042] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen and gas (500) are supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly.

[0043] More precisely, in one embodiment, a method for lowering the temperature of a sample (1) for inverted microscopy includes the following steps:

[0044] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen and gas (500) are supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly. In step (c), liquid nitrogen is supplied first, followed by helium.

[0045] Liquid nitrogen is added suddenly as a cooling medium so that the sample vitrifies without crystallization, specifically without the formation of ice crystals or ice crystals larger than 10 nm in diameter. The cooling rate for vitrification of the sample depends greatly on the properties of the sample, and mainly on the molecular components of the sample. For example, vitrification occurs when the molecular components are cooled at a rate of approximately 100,000 K / s to 1,000,000 K / s in a diluted aqueous buffer solution.

[0046] Therefore, in one embodiment, a method for lowering the temperature of a sample (1) for inverted microscopy includes the following steps.

[0047] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and reducing the temperature of the sample (1). At the very least, reduce it at a rate of -100,000 K / second.

[0048] It is well known that the cooling rate required for vitrification of a sample can be reduced by adding cryoprotective compounds such as dextran, or by applying a high pressure of approximately 200 MPa to the sample. However, the use of cryoprotective agents can also affect the properties and activity of the sample.

[0049] In a more preferred embodiment of the method of the present invention for lowering the temperature of sample (1), the temperature of sample (1) is lowered at a rate of at least -10,000 K / sec, more preferably at least -100,000 K / sec, more preferably at least -200,000 K / sec, more preferably at least -500,000 K / sec, and most preferably at least -1,000,000 K / sec.

[0050] In one embodiment, the temperature of the sample (1) is lowered at a rate between -100,000 K / second and -1,000,000 K / second.

[0051] In other embodiments, the sample originally or additionally contains cryoprotective compounds such as salts, sugars (e.g., trehalose, dextran), organic solvents (e.g., dimethyl sulfoxide, ethylene glycol), proteins, or antifreeze proteins in specific cells. In these embodiments, a cooling rate slower than the rate described above may be applied for vitrification.

[0052] Therefore, in one embodiment, a method for lowering the temperature of a sample (1) for inverted microscopy includes the following steps.

[0053] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1) at a rate of at least -10,000 K / second.

[0054] In other words, in one embodiment, a method for lowering the temperature of a sample (1) for inverted microscopy includes the following steps:

[0055] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10), thereby lowering the temperature of the sample (1) at a rate of at least -10,000 K / second. The sample contains cryoprotective compounds.

[0056] In these embodiments, the temperature of the sample (1) is lowered at a rate of preferably at least -5,000 K / sec, more preferably at least -6,000 K / sec, more preferably at least -7,000 K / sec, more preferably at least -8,000 K / sec, more preferably at least -9,000 K / sec, and most preferably at least -10,000 K / sec.

[0057] It is well known that the cooling rate achieved depends on the properties of the sample, such as its thermal conductivity and thickness. Those skilled in the art can easily select an appropriate sample thickness to ensure complete vitrification of the sample using the method according to the present invention. Accordingly, in one embodiment, a method for lowering the temperature of a sample (1) for inverted microscopy includes the following steps.

[0058] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly. The thickness of sample (1) is less than 500 μm.

[0059] The thickness of sample (1) is preferably less than 500 μm, more preferably less than 400 μm, more preferably less than 300 μm, more preferably less than 200 μm, more preferably less than 100 μm, more preferably less than 50 μm, more preferably less than 30 μm, more preferably less than 20 μm, more preferably less than 15 μm, and even more preferably less than 10 μm.

[0060] Therefore, in one embodiment, a method for lowering the temperature of a sample (1) for inverted microscopy includes the following steps.

[0061] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), at least one liquid (2) is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the at least one liquid (2) into contact with the upper side (11) of the carrier substrate (10), thereby lowering the temperature of the sample (1), preferably rapidly. The thickness of sample (1) is less than 20 μm.

[0062] In other words, a method for lowering the temperature of a sample (1) for inverted microscopy includes the following steps:

[0063] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly from room temperature to at least -120°C, preferably -130°C.

[0064] In a preferred embodiment of the present invention's method for lowering the temperature of a sample (1) for inverted microscopy, the sample (1) is vitrified (i.e., no ice crystals larger than 10 nm in diameter are formed). Nanometer-sized ice crystals typically do not affect the properties or activity of the sample (Huebinger et al., 2016, Biophysical Journal, 110:840-49). The size of the ice crystals in the sample (1) can be measured by standard methods such as cryo-electron microscopy (cryo-EM) or X-ray diffraction.

[0065] Therefore, a method for lowering the temperature of a sample (1) for inverted microscopy includes the following steps:

[0066] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) to lower the temperature of the sample (1), preferably rapidly, so as to vitrify the sample (1) without crystallization, preferably without forming ice crystals with a diameter of 80 nm or more, preferably without forming ice crystals with a diameter of 10 nm or more, and even more preferably without forming ice crystals.

[0067] All methods disclosed herein for lowering the temperature of sample (1) can lower the temperature of sample (1) without forming ice crystals or ice crystals with a diameter greater than 10 nm.

[0068] More precisely, all methods disclosed herein for lowering the temperature of sample (1) can lower the temperature of sample (1) to below -120°C, preferably below -130°C, without forming ice crystals or ice crystals with a diameter greater than 10 nm.

[0069] Therefore, the temperature of sample (1) decreases explosively, i.e., very rapidly. Defining a minimum cooling temperature to avoid the formation of ice crystals larger than 10 nm in diameter is difficult because the required cooling rate depends on the thickness and size of sample (1), the material of sample (1) or a mixture of materials, the presence of cryoprotective compounds (i.e., cryoprotectants) and other parameters disclosed herein. However, as an example, the method for lowering the temperature of sample (1) disclosed herein can lower the temperature of a 100 μm thick aqueous sample from room temperature to a temperature between -120°C and -130°C within a time frame of less than 10 milliseconds at a cooling rate of -15,000 K / sec or less (see Figure 7A). The cooling rate approaches the theoretical thermal diffusion limit of the aqueous sample and therefore corresponds to a maximum cooling rate of -200,000 K / sec for aqueous samples with a thickness of 10 μm to 15 μm containing attached mammalian cells.

[0070] Another aspect of the present invention relates to an apparatus (100) for controlling the temperature of a sample (1), wherein the apparatus (100) (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and equipped with an outlet valve (22) located at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To shorten the supply line (20) as much as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0071] Under pressurized conditions, coolant is ejected from the pressure tank (400) onto the upper side (11) of the carrier substrate (10). By supplying the coolant naturally, the formation of an adiabatic gas layer on the carrier substrate (Leidenfrost effect) is prevented. Simply using a liquid at low pressure does not prevent the formation of an adiabatic gas layer, resulting in a slow cooling rate. This problem can be solved by further pressurizing the coolant using a gas such as helium (500), which does not liquefy under these conditions. When this liquid-gas mixture is rapidly released onto the carrier substrate, the flow of the coolant on the carrier substrate becomes very large, enabling rapid convective cooling and suppressing film boiling. Furthermore, this apparatus is inverted compared to the apparatus in EP2877828B1, with the tank for storing the coolant located above the sample, and the coolant being applied to the carrier substrate from above.

[0072] To minimize the temperature rise of the coolant (2) in the supply line (20), the pressure tank (400) is positioned above (11) the carrier substrate so that the supply line (20) can be as short as possible. By shortening the line from the outlet valve (22) of the pressure tank (400) to the top (11) of the carrier substrate (10), heat exchange between the coolant (2) and the surroundings can be reduced. This can be achieved by mounting or connecting the pressure tank (400), which has an outlet valve (22) at its bottom, to the top (11) of the carrier substrate (10).

[0073] In a preferred embodiment of the apparatus (100), the temperature rise of the coolant (2) is further minimized by insulating the supply line (20). Therefore, a preferred embodiment of the apparatus (100) for controlling the temperature of the sample (1) is: (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and equipped with an outlet valve (22) located at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To make the supply line (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10). The supply line (20) is preferably made of high-density polyethylene and is insulated from the inside.

[0074] The apparatus (100) according to the present invention enables fixation without the use of cryoprotective agents and observation of the inherent non-equilibrium molecular state, such as the cytoplasmic molecular structure and reaction patterns within living cells. When the apparatus (100) is coupled to an inverted microscope, dynamic cellular processes at physiological temperatures can be continuously observed, followed by rapid freezing and measurement of the molecular patterns of the same cells in a cryopreserved state.

[0075] In other words, the apparatus (100) according to the present invention is an apparatus for lowering, preferably rapidly lowering, the temperature of a sample (1). (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and equipped with an outlet valve (22) located at the bottom, (c) A supply line (20) having an upper end connected to an outlet valve (22) and an outlet opening (21) at its lower end facing the upper side (11) of the carrier substrate (10), and configured to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) The system comprises an expansion chamber (30) surrounding the outlet opening (21).

[0076] Surprisingly, the inventors discovered that a higher cooling rate can be achieved using the apparatus of the present invention compared to the apparatus of EP2877828B1. Figure 7A shows the thickness This shows that when a 100 μm aqueous sample was cooled from above with a thermocouple element placed beneath the carrier substrate, a cooling rate of approximately -15,000 K / second was measured (theoretically, for aqueous samples with a thickness of 10 μm to 15 μm, such as adherent cells, the vitrification cooling rate of water is estimated to reach a maximum of -200,000 K / second). When the EP2877828B1 apparatus was configured so that the sample was placed beneath the carrier substrate and cooled from above, the observed temperature change rate was only -1,000 K / second (Figure 7B).

[0077] Furthermore, no ice crystals containing microcrystals were observed in a 15 μm thick cell culture medium solution of the fluorescent dye rhodamine 6G under a cooled inverted optical microscope (see Figure 18). No dehydration of the sample was observed either.

[0078] More importantly, we were able to demonstrate by wide-field fluorescence imaging that the morphology of EGFP expressed in adherent HeLa cells was preserved during cryopreservation and no ice crystals were detected within the cells (see Examples and Figure 17).

[0079] Furthermore, confocal laser scanning microscopy (CLSM) and wide-field fluorescence imaging demonstrated that the morphology of large organelles, such as the endoplasmic reticulum (ER), remained intact during cryopreservation. In this regard, the ER membrane was labeled using MCF7 cells expressing T-cell protein tyrosine phosphatase (TC-PTP) fused to fluorescent mCitrine (see Figure 19). Due to the high photostability of mCitrine at cryogenic temperatures, it was then possible to acquire 100 wide-field fluorescence images of the same resting cells to generate ER SRRF (super-resolution radial fluctuation) reconstruction, thereby demonstrating that the spatial localization of the fluorescent protein is completely preserved after cryopreservation using the apparatus according to the present invention.

[0080] Advantageously, the present invention enables the fixation ("freeze-stop") of short-lived states in a targeted and unaffected manner, and allows for the investigation of these states in ways that other methods would be too slow to study. These include many optical microscopy techniques with improved spatial and spectral resolution, such as CLSM and stimulated emission suppression microscopy (STED).

[0081] For this reason, the inventors focused on the proto-oncoprotein EGFR (epidermal growth factor receptor) and the functionally related tumor suppressor R-PTP-γ (receptor-like protein tyrosine phosphatase γ), which were co-expressed in MCF7 cells as a fluorescent fusion construct (Alexa647-SNAP-EGFR and R-PTP-γ-mCitrine).

[0082] The vesicle dynamics of both fluorescent fusion proteins at room temperature could be tracked by CLSM upon stimulation with epidermal growth factor (EGF, 100 ng / ml), and subsequent freeze-stopping allowed for precise capture of the spatial patterns in the relationships between both proteins (Figure 20A). The elimination of motion blur (Figure 20B) and improved photostability of the fluorescent markers enabled colocalization analysis using SRRF reconstruction of 100 wide-field fluorescence images from each channel. Thus, this freeze-stop analysis revealed previously unknown co-organization of EGFR and R-PTP-γ into nanoscale clusters along the plasma membrane and within endosomes, as well as their separation upon EGF stimulation. This growth factor-induced separation of EGFR nanoclusters from inhibitory phosphatases is consistent with the system dynamically switching from a dormant state to a transiently active signaling state.

[0083] STED nanoscopy is a special case among super-resolution techniques because the fluorescence intensity is directly related to the density of the fluorescent marker, and quantitative molecular patterns can be obtained. In laser scanning nanoscopy, diffraction-limit-breaking resolution is achieved by stimulated emission of fluorescent dye molecules excited using a second-highest intensity donut-shaped laser beam. However, the high demands on photostability, fluorescence quantum yield, and fluorescence label density hinder STED imaging of sparsely tagged molecules within living cells.

[0084] In fact, low depletion laser power (<42mW / μm 2 Only ) preserved enough fluorescent markers to obtain noisy STED images of Alexa647-Snap-EGFR in MCF7 cells at room temperature (see Figures 21A and 21B), and there was no statistically significant increase in information beyond the diffraction limit compared to normal CLSM (Figures 21A and 21C). Resolution could be slightly improved by reducing motion blur through chemical fixation of cells, allowing for the accumulation of photons from five consecutive STED frames before bleaching the sample (Figure 21C). However, after ultrarapid freeze-stopping of living cells, the bleaching rate decreased to approximately 1 / 45th (Figure 21B), indicating that the depletion laser power (420 mW / μm) was used. 2 ) and exposure time (radiation energy density: 35 mJ / μm 2 Both of these capabilities were increased tenfold, and endosomal structures containing Alexa647-Snap-EGFR were clearly resolved (Figure 21D). The amount of information increased significantly from a scale of less than 125 nm to a digital (pixel-limited) resolution of 80 nm (Figure 21C).

[0085] Furthermore, because there is no motion blur and photochemistry is significantly reduced, CLSM allows for 3D scanning of the entire cell, followed by recording of super-resolution STED images of Alexa647-Snap-EGFR in various regions at different z levels (Figure 21D). This enables the identification of vesicle clusters beyond the diffraction limit that cannot be resolved at room temperature. Here, the total acquisition time exceeded 15 minutes, and the average irradiation energy density across the entire cell region was 20 mJ / μm 2 Exceeding approximately 1 mJ / μm, the lethal ray dose for mammalian cell lines at positive temperatures is approximately 1 mJ / μm2 This far exceeded the previous limit. Therefore, the inventors were able to demonstrate that freezing fixation using the apparatus according to the present invention enables 3D imaging of entire living cells with improved spatial and spectral resolution in the absence of molecular motion, with significantly extended exposure times due to reduced motion blur and greatly improved photostability.

[0086] As a result, the method of the invention described herein enables the freezing and fixation of diluted aqueous media and cells without ice crystals, even for cellular structures imaged beyond the diffraction limit by STED or SRRF, which are detectable at this resolution as non-fluorescent regions within the cytoplasm of the cells (Figure 26). Furthermore, after repeating STED scans at maximum depletion laser intensity, the structure and arrangement of endocytosis structures were preserved, but no ice crystals were observed, demonstrating that high-intensity STED illumination does not heat the biological sample enough to generate ice crystals. In addition, the distribution of molecular three-dimensional structures of the LIFEA2 sensor measured by FRET-FLIM was preserved, indicating that the protein structure remained intact even after freezing and stopping. Therefore, ultrafast freeze-fixation by the method described herein faithfully fixes the transient state of dynamic molecular patterns within cells that can be observed at multiple resolutions.

[0087] A further important advantage of the present invention is that, compared to conventional techniques, the temperature of the sample can be changed at a faster tempering rate, and / or the sample can be monitored without drawing the sample out of its natural state, moving it, or manipulating it with additives or, for example, excessive pressure increases.

[0088] Furthermore, the inventors evaluated the functional imaging of molecular reactions in cells after ultra-rapid freeze-stopping. FLIM adds a nanosecond fluorescence decay kinetics spectral dimension to fluorescence microscopy. This allows for imaging of excited state reactions between fluorescent dye molecules (e.g., FRET) caused by molecular proximity, enabling spatial resolution of macromolecular reactions such as interactions and conformational changes. The genetically encoded biosensor LIFEA2 analyzes molecular conformation, and its This report describes the kinase activity of ephrin receptor type A (EphA2) mediated by FRET between the fluorescent proteins mCitrine and mCherry, which facilitates cellular guidance. At room temperature, receptor activation by ligand-clustered ephrin A1 was observed in the plasma membrane of Cos7 cells by a slight (approximately 0.2 ns) decrease in the mean fluorescence lifetime of FRET donor mCitrine in LIFEA2 (Figure 22). However, motion blur and poor separation of fluorescence decay profiles and derived phasers significantly limited the spatial resolution and fluorescence lifetime resolution of intracellular membrane structures (Figure 22). However, at cryogenic temperatures, the increased quantum yield of mCitrine significantly improved the decay profile, mean fluorescence lifetime (τ), and separation of associated phasers in LIFEA2 between stimulated and unstimulated cells (Figure 22). The activity of clustered receptors was degradable in plasma membrane patches and endocytosis structures (Figure 22), and the basal activity in the absence of exogenous stimuli was likely induced by endogenous ephrin in adjacent cells. This enhanced contrast in molecular activity imaging was particularly evident when comparing the same ephrin A1-stimulated cells before and during freeze-stopping (Figure 22).

[0089] Under freeze-stop conditions, the cumulative phasors are largely separated (Figure 24C), allowing for the derivation of the spatially invariant fluorescence lifetimes of active (τ=0.9±0.2ns) and inactive (τ=3.5±0.1ns) three-dimensional structures, as well as the proportion of LIFEA2 containing non-absorbable mCherry acceptors (approximately 0.2) through global analysis. Using these constraints, the proportion of spatially varying molecules (α) of active LIFEA2 can be derived from the phasors within each voxel of the image. This enables the acquisition (time > 10 min) and reconstruction of a 3D molecular activity map of LIFEA2 in stimulated Cos7 cells (Figure 25), allowing for the differentiation of individual vesicles with activated receptors emerging from the plasma membrane from gradually inactivated receptors in the perinuclear endosomal compartment.

[0090] Therefore, we have demonstrated that freeze-fixation using the apparatus of the present invention allows for measurement of the spatial distribution of fluorescence decay kinetics by fluorescence lifetime imaging microscopy (FLIM) with significantly improved resolution, thereby enabling unprecedented mapping of the reaction state of fluorescent proteins of one or two genetically encoded components, including a fluorescently labeled molecule, preferably an indicator. An example of a genetically encoded structural FRET sensor, LIFEA2, is shown, which can be used to accurately measure the activity state of EphA2 receptors in numerous individual endosomes and inner membrane structures within cells (Figure 25).

[0091] Therefore, further aspects of the present invention relate to a fluorescence microscopy method comprising the following steps.

[0092] (a) To provide a sample (1) containing fluorescent molecules on the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into a pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly. (d) Irradiating sample (1) with laser light, (e) Detect the fluorescence generated by sample (1).

[0093] Therefore, a further aspect of the present invention relates to a method for performing optical microscopy by suppressing stimulated emission of a freeze-stopped sample (1), and includes the following steps.

[0094] (a) To provide a sample (1) containing fluorescent molecules on the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly. (d1) Irradiating the sample (1) with a first pulsed laser beam focused to at least one focal region to excite fluorescent molecules in the sample (1) and cause them to emit fluorescence, (d2) Irradiate the sample (1) with a second torus-shaped laser beam containing an intensity zero point within at least one focal region to de-excite the fluorescent molecules in the sample (1), (e) Detect the fluorescence generated by sample (1), (f) Optionally, repeat steps d1, d2, and e at various positions in the focal region of the first pulsed laser beam and at various positions in the intensity zero point of the second torus-shaped laser beam.

[0095] Therefore, a further aspect of the present invention relates to a method for performing fluorescence lifetime imaging microscopy using time-correlated single-photon counting on a freeze-stopped sample (1), and includes the following steps.

[0096] (a) To provide a sample (1) containing fluorescent molecules on the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly. (d) By irradiating the sample with a pulsed laser beam, fluorescent molecules are periodically excited and fluorescent photons are emitted, (e) Detecting the fluorescent photons produced by sample (1), (f) Determine the fluorescence lifetime from the detected fluorescent photons.

[0097] Accordingly, in all embodiments disclosed herein, the phrase "a method for lowering the temperature of a sample (1) for inverted microscopy" can be replaced with the phrase "a method for lowering the temperature of a living sample (1) for inverted microscopy," or more precisely, "a method for lowering the temperature of a living cell (1) for inverted microscopy."

[0098] Therefore, the phrase "device (100) for controlling the temperature of sample (1)" can be replaced with "device (100) for controlling the temperature of a living sample (1)," or, more precisely, with "device (100) for controlling the temperature of a living cell (1)."

[0099] Therefore, this application relates to a method for lowering the temperature of a living sample (1), preferably living cells (1) for inverted microscopy, comprising the following steps.

[0100] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Liquid nitrogen having a temperature below its critical temperature and a pressure of at least 2.5 MPa The helium is supplied into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly.

[0101] Therefore, this application relates to an apparatus (100) for controlling the temperature of a living sample (1), preferably living cells (1), (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and equipped with an outlet valve (22) located at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) The system comprises an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), and a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10) in order to shorten the supply line (20) as much as possible. [Description of the invention] The term "lowering the temperature" of a sample is typically used to describe a temperature change that occurs in response to the temperature difference between the tempering solution and the carrier substrate containing the sample, including a decrease in temperature (cooling).

[0102] Therefore, tempering solutions are also called refrigerants or coolants. When a carrier substrate containing a sample is placed at a temperature above the freezing point of water, the use of a refrigerant causes the sample to change into a solid state. This transition to a solid state is also called freezing of the sample.

[0103] The term "point spread function" (PSF) describes the spread of a small point source due to the impulse response of a focusing optical system, and corresponds to the optical transfer function of an image system. The acquired image of an object in an optical system is the convolution of the object and the PSF.

[0104] The term "positioning accuracy" refers to the deviation of the detected position of a single object or point source within an optical system, and is expressed as variance or standard deviation.

[0105] The term "resolution" is defined as the minimum distance at which two objects or point sources can be distinguished within an optical system, and can be defined, for example, by the Rayleigh criterion.

[0106] As used herein, the term “freeze-fixation” refers to the (near) instantaneous (rapid or explosive) freezing of a sample, which halts molecular motion within the sample and prevents water molecules in the sample from forming a hydrogen bond network of ice crystals. The resulting amorphous ice is called “glass ice” and remains in a disordered, liquid-water-like arrangement that does not disrupt the original structure of the sample. Thus, freeze-fixation avoids ice crystal growth and / or only causes the formation of nano-dispersed ice (vitrification), without affecting the structure of the sample. Nano-dispersed ice includes ice particles smaller than 100 nm, particularly smaller than 10 nm, e.g., smaller than 5 nm.

[0107] In the case of living cells, cryopreservation generally preserves the cells very well, and many cells will survive if thawed at an appropriate rate later. Therefore, this process can be considered reversible.

[0108] In this specification, "freeze stop" is used synonymously with "freeze fixation."

[0109] A tempering solution is typically a substance containing a chemical element, compound, or composition that can be provided in a liquid state within a target temperature range for cooling the sample. Preferably, the liquid contains nitrogen. Further examples of liquids as temperature-controlling media include hydrogen, argon, helium, or methane.

[0110] As used herein, “gas” (500) refers to a gas that does not liquefy under the conditions present in the pressure tank (400), i.e., under a pressure higher than atmospheric pressure and at the boiling point of the tempering liquid. Furthermore, the gas (500) is preferably inert; that is, the gas (500) does not react with or dissolve in the tempering liquid, or react with the pressure tank or other components of the apparatus (100). The gas functions as a pressurizing means; that is, it is used to increase the pressure in the pressure tank. In this respect, the gas must be compressible. Preferably, the gas contains or consists of helium or hydrogen.

[0111] As used herein, the “upper side” (11) of the carrier substrate (10) refers to the side of the carrier substrate (10) facing the outlet opening (21) of the supply line (20), (10), upward, that is, facing away from the direction of Earth’s gravity.

[0112] As used herein, “lower side” (12) refers to the side of the carrier substrate (10) opposite to the outlet opening (21) of the supply line (20), and is facing downward, i.e., in the direction of Earth's gravity.

[0113] For clarity, the carrier substrate (10) has only one upper side and one lower side. The upper side is opposite the lower side, and vice versa.

[0114] As used herein, “room temperature” refers to temperatures above 4°C, preferably 15 to 40°C, 15 to 30°C, 15 to 24°C, and 16 to 21°C. Such temperatures include 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, and 21°C.

[0115] As used herein, “positive Celsius temperature” refers to a temperature above the melting point of water, i.e., a temperature above 0°C. Such temperatures include, for example, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 25°C, 30°C, 35°C, 37°C, and 40°C.

[0116] As used herein, “inverted microscopy” refers to any type of microscopy in which the microscope's objective lens is located below the stage and facing upwards, allowing the sample to be observed from below. The sample may be placed at the bottom of a container with the best optical properties, or attached to the underside of a carrier substrate. Therefore, inverted microscopy includes, but is not limited to, confocal microscopy, (multimodal) epifluorescence microscopy, wide-field fluorescence microscopy, super-resolution microscopy such as single-molecule localization microscopy, stimulated emission suppression microscopy (STED), time-correlated single-photon counting fluorescence lifetime imaging microscopy, and electron microscopy.

[0117] As used herein, “critical temperature” refers to the temperature below which a gas can be liquefied by pressure, but above which this is not possible. In embodiments involving two or more liquids (2), the critical temperature refers to the lowest critical temperature of each individual liquid.

[0118] According to the present invention, the supply line (20) is configured to guide the tempering liquid to the carrier substrate at a pressure higher than atmospheric pressure. Furthermore, the lower side (12) of the carrier substrate is configured to receive, contain, or hold the sample at atmospheric pressure.

[0119] It was found that when the pressure of the gas (500) in the pressure tank (400) is at least 50 bar, the cooling rate is limited by the thermal conductivity of the sample and no longer limited by the Leidenfrost effect. Accordingly, one embodiment of the present invention relates to a method for lowering the temperature of a sample when the pressure of the gas (500) is between 50 bar and 1000 bar. Accordingly, embodiments of the present invention relate to a method for lowering the temperature of a sample (1) for inverted microscopy, preferably a living sample (1), more preferably living cells (1), comprising the following steps.

[0120] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) supplying at least one liquid (2) having a temperature below the critical temperature and a gas (500) having a pressure of at least 2.5 MPa into a pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), at least one liquid (2) is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing at least one liquid (2) into contact with the upper side (11) of the carrier substrate (10), thereby lowering the temperature of the sample (1), preferably rapidly. At least one liquid (2) is liquid nitrogen, and the gas (500) is helium.

[0121] Accordingly, one embodiment of the present invention relates to a method for lowering the temperature of a sample, wherein the helium pressure is between 5 MPa and 100 MPa. Accordingly, embodiments of the present invention relate to a method for lowering the temperature of a sample (1), preferably a living sample (1), more preferably a living cell (1), for inverted microscopy, comprising the following steps.

[0122] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen having a temperature below the critical temperature and helium having a pressure between 5 MPa and 100 MPa into a pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen (2) into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly.

[0123] In principle, a liquid or mixture of liquids that can cool the sample to below the glass transition temperature of water (approximately -136°C) within a few milliseconds can be used as the liquid (2). However, the desired cooling rate is only achieved when liquid nitrogen is used in the method of the present invention.

[0124] In step b) of the method of the invention described herein, it is preferable that at least one liquid (2) is first filled into the pressure tank (400), and then a gas (500) is added to generate high pressure in the pressure tank (400).

[0125] In step b) of the method of the invention described herein, at least one liquid (2) can be supplied into the pressure tank (400) by filling it with a liquid cooled to a temperature below its critical temperature, that is, by filling the pressure tank with a pre-cooled liquid. Alternatively, at least one liquid (2) can be filled into the pressure tank (400) as a gas and then cooled to a temperature below its critical temperature so that the gas condenses into a liquid. Cooling of the gas in the pressure tank can be achieved, for example, by a cooling tank (300) surrounding the pressure tank.

[0126] Preferably, at least one liquid (2) is liquid nitrogen. In some embodiments, at least one liquid (2) is a further coolant such as liquid argon or liquid methane. This may include the following. Accordingly, embodiments of the present invention relate to a method for lowering the temperature of a sample (1), preferably a living sample (1), more preferably living cells (1), for inverted microscopy, comprising the following steps.

[0127] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) supplying at least one liquid (2) having a temperature below critical temperature and helium having a pressure of at least 2.5 MPa into a pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), at least one liquid (2) is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, and liquid nitrogen is brought into contact with the upper side (11) of the carrier substrate (10) to lower the temperature of the sample (1), preferably rapidly. At least one of the liquids (2) contains liquid nitrogen.

[0128] In another embodiment, a method for lowering the temperature of a sample (1) for inverted microscopy, preferably a living sample (1), more preferably living cells (1), includes the following steps:

[0129] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) supplying at least one liquid (2) having a temperature below critical temperature and helium having a pressure of at least 2.5 MPa into a pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), at least one liquid (2) is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, and liquid nitrogen is brought into contact with the upper side (11) of the carrier substrate (10) to lower the temperature of the sample (1), preferably rapidly. At least one liquid (2) comprises (i) liquid nitrogen and liquid argon, or (ii) liquid nitrogen and liquid methane, or (iii) a mixture of liquid nitrogen, liquid argon, and liquid methane.

[0130] In another embodiment, a method for lowering the temperature of a sample (1) for inverted microscopy, preferably a living sample (1), more preferably living cells (1), includes the following steps:

[0131] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) supplying at least one liquid (2) having a temperature below critical temperature and helium having a pressure between 5 MPa and 100 MPa into a pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), at least one liquid (2) is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, and liquid nitrogen is brought into contact with the upper side (11) of the carrier substrate (10) to lower the temperature of the sample (1), preferably rapidly. At least one liquid (2) comprises (i) liquid nitrogen and liquid argon, or (ii) liquid nitrogen and liquid methane, or (iii) a mixture of liquid nitrogen, liquid argon, and liquid methane.

[0132] In a preferred embodiment, a method for lowering the temperature of a sample (1) for inverted microscopy includes the following steps:

[0133] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1). To reduce the temperature, preferably rapidly, without forming ice crystals within the sample (1), or without forming ice crystals with a diameter greater than 80 nm, or without forming ice crystals with a diameter greater than 10 nm.

[0134] In a preferred embodiment, a method for lowering the temperature of a sample (1) for inverted microscopy includes the following steps:

[0135] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen having a temperature below the critical temperature and helium having a pressure between 5 MPa and 100 MPa into a pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) to lower the temperature of the sample (1), preferably rapidly, and without forming ice crystals in the sample (1), or without forming ice crystals with a diameter of 80 nm or more than 10 nm in the sample (1).

[0136] The temperature of at least one liquid (2) used in the method of the present invention is at least 5°C, preferably 10°C or more, below the critical temperature of the liquid. Accordingly, embodiments of the present invention relate to a method for lowering the temperature of a sample, wherein the temperature of at least one liquid (2) is at least 10°C below its critical temperature.

[0137] Accordingly, embodiments of the present invention relate to a method for lowering the temperature of a sample (1), preferably a living sample (1), more preferably living cells (1), for inverted microscopy, comprising the following steps.

[0138] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) supplying at least one liquid (2) having a temperature at least 10°C below the critical temperature and a gas (500) having a pressure of at least 2.5 MPa into a pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), at least one liquid (2) is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the at least one liquid (2) into contact with the upper side (11) of the carrier substrate (10), thereby lowering the temperature of the sample (1), preferably rapidly. At least one liquid (2) is liquid nitrogen, and the gas (500) is helium.

[0139] In further embodiments, a method for lowering the temperature of a sample (1) for inverted microscopy, preferably a living sample (1), more preferably living cells (1), includes the following steps:

[0140] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen having a temperature at least 10°C below its critical temperature and helium having a pressure between 5 MPa and 100 MPa into a pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), at least one liquid (2) is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the at least one liquid (2) into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly.

[0141] In principle, any gas such as helium or hydrogen (500) can be used in the method of the invention described herein. This gas is a compressible gas and is present in a pressure tank. Under certain conditions, it does not liquefy, does not react with the liquid, or dissolves in the liquid. However, when helium was used as a gas (500), the desired cooling rate was achieved.

[0142] In one embodiment, a mixture of gases (500) is used in the method of the present invention. The mixture of gases may be advantageous for adjusting the pressure in the pressure tank (500). Accordingly, one embodiment of the present invention relates to a method for lowering the temperature of a sample (1), preferably a living sample (1), more preferably living cells (1), for inverted microscopy, comprising the following steps.

[0143] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) A mixture of liquid nitrogen having a temperature below critical temperature and gas (500) having a pressure of at least 2.5 MPa is supplied into a pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly. The mixture of gases (500) contains helium.

[0144] Further embodiments of the present invention relate to a method for lowering the temperature of a sample (1), preferably a living sample (1), more preferably living cells (1), for use in an inverted microscope, comprising the following steps:

[0145] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) A mixture of liquid nitrogen having a temperature below critical temperature and gas (500) having a pressure of at least 2.5 MPa is supplied into a pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly. The gaseous (500) mixture contains helium and hydrogen.

[0146] Furthermore, one embodiment of the present invention relates to a method for lowering the temperature of a sample (1) for inverted microscopy, preferably a living sample (1), more preferably living cells (1), comprising the following steps.

[0147] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) A mixture of liquid nitrogen having a temperature below critical temperature and gas (500) having a pressure between 2.5 MPa and 1000 MPa is supplied into a pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly. The mixture of gases (500) contains helium.

[0148] Furthermore, one embodiment of the present invention relates to a method for lowering the temperature of a sample (1) for inverted microscopy, preferably a living sample (1), more preferably living cells (1), comprising the following steps.

[0149] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) A mixture of liquid nitrogen having a temperature below critical temperature and gas (500) having a pressure of at least 2.5 MPa is supplied into a pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) to lower the temperature of the sample (1), preferably rapidly, and without forming ice crystals in the sample (1), or without forming ice crystals with a diameter of 80 nm or more than 10 nm in the sample (1).

[0150] As used herein, “sample” refers to any specimen that is freeze-fixed or freeze-stopped and analyzed using the methods and apparatus of the present invention. Generally, each type of sample can be cooled. Samples typically contain a variable liquid composition of substances such as soft materials, materials, chemical reaction partners, or, in particular, biological materials.

[0151] The sample may include (polymer) materials such as peptides, proteins, oligonucleotides, sugars, or aqueous solutions of biochemical systems, or chemical molecular systems such as biopolymers. The aforementioned systems may be in their unaffected original state, modified state, excited state, non-equilibrium state, or equilibrium state. The sample may also be a biological sample such as (mammalian) cells, tissues, or bodily fluids (blood, serum, plasma, etc.). Preferably, the sample includes living biological material, more preferably living cells, living living cells, or living cellular components. The cells form an adherent cell culture on the underside (12) of the carrier substrate, preferably in the form of a cell monolayer. Adhesion is not absolutely necessary if the sample consists of a very thin layer between the carrier substrate and the observation window.

[0152] According to the present invention, the sample is prepared at normal pressure (atmospheric pressure) and tempered (frozen or thawed). Accordingly, embodiments of the present invention relate to a method for lowering the temperature of a sample (1) which includes living cells, preferably living living cells. Accordingly, embodiments of the present invention relate to a method for lowering the temperature of a sample (1), preferably a living sample (1), more preferably living cells (1) for inverted microscopy, comprising the following steps.

[0153] (a) To provide living cells (1), preferably living living cells (1), on the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Provide in a pressure tank (400) at least one liquid (2) having a temperature below the critical temperature and a gas (500) having a pressure of at least 2.5 MPa, (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), at least one liquid (2) is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, thereby bringing the at least one liquid (2) into contact with the upper side (11) of the carrier substrate (10), thereby lowering, preferably rapidly lowering, the temperature of the living cells (1), preferably living living cells (1). At least one liquid (2) is nitrogen and the gas (500) is helium.

[0154] Further embodiments of the present invention relate to a method for lowering the temperature of a sample (1), preferably a living sample (1), more preferably living cells (1), for use in an inverted microscope, comprising the following steps:

[0155] (a) To provide living cells (1), preferably living living cells (1), on the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Providing liquid nitrogen at a temperature below the critical temperature and helium at a pressure between 2.5 MPa and 1000 MPa in a pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10), bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10), and the living cells (1), preferably Or, to lower the temperature of living biological cells (1), preferably rapidly.

[0156] Further embodiments of the present invention relate to a method for lowering the temperature of a sample (1), preferably a living sample (1), more preferably living cells (1), for use in an inverted microscope, comprising the following steps:

[0157] (a) To provide living cells (1), preferably living living cells (1), on the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below the critical temperature and helium at a pressure between 2.5 MPa and 1000 MPa into a pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) to lower the temperature of the living cells (1), preferably living living cells (1), preferably rapidly lowering the temperature without forming ice crystals within the living cells (1), or without forming ice crystals with a diameter of 80 nm or more, or without forming ice crystals with a diameter of 10 nm.

[0158] According to the present invention, the liquid (2) comes into contact with the carrier substrate (10). To achieve a rapid temperature change, the liquid is released from a pressure tank (400) under pressure. A flow of liquid is formed on the upper side of the carrier substrate. This flow can be achieved with a defined start time and a predetermined flow velocity. The flow rate is selected according to the properties of the liquid (such as viscosity) and the desired temperature.

[0159] Advantageously, there are various ways to direct the flow of the tempering fluid relative to the upper surface of the carrier substrate. The flow can be directed by the design of the supply line (20) and its outlet opening (21). According to the first modification, a flow direction parallel to the surface normal of the upper surface of the carrier substrate is provided. The tempering fluid flows perpendicularly upward and then laterally away from the carrier substrate. In this case, there may be an advantage in that particularly efficient heat transfer can be obtained. Alternatively, in the second modification, the flow direction is perpendicular to the surface normal of the upper surface of the carrier substrate. The tempering fluid flows parallel to the upper surface so as to pass over the carrier substrate. In this case, there is an advantage in that flow resistance is low.

[0160] In a preferred embodiment, the flow is directed parallel to the surface normal of the upper side of the carrier substrate, resulting in the liquid flowing in the direction of gravity, thereby increasing the flow velocity to a certain extent.

[0161] According to the present invention, a rapid temperature drop is achieved. The terms "rapid temperature drop" (or "rapid temperature drop," "instantaneous temperature drop," "instantaneous temperature drop," "rapid temperature drop," or "explosive temperature drop") are used to describe a situation where the rate at which the sample temperature drops from the initial temperature (start temperature) to the final temperature (target temperature) (cooling rate, tempering rate) is lower than or negligible than the rate of molecular processes within the sample (such as ice crystal formation or denaturation of biomolecules or biological systems).

[0162] As a result, the method disclosed herein can lower the temperature of sample (1) in a manner that does not cause ice crystals to form within sample (1), or ice crystals larger than 10 nm to form within sample (1), or ice crystals larger than 80 nm in diameter to form within sample (1), or biomolecules or biological systems such as cells to denature (i.e., very rapidly or explosively).

[0163] In the investigation of samples, especially biological samples, it is sometimes desirable to keep the sample at a low temperature after the rapid (or explosive) cooling in step (c) to avoid the formation of ice crystals. In this regard, the upper side (11) of the carrier substrate (10) is permanently placed in at least one liquid (2) (liquid nitrogen). It may be passed through the target. Accordingly, embodiments of the present invention relate to a method for lowering the temperature of a sample, comprising an additional step (d).

[0164] (d) Temper the sample (1) on the lower side (12) of the carrier substrate (10) by passing it through at least one liquid over the upper side (11) of the carrier substrate (10).

[0165] Accordingly, one embodiment of the present invention relates to a method for lowering the temperature of a sample (1), preferably a living sample (1), more preferably living cells (1), for inverted microscopy, comprising the following steps.

[0166] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) supplying at least one liquid (2) having a temperature below the critical temperature and a gas (500) having a pressure of at least 2.5 MPa into a pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), at least one liquid (2) is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the at least one liquid (2) into contact with the upper side (11) of the carrier substrate (10), thereby lowering the temperature of the sample (1), preferably rapidly. (d) Tempering the sample (1) on the lower side (12) of the carrier substrate (10) by passing it through at least one liquid, At least one liquid (2) is liquid nitrogen, and the gas (500) is helium.

[0167] Further embodiments of the present invention relate to a method for lowering the temperature of a sample (1), preferably a living sample (1), more preferably living cells (1), for use in an inverted microscope, comprising the following steps:

[0168] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen having a temperature below the critical temperature and helium having a pressure between 5 MPa and 100 MPa into a pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly. (d) Temper the sample (1) on the lower side (12) of the carrier substrate (10) by passing liquid nitrogen over the upper side (11) of the carrier substrate (10).

[0169] A preferred embodiment of the present invention relates to a method for lowering the temperature of a sample (1), preferably a living sample (1), more preferably living cells (1), for use in an inverted microscope, comprising the following steps:

[0170] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly, and without forming ice crystals in the sample (1), or without forming ice crystals with a diameter of 10 nm or more in the sample (1), or without forming ice crystals with a diameter of 80 nm. (d) By passing liquid nitrogen over the carrier substrate (10) (11), Temper the sample (1) on the lower side (12) of the carrier substrate (10).

[0171] A preferred embodiment of the present invention relates to a method for lowering the temperature of a sample (1), preferably a living sample (1), more preferably living cells (1), for use in an inverted microscope, comprising the following steps:

[0172] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly, and without forming ice crystals within the sample (1), or without forming ice crystals with a diameter of 10 nm or more within the sample (1), or without forming ice crystals with a diameter of 80 nm or more within the sample (1), (d) Temper the sample (1) without forming ice crystals in the sample (1) on the lower side (12) of the carrier substrate (10) by passing liquid nitrogen over the upper side (11) of the carrier substrate (10).

[0173] The tempering solution used in step (d) may be the same liquid as the one used in step (b). The tempering solution may be at the same temperature below the critical temperature, or at a different temperature. Alternatively, the tempering solution used in step (d) may be a different liquid from the one used in step (b), having the same or a different temperature below its critical temperature.

[0174] Accordingly, one embodiment of the present invention relates to a method for lowering the temperature of a sample (1), preferably a living sample (1), more preferably living cells (1), for inverted microscopy, comprising the following steps.

[0175] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) A first liquid (2) having a temperature below the critical temperature and a gas (500) having a pressure of at least 2.5 MPa are supplied into a pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), the first liquid (2) is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing at least one liquid (2) into contact with the upper side (11) of the carrier substrate (10), thereby lowering the temperature of the sample (1), preferably rapidly. (d) Tempering the sample (1) on the lower side (12) of the carrier substrate (10) by passing the upper side (11) of the carrier substrate (10) through the second liquid, The first liquid (2) is liquid nitrogen, and the gas (500) is helium.

[0176] In other words, one embodiment of the present invention relates to a method for lowering the temperature of a sample (1), preferably a living sample (1), more preferably living cells (1), for use in an inverted microscope, comprising the following steps:

[0177] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into a pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1). To cause to decrease, preferably rapidly, (d) Temper the sample (1) on the lower side (12) of the carrier substrate (10) by passing it over the upper side (11) of the carrier substrate (10) through a further liquid.

[0178] In other words, one embodiment of the present invention relates to a method for lowering the temperature of a sample (1), preferably a living sample (1), more preferably living cells (1), for use in an inverted microscope, comprising the following steps:

[0179] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen having a temperature below the critical temperature and helium having a pressure between 5 MPa and 100 MPa into a pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly. (d) Temper the sample (1) on the lower side (12) of the carrier substrate (10) by passing it over the upper side (11) of the carrier substrate (10) through a further liquid.

[0180] In other words, one embodiment of the present invention relates to a method for lowering the temperature of a sample (1), preferably a living sample (1), more preferably living cells (1), for use in an inverted microscope, comprising the following steps:

[0181] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly, and without forming ice crystals within the sample (1), or without forming ice crystals with a diameter of 10 nm or more within the sample (1), or without forming ice crystals with a diameter of 80 nm or more within the sample (1), (d) Temper the sample (1) on the lower side (12) of the carrier substrate (10) by passing it over the upper side (11) of the carrier substrate (10) through a further liquid.

[0182] Accordingly, one embodiment of the present invention relates to a method for lowering the temperature of a sample (1), preferably a living sample (1), more preferably living cells (1), for inverted microscopy, comprising the following steps.

[0183] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supply liquid nitrogen at temperature T1 and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly. (d) Tempering the sample (1) on the lower side (12) of the carrier substrate (10) by passing it through a further liquid at temperature T2, Temperature T1 is lower than the critical temperature of liquid nitrogen, and optionally, temperature T2 is lower than the critical temperature of another liquid.

[0184] In a preferred embodiment, to avoid rapid temperature changes in the sample, the difference between T1 and T2 does not exceed 10K. Preferably, T1 and T2 are the same or nearly the same; that is, the difference between T1 and T2 does not exceed 5K.

[0185] Accordingly, one embodiment of the present invention relates to a method for lowering the temperature of a sample (1), preferably a living sample (1), more preferably living cells (1), for inverted microscopy, comprising the following steps.

[0186] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly. (d) Temper the sample (1) on the lower side (12) of the carrier substrate (10) by passing liquid nitrogen over the upper side (11) of the carrier substrate (10).

[0187] The tempering fluid used in step (d) may be supplied from a pressure tank (400) or via another line connected to the supply line.

[0188] To maintain sufficient cooling of the sample over a long period of time, step (d) of the method of the present invention involves passing the tempering solution over the carrier substrate at a pressure higher than atmospheric pressure. Preferably, the pressure is between 0.1 MPa and 1.5 MPa, more preferably between 0.2 MPa and 1 MPa, and most preferably between 0.5 MPa and 1 MPa. Accordingly, one embodiment of the present invention relates to a method for lowering the temperature of a sample (1), preferably a living sample (1), more preferably a living cell (1), for inverted microscopy, comprising the following steps.

[0189] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly. (d) Tempering the sample (1) on the lower side (12) of the carrier substrate (10) by passing liquid nitrogen over the upper side (11) of the carrier substrate (10) at a pressure higher than atmospheric pressure.

[0190] Furthermore, one embodiment of the present invention relates to a method for lowering the temperature of a sample (1) for inverted microscopy, preferably a living sample (1), more preferably living cells (1), comprising the following steps.

[0191] (a) To provide the sample (1) to the lower surface (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen having a temperature below the critical temperature and helium having a pressure between 5 MPa and 100 MPa into a pressure tank (400), (c) Liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressure by opening the outlet valve (22) located at the bottom of the pressure tank (400). The process involves bringing liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) to lower the temperature of the sample (1), preferably rapidly. (d) Temper the sample (1) on the lower side (12) of the carrier substrate (10) by passing liquid nitrogen over the upper side (11) of the carrier substrate (10) at a pressure between 0.5 MPa and 1 MPa.

[0192] Therefore, further embodiments of the present invention relate to a method for lowering the temperature of a sample (1), preferably a living sample (1), more preferably living cells (1), for inverted microscopy, comprising the following steps.

[0193] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly, and without forming ice crystals within the sample (1), or without forming ice crystals with a diameter of 10 nm or more within the sample (1), or without forming ice crystals with a diameter of 80 nm or more within the sample (1), d) Tempering the sample (1) on the lower side (12) of the carrier substrate (10) by passing it through liquid nitrogen at a pressure higher than atmospheric pressure over the upper side (11) of the carrier substrate (10).

[0194] Furthermore, one embodiment of the present invention relates to a method for lowering the temperature of a sample (1) for inverted microscopy, preferably a living sample (1), more preferably living cells (1), comprising the following steps.

[0195] (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly. (d) Tempering the sample (1) on the lower side (12) of the carrier substrate (10) by passing liquid nitrogen over the upper side (11) of the carrier substrate (10) at a pressure higher than atmospheric pressure. [Device] The present invention relates to a device (100) for lowering the temperature of a sample (1) in a manner that prevents the formation of ice crystals within the sample (1), or ice crystals with a diameter greater than 10 nm within the sample (1), or ice crystals with a diameter greater than 80 nm within the sample (1), or biomolecules or biological systems such as cells from denaturing (i.e., very rapidly or explosively), and the device (100) is: (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10) , To shorten the supply line (20) as much as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0196] In other words, the present invention also relates to an apparatus (100) for controlling the temperature of a sample (1), (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To keep the supply line (20) as short as possible, the pressure tank (400) is connected to the upper side (11) of the carrier substrate (10).

[0197] Furthermore, the present invention relates to an apparatus (100) for controlling the temperature of a sample (1), (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To keep the supply line (20) as short as possible, the pressure tank (400) is mounted directly to the upper side (11) of the carrier substrate (10).

[0198] Furthermore, the present invention relates to an apparatus (100) for controlling the temperature of a sample (1), (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To keep the supply line (20) as short as possible, the pressure tank (400) is connected directly to the upper side (11) of the carrier substrate (10).

[0199] More precisely, the present invention also relates to an apparatus (100) for controlling the temperature of a sample (1), the apparatus (100) is (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10) , To make the supply line (20) as short as possible, the bottom of the pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0200] In other words, the present invention also relates to an apparatus (100) for controlling the temperature of a sample (1), (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To make the supply line (20) as short as possible, the bottom of the pressure tank (400) is directly attached to the upper side (11) of the carrier substrate (10). [Supply line] The supply line (20) has its upper end connected to the outlet valve (22) of the pressure tank (400). The supply line has an outlet opening (21) at its lower end for releasing at least one liquid (2), as shown in Figure 2. The supply line (20) is positioned between the pressure tank (400) and the upper side (11) of the carrier substrate, thereby allowing at least one liquid (2) (and optionally a gas (500)) to be supplied to the upper side (11) of the carrier substrate.

[0201] The supply line (20) is preferably as short as possible to reduce unwanted temperature changes in at least one liquid (2), which could result in a slower cooling rate of the sample. On the other hand, the supply line needs to be long enough so that the outlet opening (21) is positioned optimally for supplying the liquid to the upper side of the carrier substrate. In other words, as shown in Figure 3, the discharged liquid (2) needs to be in direct contact with the upper side of the carrier substrate and not with other components of the apparatus.

[0202] To minimize the temperature rise of the coolant (2) within the supply line (20), the supply line (20) may be insulated. In principle, the supply line can be insulated from the inside with a suitable inert polymer material such as low-density polyethylene (LDPE), high-density polyethylene (HDPE), polypropylene, polysulfone, polycarbonate, polyethylene terephthalate, polyimide, polytetrafluoroethylene (PTFE), or polyurethane. The inventors have found that high-density polyethylene is the most stable and long-lasting insulator under low temperature and high pressure. Therefore, it is most preferable that the supply line be insulated from the inside with high-density polyethylene.

[0203] The supply line (20) can have any shape, such as cylindrical or funnel-shaped. In this specification, funnel-shaped means that the diameter of the upper part of the supply line is greater than the diameter of the lower part of the supply line, specifically that the diameter of the upper part of the supply line is greater than the diameter of the outlet opening (21) of the supply line. Preferably, the lower end (outlet opening (21)) of the supply line (20) is funnel-shaped, as shown in Figures 3C and 3D.

[0204] Therefore, embodiments of the present invention relate to a device (100) for lowering the temperature of a sample for inverted microscopy, wherein the supply line (20) is funnel-shaped.

[0205] Therefore, an embodiment of the present invention is an apparatus (100) for lowering the temperature of a sample. Regarding (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) An outlet valve (22) is connected to the upper side (11) of the carrier substrate (10), and a funnel-shaped supply line (20) is configured to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To shorten the supply line (20) as much as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0206] Further embodiments of the present invention relate to an apparatus (100) for lowering the temperature of a sample, (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) An outlet valve (22) is connected to the upper side (11) of the carrier substrate (10), and a funnel-shaped supply line (20) is configured to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To keep the supply line (20) as short as possible, the pressure tank (400) is mounted directly to the upper side (11) of the carrier substrate (10).

[0207] Further embodiments of the present invention relate to an apparatus (100) for lowering the temperature of a sample, (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) a funnel-shaped supply line (20) configured to connect the outlet valve (22) to an upper side (11) of a carrier substrate (10) and allow a liquid (2) to pass through under a pressure higher than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), in order to make the supply line (20) as short as possible, the pressure tank (400) is attached to the upper side (11) of the carrier substrate (10), and the supply line (20) is preferably made of high-density polyethylene and insulated from the inside.

[0208] Preferably, the apparatus (100) of the present invention for lowering the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for accommodating a sample (1) and an upper side (11) exposed for supplying a liquid (2); (b) a pressure tank (400) configured to hold a fluid and provided with an outlet valve (22) at a bottom portion thereof; (c) a supply line (20) configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10), allow the liquid (2) to pass through under a pressure higher than atmospheric pressure, and having a funnel-shaped lower end; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), in order to make the supply line (20) as short as possible, the pressure tank (400) is attached to the upper side (11) of said carrier substrate (10).

[0209] Preferably, the apparatus (100) of the present invention for lowering the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for accommodating a sample (1) and an upper side (11) exposed for supplying a liquid (2); (b) a pressure tank (400) configured to hold a fluid and provided with an outlet valve (22) at a bottom portion thereof; (c) a supply line (20) configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10), allow the liquid (2) to pass through under a pressure higher than atmospheric pressure, and having a funnel-shaped lower end; (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To keep the supply line (20) as short as possible, the pressure tank (400) is mounted directly to the upper side (11) of the carrier substrate (10).

[0210] Preferably, the apparatus (100) of the present invention for lowering the temperature of the sample is (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) The outlet valve (22) is connected to the upper side (11) of the carrier substrate (10), and the supply line (20) is configured to pass the liquid (2) through under a pressure higher than atmospheric pressure, with its lower end being funnel-shaped. (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To make the supply line (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10), and the supply line (20) is insulated from the inside, preferably with high-density polyethylene.

[0211] Furthermore, the supply line (20) of the apparatus (100) of the invention described herein is configured to pass the liquid (2) through under a pressure higher than atmospheric pressure. Therefore, the supply line is made of a material that is mechanically stable at the temperature of the liquid (2) and does not deform even when the liquid passes through under high pressure.

[0212] Furthermore, the supply line (20) is preferably straight (as shown in Figure 3) and does not contain corners, edges, or swirls in order to supply at least one liquid (2) at a higher flow rate. [Pressure tank (400)] The apparatus (100) of the present invention includes at least one pressure tank (400). The pressure tank (400) of the apparatus (100) is preferably a pressure vessel configured to hold a tempering liquid (2) and a gas (500), and / or an insulated storage container configured to hold a tempering liquid (2) and a gas (500). Since the term “fluid” includes liquids and gases, it is also preferable that the pressure tank (400) of the apparatus (100) is a pressure vessel configured to hold a fluid and / or an insulated storage container configured to hold a fluid. More preferably, the pressure tank (400) is an insulated pressure vessel configured to hold a tempering liquid (2) and a gas (500).

[0213] The volume of the pressure tank (400) is preferably 0.5L to 10L, more preferably 0.5L to 9L, more preferably 0.5L to 8L, more preferably 1L to 7L, and most preferably 1L to 5L.

[0214] Therefore, in one embodiment, the apparatus (100) of the present invention for lowering the temperature of a sample is, (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) with a volume of 1 L to 5 L, configured to hold fluid and equipped with an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To shorten the supply line (20) as much as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0215] Preferably, the apparatus (100) of the present invention for lowering the temperature of the sample is (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) with a volume of 1 L to 5 L, configured to hold fluid and equipped with an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To keep the supply line (20) as short as possible, the pressure tank (400) is mounted directly to the upper side (11) of the carrier substrate (10).

[0216] Preferably, the apparatus (100) of the present invention for lowering the temperature of the sample is (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) with a volume of 1 L to 5 L, configured to hold fluid and equipped with an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To make the supply line (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10), and the supply line (20) is insulated from the inside, preferably with high-density polyethylene.

[0217] In a preferred embodiment, the pressure tank (400) has a rounded bottom as shown in Figure 4A. Therefore, in one embodiment, the apparatus (100) of the present invention for lowering the temperature of a sample is (a) a carrier substrate (10) having a lower side (12) for accommodating a sample (1) and an upper side (11) exposed for supplying a liquid (2); (b) a pressure tank (400) configured to hold a fluid, provided with an outlet valve (22) at a bottom portion thereof and having a rounded bottom; (c) a supply line (20) connecting the outlet valve (22) to the upper side (11) of the carrier substrate (10) and configured to apply pressure higher than atmospheric pressure to allow the liquid (2) to pass therethrough; and (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), wherein, in order to make the supply line (20) as short as possible, the pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0218] Preferably, the apparatus (100) of the present invention for lowering the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for accommodating a sample (1) and an upper side (11) exposed for supplying a liquid (2); (b) a pressure tank (400) configured to hold a fluid, provided with an outlet valve (22) at a bottom portion thereof and having a rounded bottom; (c) a supply line (20) connecting the outlet valve (22) to the upper side (11) of the carrier substrate (10) and configured to apply pressure higher than atmospheric pressure to allow the liquid (2) to pass therethrough; and (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), wherein, in order to make the supply line (20) as short as possible, the pressure tank (400) is directly attached to the upper side (11) of the carrier substrate (10).

[0219] Preferably, the apparatus (100) of the present invention for lowering the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for accommodating a sample (1) and an upper side (11) exposed for supplying a liquid (2); (b) A pressure tank (400) configured to hold fluid, having an outlet valve (22) at the bottom and a rounded bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To make the supply line (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10), and the supply line (20) is insulated from the inside, preferably with high-density polyethylene.

[0220] In a preferred embodiment, the pressure tank (400) has a funnel-shaped bottom, which is advantageous for sealing the connection to the outlet valve (22) and the supply line (20). Thus, in one embodiment, the apparatus (100) of the present invention for lowering the temperature of a sample is, (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid, equipped with an outlet valve (22) at the bottom and having a funnel-shaped bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To shorten the supply line (20) as much as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0221] Preferably, the apparatus (100) of the present invention for lowering the temperature of the sample is (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid, equipped with an outlet valve (22) at the bottom and having a funnel-shaped bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To keep the supply line (20) as short as possible, the pressure tank (400) is mounted directly to the upper side (11) of the carrier substrate (10).

[0222] Preferably, the apparatus (100) of the present invention for lowering the temperature of the sample is (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid, equipped with an outlet valve (22) at the bottom and having a funnel-shaped bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To make the supply line (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10), and the supply line (20) is insulated from the inside, preferably with high-density polyethylene.

[0223] In a preferred embodiment, the apparatus (100) of the present invention for lowering the temperature of the sample is: (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid, equipped with an outlet valve (22) at the bottom and having a funnel-shaped bottom, (c) The outlet valve (22) is connected to the upper side (11) of the carrier substrate (10), and the supply line (20) is configured to pass the liquid (2) through under a pressure higher than atmospheric pressure, with its lower end being funnel-shaped. (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To shorten the supply line (20) as much as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10). [Cooling tank (300)] The apparatus (100) preferably further comprises at least one cooling tank (300) for cooling or tempering at least one liquid (2) in the pressure tank (400). The cooling tank (300) of the apparatus (100) of the present invention is preferably made of stainless steel, but may be made of other materials. The cooling tank (300) preferably has an opening at the top to conveniently supply a cooling medium. Thus, the cooling tank (300) surrounds the pressure tank (400) for cooling the liquid.

[0224] Therefore, in one embodiment, the apparatus (100) of the present invention for lowering the temperature of a sample is, (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) An expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), (e) comprising a cooling tank (300) for cooling the liquid (2), To shorten the supply line (20) as much as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0225] In a preferred embodiment, the apparatus (100) of the present invention for lowering the temperature of the sample is: (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) An expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), (e) comprising a cooling tank (300) for cooling the liquid (2), To keep the supply line (20) as short as possible, the pressure tank (400) is mounted directly to the upper side (11) of the carrier substrate (10).

[0226] In a further embodiment, the apparatus (100) of the present invention for lowering the temperature of a sample is: (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) An expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), (e) comprising a cooling tank (300) for cooling the liquid (2), To make the supply line (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10), and the supply line is insulated from the inside, preferably with high-density polyethylene.

[0227] In a preferred embodiment, the apparatus (100) of the present invention for lowering the temperature of the sample is: (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) An expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), (e) comprising a cooling tank (300) surrounding a pressure tank (400) for cooling the liquid (2).

[0228] In a preferred embodiment, the apparatus (100) of the present invention for lowering the temperature of the sample is: (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) An expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), (e) comprising a cooling tank (300) surrounding a pressure tank (400) for cooling the liquid (2), To make the supply line (20) as short as possible, the pressure tank (400) is the carrier base It is attached directly to the upper side (11) of the board (10).

[0229] In a further embodiment, the apparatus (100) of the present invention for lowering the temperature of a sample is: (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) An expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), (e) comprising a cooling tank (300) surrounding a pressure tank (400) for cooling the liquid (2), To make the supply line (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10), and the supply line is insulated from the inside, preferably with high-density polyethylene.

[0230] Cooling of the liquid (2) in the pressure tank (400) is achieved by heat exchange with a cooling medium contained in the cooling tank (300). In this respect, the temperature of the cooling medium is lower than the critical temperature of the liquid (2). The cooling medium may be the same substance as the liquid (2), for example, liquid nitrogen. Therefore, in one embodiment, the apparatus (100) of the present invention for lowering the temperature of a sample is: (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) An expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), (e) A cooling tank (300) configured to hold a cooling medium for cooling the liquid (2), To shorten the supply line (20) as much as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0231] In a preferred embodiment, the apparatus (100) of the present invention for lowering the temperature of the sample is: (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) An expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), (e) comprising a cooling tank (300) for cooling the liquid (2), The cooling tank (300) and the pressure tank (400) are not insulated from each other, and in order to shorten the supply line (20) as much as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10).

[0232] In a preferred embodiment, the apparatus (100) of the present invention for lowering the temperature of the sample is: (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) configured to hold fluid, equipped with an outlet valve (22) at the bottom, and having a funnel-shaped bottom It has a pressure tank (400) and (c) The outlet valve (22) is connected to the upper side (11) of the carrier substrate (10), and the supply line (20) is configured to pass the liquid (2) through under a pressure higher than atmospheric pressure, with its lower end being funnel-shaped. (d) An expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), (e) comprising a cooling tank (300) for cooling the liquid (2), To shorten the supply line (20) as much as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0233] In a preferred embodiment, the apparatus (100) of the present invention for lowering the temperature of the sample is: (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid, equipped with an outlet valve (22) at the bottom and having a funnel-shaped bottom, (c) The outlet valve (22) is connected to the upper side (11) of the carrier substrate (10), and the supply line (20) is configured to pass the liquid (2) through under a pressure higher than atmospheric pressure, with its lower end being funnel-shaped. (d) An expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), (e) comprising a cooling tank (300) for cooling the liquid (2), To keep the supply line (20) as short as possible, the pressure tank (400) is mounted directly to the upper side (11) of the carrier substrate (10).

[0234] In a preferred embodiment, the apparatus (100) of the present invention for lowering the temperature of the sample is: (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid, equipped with an outlet valve (22) at the bottom and having a funnel-shaped bottom, (c) The outlet valve (22) is connected to the upper side (11) of the carrier substrate (10), and the supply line (20) is configured to pass the liquid (2) through under a pressure higher than atmospheric pressure, with its lower end being funnel-shaped. (d) An expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), (e) comprising a cooling tank (300) for cooling the liquid (2), To make the supply line (20) as short as possible, the pressure tank (400) is mounted directly to the upper side (11) of the carrier substrate (10), and the supply line is insulated from the inside, preferably with high-density polyethylene. [Exit valve] According to the present invention, the supply of tempering solution to the upper side of the carrier substrate is activated (started) by an outlet valve, for example, an electric or pneumatic valve, located in the tempering solution supply line (20). A control element for opening and closing the outlet valve is located separately from the heat exchanger (carrier substrate). By activating the final control element, defined temperature control can be initiated. When the outlet valve is open, the tempering solution strikes the carrier substrate (10) with a predetermined defined delay. This allows the selected state of the sample, which has been identified in advance, to be fixed during observation with an inverted microscope. Therefore, the apparatus (100) is equipped with an outlet valve (22), preferably a switchable outlet valve, more preferably a switchable outlet valve that opens quickly. The switchable outlet valve (22) may be a pneumatically or electrically switchable valve and / or a fluid rotary valve.

[0235] Therefore, embodiments of the present invention relate to a device (100) for lowering the temperature of a sample, wherein the pressure tank (400) is equipped with a switchable outlet valve, more preferably a switchable outlet valve that opens quickly. Therefore, embodiments of the present invention relate to lowering the temperature of a sample Regarding the apparatus (100) for doing so, (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To make the supply line (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10), and the outlet valve is a switchable outlet valve, more preferably a quick-opening switchable outlet valve.

[0236] Preferably, the apparatus (100) for lowering the temperature of the sample is (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To keep the supply line (20) as short as possible, the pressure tank (400) is mounted directly to the upper side (11) of the carrier substrate (10), and the outlet valve is a switchable outlet valve, more preferably a quick-opening switchable outlet valve.

[0237] Preferably, the apparatus (100) for lowering the temperature of the sample is (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To make the supply line (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10). The outlet valve is a switchable outlet valve, more precisely, a switchable outlet valve that opens quickly. The supply line is preferably insulated from the inside with high-density polyethylene.

[0238] In another embodiment of the present invention, the apparatus (100) for lowering the temperature of the sample is, (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To make the supply line (20) as short as possible, the pressure tank (400) is the carrier base Attached to the upper side (11) of the board (10), The outlet valve is a valve and / or fluid rotary valve that can be switched by pneumatic or electrical means.

[0239] Any valve is suitable for the apparatus (100) and method of the invention disclosed herein, as long as it can open a large cross-section very quickly (switchably), i.e., within a few milliseconds, at -196°C and at least 50 bar. The outlet valve (22) may be an electrically or pneumatically switchable valve. Preferably, the outlet valve (22) is a pneumatically switchable valve. The switchable outlet valve (22) may consist of a lifting cylinder (200), a piston (501), and a piston tip (510), as shown in Figure 5. The piston tip is preferably made of high-density polyethylene (HDPE).

[0240] Furthermore, the switchable outlet valve (22) may further include a piston guide (520). Preferably, the switchable outlet valve (22) is a pneumatically switchable valve composed of a lifting cylinder (200), a piston (501), and a piston tip (510). Furthermore, the pneumatically switchable valve may further include a piston guide (520). This pneumatically switchable valve allows for quick opening of a large cross-section.

[0241] The piston (501) is movable. The vertically movable piston (501) is inserted into the pressure tank (400) via a sealed piston guide (520). The piston tip (510) is designed to seal the bottom of the pressure tank (400). The piston can be adjusted by a pneumatically driven lifting cylinder (200), thereby functioning as an outlet valve for the pressure tank (400). The pressure tank (400) can be filled with liquid and gas through a tube threaded into a screw hole. The gas may be cooled and condensed by cooling the pressure tank (400) by the surrounding cooling tank (300).

[0242] To achieve high cooling capacity immediately, it is advantageous for the piston (501) to move rapidly, i.e., within a few microseconds.

[0243] Therefore, a preferred embodiment of the present invention relates to a device (100) for lowering the temperature of a sample, in which a pressure tank (400) is provided with a pneumatically switchable valve comprising a lifting cylinder (200), a piston (501), and a piston tip (510). Therefore, a preferred embodiment of the present invention relates to a device (100) for lowering the temperature of a sample, (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To make the supply line (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10). The outlet valve (22) is a pneumatically switchable valve equipped with a lifting cylinder (200), a piston (501), and a piston tip (510).

[0244] Preferably, the apparatus (100) for lowering the temperature of the sample is (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (4) configured to hold fluid and equipped with an outlet valve (22) at the bottom. 00) and, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To make the supply line (20) as short as possible, the pressure tank (400) is mounted directly to the upper side (11) of the carrier substrate (10). The outlet valve (22) is a pneumatically switchable valve equipped with a lifting cylinder (200), a piston (501), and a piston tip (510).

[0245] Preferably, the apparatus (100) for lowering the temperature of the sample is (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To make the supply line (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10). The outlet valve (22) is a pneumatically switchable valve comprising a lifting cylinder (200), a piston (501), and a piston tip (510), and the supply line is preferably insulated from the inside with high-density polyethylene.

[0246] Another preferred embodiment of the present invention relates to a device (100) for lowering the temperature of a sample, wherein a pressure tank (400) comprises a lifting cylinder (200), a piston (501), a piston tip (510), and a pneumatically switchable valve having a piston guide (520). Therefore, a preferred embodiment of the present invention relates to a device (100) for lowering the temperature of a sample, (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To make the supply line (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10). The outlet valve (22) is a pneumatically switchable valve comprising a lifting cylinder (200), a piston (501), a piston tip (510), and a piston guide (520).

[0247] In a preferred embodiment, the apparatus (100) of the present invention for lowering the temperature of the sample is: (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid, equipped with an outlet valve (22) at the bottom and having a funnel-shaped bottom, (c) The outlet valve (22) is connected to the upper side (11) of the carrier substrate (10) and is configured to pass the liquid (2) through under a pressure higher than atmospheric pressure, and its lower end is funnel-shaped. Line (20) and, (d) An expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), (e) comprising a cooling tank (300) for cooling the liquid (2), To make the supply line (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10). The outlet valve (22) is a pneumatically switchable valve comprising a lifting cylinder (200), a piston (501), a piston tip (510), and a piston guide (520).

[0248] Preferably, the apparatus (100) for lowering the temperature of the sample is (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid, equipped with an outlet valve (22) at the bottom and having a funnel-shaped bottom, (c) The outlet valve (22) is connected to the upper side (11) of the carrier substrate (10), and the supply line (20) is configured to pass the liquid (2) through under a pressure higher than atmospheric pressure, with its lower end being funnel-shaped. (d) An expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), (e) comprising a cooling tank (300) for cooling the liquid (2), To make the supply line (20) as short as possible, the pressure tank (400) is mounted directly to the upper side (11) of the carrier substrate (10). The outlet valve is a pneumatically switchable valve comprising a lifting cylinder (200), a piston (501), a piston tip (510), and a piston guide (520).

[0249] Preferably, the apparatus (100) for lowering the temperature of the sample is (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid, equipped with an outlet valve (22) at the bottom and having a funnel-shaped bottom, (c) The outlet valve (22) is connected to the upper side (11) of the carrier substrate (10), and the supply line (20) is configured to pass the liquid (2) through under a pressure higher than atmospheric pressure, with its lower end being funnel-shaped. (d) An expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), (e) comprising a cooling tank (300) for cooling the liquid (2), To make the supply line (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10). The outlet valve (22) is a pneumatically switchable valve comprising a lifting cylinder (200), a piston (501), a piston tip (510), and a piston guide (520). The supply line (20) is preferably made of high-density polyethylene and is insulated from the inside. [Exit opening] Further advantages of the present invention arise when the lower end of the supply line (20) forming the outlet opening (21) has at least one of the following features, particularly in designs involving the expansion of the tempering medium. According to a first modification, the end may be positioned such that the distance of the outlet opening (21) from the upper side (11) of the carrier substrate is variable. For example, the supply line may be movable and the end may be adjustable relative to the carrier substrate. Alternatively or additionally, the end may be designed so that the diameter of the outlet opening is variable. The distance and / or diameter mentioned can be selected depending on the specific application of the present invention for optimal heat transfer and / or a given temperature setting of the tempering medium. According to another particularly preferred modification, the outlet opening (21) has a nozzle shape. The nozzle forms the outlet opening (21) of the supply line (20) and is shaped (funnel-shaped) such that the outlet opening (21) has a smaller diameter than the supply line (20). In another embodiment of the present invention, the lower end is a supply It is positioned so as to be separable from the line (20). For example, a replaceable nozzle (24) can be provided. If the end, in particular the nozzle, is connected to the supply line via a screw connection, both replaceability (to change the nozzle diameter) and positionability (to change the distance to the carrier substrate) can be advantageously simplified.

[0250] Preferably, the shape of the outlet opening (21) is that of a nozzle. Therefore, embodiments of the present invention relate to an apparatus (100) for lowering the temperature of a sample, wherein the outlet opening (21) is that of a nozzle. Therefore, embodiments of the present invention relate to an apparatus (100) for lowering the temperature of a sample. (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To make the supply line (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10). The outlet opening (21) has the shape of a nozzle.

[0251] Preferably, the apparatus (100) for lowering the temperature of the sample is (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To make the supply line (20) as short as possible, the pressure tank (400) is mounted directly to the upper side (11) of the carrier substrate (10). The outlet opening (21) has the shape of a nozzle.

[0252] Preferably, the apparatus (100) for lowering the temperature of the sample is (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To make the supply line (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10), the supply line (20) is preferably made of high-density polyethylene and insulated from the inside, and the outlet opening (21) has a nozzle shape.

[0253] In a preferred embodiment, the apparatus (100) of the present invention for lowering the temperature of the sample is: (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid, equipped with an outlet valve (22) at the bottom and having a funnel-shaped bottom, (c) The outlet valve (22) is connected to the upper side (11) of the carrier substrate (10), and the supply line (20) is configured to pass the liquid (2) through under a pressure higher than atmospheric pressure, with its lower end being funnel-shaped. (d) An expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), (e) comprising a cooling tank (300) for cooling the liquid (2), To make the supply line (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10). The outlet valve (22) is a pneumatically switchable valve comprising a lifting cylinder (200), a piston (501), a piston tip (510), and a piston guide (520). The outlet opening (21) has the shape of a nozzle.

[0254] Preferably, the nozzle diameter is between 1 mm and 15 mm, more preferably between 1 mm and 12 mm, more preferably between 1 mm and 10 mm, more preferably between 1 mm and 5 mm, and most preferably between 1.5 mm and 5 mm. Preferably, the nozzle diameter is 3 mm. Therefore, one embodiment of the present invention relates to an apparatus (100) for lowering the temperature of a sample, (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To make the supply line (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10). The outlet opening (21) has a nozzle shape with a diameter between 1.5 mm and 5 mm. [Exit opening and carrier substrate] The rate of temperature change can be further improved by adjusting the distance between the outlet opening (21) and the upper side (11) of the carrier substrate (10). The distance may be in the range of 0.1 mm to 5 mm, preferably in the range of 0.1 mm to 3 mm, more preferably in the range of 0.5 mm to 1.5 mm, and most preferably in the range of 1 mm.

[0255] Therefore, embodiments of the present invention relate to an apparatus (100) for lowering the temperature of a sample, wherein the distance between the outlet opening (21) and the upper side (11) of the carrier substrate (10) is in the range of 0.5 mm to 1.5 mm. Therefore, embodiments of the present invention relate to an apparatus (100) for lowering the temperature of a sample, (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To make the supply line (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10). The distance between the exit opening (21) and the upper side (11) of the carrier substrate (10) is 0.5 mm It is within a range of 1.5 mm.

[0256] It was also found that the ratio of the diameter of the outlet opening (21) to the diameter of the carrier substrate (10) may have a positive effect on the rate of temperature change. Therefore, the embodiment of the present invention relates to an apparatus (100) for lowering the temperature of a sample, (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To make the supply line (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10). The diameter of the exit opening (21) is smaller than the diameter of the carrier substrate (10).

[0257] In a preferred embodiment, the apparatus (100) of the present invention for lowering the temperature of the sample is: (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid, equipped with an outlet valve (22) at the bottom and having a funnel-shaped bottom, (c) The outlet valve (22) is connected to the upper side (11) of the carrier substrate (10), and the supply line (20) is configured to pass the liquid (2) through under a pressure higher than atmospheric pressure, with its lower end being funnel-shaped. (d) An expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), (e) comprising a cooling tank (300) for cooling the liquid (2), To make the supply line (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10). The outlet valve (22) is a pneumatically switchable valve comprising a lifting cylinder (200), a piston (501), a piston tip (510), and a piston guide (520). The outlet opening (21) has a nozzle shape, and the diameter of the outlet opening (21) is smaller than the diameter of the carrier substrate (10). [Carrier substrate] The carrier substrate (10) (or cooling substrate) forms the sample holder and the heat exchanger. The carrier substrate (10) is typically a plate-like, flat or curved component, with one surface (referred to here as the lower side) forming the sample container and the other surface (referred to here as the upper side) forming the heat exchanger surface. In the case of self-adhesive samples such as adherent cell cultures, the exposed lower surface, which may support a modification layer, provides the sample holder. Otherwise, for example in the case of non-adhesive samples, a receiving structure may be provided on the lower side to form a container for receiving a sample, for example, a liquid reaction partner. The upper surface is preferably a flat surface exposed for moistening with a cooling medium. However, the upper side may also have surface structures such as protrusions or indentations to which the tempering fluid can come into contact.

[0258] The tempering rate (cooling rate or heating rate) is preferably greater than 5,000 K / sec, particularly greater than 20,000 K / sec, for example, exceeding 50,000 K / sec, and even more preferably exceeding 100,000 K / sec. The temperature change of the sample occurs essentially simultaneously with the temperature change of the carrier substrate caused by the tempering fluid. For this purpose, the carrier substrate preferably includes a material whose thermal conductivity is selected such that the temperature of the sample changes at a tempering rate (temperature gradient) that exceeds the limits mentioned. The conductivity is particularly preferably greater than 1,000 W / Km, and especially preferably greater than 1,500 W / Km.

[0259] The thickness of the carrier substrate (10) is preferably less than 2 mm, preferably less than 1 mm, and more preferably less than 500 μm. Rapid heat transfer combined with the appropriate stability required for the high pressure applied to the carrier substrate can be achieved when the thickness is in the range of 0.1 mm to 10 mm, more preferably 0.1 mm to 2 mm, and most preferably 0.5 mm to 1.5 mm.

[0260] Therefore, embodiments of the present invention relate to an apparatus (100) for lowering the temperature of a sample, wherein the thickness of the carrier substrate (10) is in the range of 0.1 mm to 10 mm. (a) A carrier substrate (10) having a thickness ranging from 0.1 mm to 10 mm, having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To shorten the supply line (20) as much as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0261] The carrier substrate (10) can be made of any material having the desired thermal conductivity and sufficient strength under the pressure of the tempering fluid. Thus, the carrier substrate (10) functions as a thermal conductor. For example, the carrier substrate can be made of copper (400 W / Km) or silver (430 W / Km). In such cases, the thickness of the carrier substrate required to obtain sufficient strength may adversely affect the value of the thermal conductivity and the achievable rate of temperature change (tempering rate), but it is important that the carrier substrate (10) is ensured to be sufficiently thick. However, this is not critical when the requirement for the tempering rate is low and / or when the sample is placed under a pressure higher than atmospheric pressure.

[0262] The carrier substrate includes diamond disks, such as synthetic diamond disks with a thickness of less than 2 mm, and especially less than 500 μm. The use of diamond offers special advantages in terms of optimal thermal conductivity (which varies depending on the product, e.g., 1,500 W / Km) and strength when the substrate thickness is reduced.

[0263] Accordingly, embodiments of the present invention relate to a device (100) for lowering the temperature of a sample, wherein the carrier substrate (10) is diamond, particularly CVD diamond. Preferably, the impurities in the CVD diamond are less than 6 ppb. The carrier substrate may include a diamond disk, for example, a synthetic diamond disk with a thickness of less than 2 mm, preferably less than 1 mm, and more preferably less than 500 μm. The use of diamond offers special advantages in terms of optimal values ​​of thermal conductivity (which varies depending on the product, e.g., 1,500 W / Km) and strength when the substrate thickness is reduced. Particularly preferred is single-crystal CVD diamond exhibiting a thermal conductivity of more than 2200 W / Km.

[0264] Therefore, an embodiment of the present invention is a temperature control device (1) for lowering the temperature of a sample, wherein the carrier substrate (10) is made of CVD diamond with an impurity of less than 6 ppb. This applies to 00). Using such CVD diamond increases thermal conductivity and also increases the rate of temperature change.

[0265] Therefore, embodiments of the present invention relate to an apparatus (100) for lowering the temperature of a sample. (a) A carrier substrate (10) made of CVD diamond with impurities of less than 6 ppb, having a lower side (12) for containing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To shorten the supply line (20) as much as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0266] Preferably, the apparatus (100) for lowering the temperature of the sample is (a) A carrier substrate (10) made of CVD diamond with impurities of less than 6 ppb, having a lower side (12) for containing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To keep the supply line (20) as short as possible, the pressure tank (400) is mounted directly to the upper side (11) of the carrier substrate (10).

[0267] Preferably, the apparatus (100) for lowering the temperature of the sample is (a) A carrier substrate (10) made of CVD diamond with impurities of less than 6 ppb, having a lower side (12) for containing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To make the supply line (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10). The supply line (20) is preferably made of high-density polyethylene and is insulated from the inside.

[0268] Another embodiment of the present invention relates to an apparatus (100) for lowering the temperature of a sample, wherein the apparatus (100) is (a) A carrier substrate (10) made of single-crystal CVD diamond having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (4) configured to hold fluid and equipped with an outlet valve (22) at the bottom. 00) and, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To shorten the supply line (20) as much as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0269] A further advantage of the present invention is brought about by a temperature decrease from the upper side of the carrier substrate (10), thereby exposing the sample to the lower side. For example, optical and / or electrical measurements of the sample can be performed before, during, and / or after the supply of the tempering medium. For example, a measuring device (1000) and / or impedance measuring device (1000) including an optical microscope, confocal microscope, inverted confocal microscope, or observation optical system can be arranged, specifically an inverted optical microscope, more specifically an inverted confocal microscope. Using a microscope, the sample can be observed at high resolution while remaining in an unchanging position on the carrier substrate. The impedance measuring device may include measuring electrodes positioned on the front surface of the carrier substrate.

[0270] Therefore, the apparatus (100) may include a measuring device (1000), preferably a microscope, and more preferably an inverted microscope.

[0271] Therefore, one embodiment of the present invention relates to an apparatus (100) for lowering the temperature of a sample, (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) An expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), (f) Equipped with a measuring device (1000), To shorten the supply line (20) as much as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0272] Preferably, the apparatus (100) for lowering the temperature of the sample is (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) An expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), (f) Equipped with a measuring device (1000), To keep the supply line (20) as short as possible, the pressure tank (400) is mounted directly to the upper side (11) of the carrier substrate (10).

[0273] Preferably, the apparatus (100) for lowering the temperature of the sample is (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (4) configured to hold fluid and equipped with an outlet valve (22) at the bottom. 00) and, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) An expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), (f) Equipped with a measuring device (1000), To make the supply line (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10). The supply line (20) is preferably made of high-density polyethylene and is insulated from the inside.

[0274] In a preferred embodiment of the apparatus (100) according to the present invention, the measuring device (1000) is an inverted microscope. Therefore, another aspect of the present invention relates to an inverted cryomicroscope (2000), (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) An expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), (f) Equipped with a measuring device (1000), To shorten the supply line (20) as much as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0275] In a preferred embodiment, the inverted cryomicroscope (2000) of the present invention is: (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid, equipped with an outlet valve (22) at the bottom and having a funnel-shaped bottom, (c) A supply line (20) having an upper end connected to an outlet valve (22) and an outlet opening (21) at its lower end facing the upper side (11) of the carrier substrate (10), configured to pass liquid (2) through under a pressure higher than atmospheric pressure, and having a funnel shape at its lower end, (d) Expansion chamber (30) surrounding the outlet opening (21), (c) The outlet valve (22) is connected to the upper side (11) of the carrier substrate (10), and the supply line (20) is configured to pass the liquid (2) through under a pressure higher than atmospheric pressure, with its lower end being funnel-shaped. (d) An expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), (e) A cooling tank (300) for cooling the liquid (2), (f) Equipped with a measuring device (1000), To make the supply line (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10). The outlet valve (22) is a pneumatically switchable valve comprising a lifting cylinder (200), a piston (501), a piston tip (510), and a piston guide (520). The outlet opening (21) has a nozzle shape, and the diameter of the outlet opening (21) is smaller than the diameter of the carrier substrate (10). [Sample Chamber (14)] The apparatus may further include a sample chamber (14). Therefore, embodiments of the present invention relate to an apparatus (100) for lowering the temperature of a sample, further comprising a sample chamber (14). The sample chamber (14) may include a spacer, but it is preferable that it does not include a spacer. This further significantly improves the rate of temperature change, i.e., increases the rate of temperature change.

[0276] Therefore, embodiments of the present invention relate to an apparatus (100) for lowering the temperature of a sample. (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) An expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), (g) comprising a sample chamber (14), To shorten the supply line (20) as much as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0277] Preferably, the apparatus (100) for lowering the temperature of the sample is (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) An expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), (g) comprising a sample chamber (14), To keep the supply line (20) as short as possible, the pressure tank (400) is mounted directly to the upper side (11) of the carrier substrate (10).

[0278] Preferably, the apparatus (100) for lowering the temperature of the sample is (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) An expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), (g) comprising a sample chamber (14), To make the supply line (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10). The supply line (20) is preferably made of high-density polyethylene and is insulated from the inside.

[0279] In one embodiment, as shown in Figure 3E, the sample chamber (14) is formed by the underside of the carrier substrate and an observation window (15). This sample chamber may or may not include additional spacers.

[0280] A preferred embodiment of the present invention relates to an apparatus (100) for lowering the temperature of a sample, which includes a sample chamber (14) without a spacer. Therefore, a preferred embodiment of the present invention relates to an apparatus (100) for lowering the temperature of a sample. (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) An expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), (g) A sample chamber (14) without a spacer is provided. To shorten the supply line (20) as much as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0281] If it is necessary to further increase the rate of temperature change, it is even more preferable that the apparatus according to the present invention does not even include a sample chamber (14). Accordingly, embodiments of the present invention relate to an apparatus (100) for lowering the temperature of a sample that does not include a sample chamber (14) and does not include a spacer.

[0282] Therefore, embodiments of the present invention relate to an apparatus (100) for lowering the temperature of a sample. (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) a pressure tank (400) configured to hold a fluid and provided with an outlet valve (22) at a bottom portion thereof, (c) a supply line (20) connecting the outlet valve (22) to an upper side (11) of a carrier substrate (10) and configured to apply a pressure higher than atmospheric pressure to allow the liquid (2) to pass therethrough, (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), and comprising: in order to make the supply line (20) as short as possible, the pressure tank (400) is attached to the upper side (11) of the carrier substrate (10), said apparatus (100) does not include a sample chamber (14) or a spacer. [Expansion chamber] It has been found that the high pressure difference between an outlet opening and the expansion chamber enables rapid transport of the liquid (2) on the carrier substrate (10). The larger the expansion chamber (30) is, the larger the pressure difference between the outlet opening (21) and the expansion chamber (30) becomes, which can increase the transport speed of the liquid on the carrier substrate (10).

[0283] Accordingly, embodiments according to the present invention are directed to an apparatus (100) for lowering the temperature of a sample, wherein the expansion chamber (30) has a dimension of 50 cm 3 to 100 cm 3 in the range of

[0284] Accordingly, an embodiment according to the present invention relates to an apparatus (100) for lowering the temperature of a sample, comprising: (a) a carrier substrate (10) having a lower side (12) for accommodating a sample (1) and an upper side (11) exposed for supply of a liquid (2), (b) a pressure tank (400) configured to hold a fluid and provided with an outlet valve (22) at a bottom portion thereof, (c) a supply line (20) connecting the outlet valve (22) to an upper side (11) of a carrier substrate (10) and configured to apply a pressure higher than atmospheric pressure to allow the liquid (2) to pass therethrough, (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10) and having a dimension of 50 cm3 From 100cm 3 It comprises an expansion chamber (30) which is within the range, To shorten the supply line (20) as much as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0285] To achieve the maximum cooling rate, the carrier substrate (10) is made of CVD diamond, and the dimensions of the expansion chamber (30) are 50 cm². 3 From 100cm 3 It is within the range of supplying The (20) is funnel-shaped, and the distance between the outlet opening (21) and the upper side (11) of the carrier substrate (10) is in the range of 0.5 mm to 1.5 mm.

[0286] Therefore, in a preferred embodiment of the present invention, the carrier substrate (10) is made of CVD diamond, and the dimensions of the expansion chamber (30) are 50 cm. 3 From 100cm 3 The present invention relates to a device (100) for lowering the temperature of a sample, wherein the supply line (20) is funnel-shaped, and the distance between the outlet opening (21) and the front surface (11) of the carrier substrate (10) is in the range of 0.5 mm to 1.5 mm.

[0287] Therefore, a preferred embodiment of the present invention relates to an apparatus (100) for lowering the temperature of a sample, (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) The distance between the exit opening (21) surrounding the upper side (11) of the carrier substrate (10) and the front surface (11) of the carrier substrate (10) is in the range of 0.5 mm to 1.5 mm, and the dimensions are 50 cm3 From 100cm 3 It comprises an expansion chamber (30) which is within the range, To shorten the supply line (20) as much as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0288] The expansion chamber (30) of the apparatus (100) of the present invention is preferably open so that the pressure inside the chamber can be reliably kept low when the tempering fluid is supplied. The expansion chamber may be located in a housing equipped with a gas outlet (23), as shown in Figures 3B, 3C, and 3D. Thus, the apparatus according to the present invention may further include a gas outlet (23). The gas outlet (23) is preferably connected to an exhaust hose via a quick coupling for an exhaust hose. The exhaust hose is preferably connected to the housing of the expansion chamber (30).

[0289] Therefore, embodiments of the present invention relate to an apparatus (100) for lowering the temperature of a sample. (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an open expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To shorten the supply line (20) as much as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0290] Another embodiment of the present invention relates to an apparatus (100) for lowering the temperature of a sample, (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) The outlet valve (22) is connected to the upper side (11) of the carrier substrate (10), and the pressure is greater than atmospheric pressure. A supply line (20) configured to pass liquid (2) through under high pressure, (d) an open expansion chamber (30) with a gas outlet (23) surrounding the upper side (11) of the carrier substrate (10), To shorten the supply line (20) as much as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0291] Preferably, the apparatus (100) for lowering the temperature of the sample is (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) an open expansion chamber (30) with a gas outlet (23) surrounding the upper side (11) of the carrier substrate (10), To keep the supply line (20) as short as possible, the pressure tank (400) is mounted directly to the upper side (11) of the carrier substrate (10).

[0292] Preferably, the apparatus (100) for lowering the temperature of the sample is (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) an open expansion chamber (30) with a gas outlet (23) surrounding the upper side (11) of the carrier substrate (10), To make the supply line (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10). The supply line (20) is preferably made of high-density polyethylene and is insulated from the inside.

[0293] In a preferred embodiment, the apparatus (100) of the present invention is (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid, equipped with an outlet valve (22) at the bottom and having a funnel-shaped bottom, (c) The outlet valve (22) is connected to the upper side (11) of the carrier substrate (10), and the supply line (20) is configured to pass the liquid (2) through under a pressure higher than atmospheric pressure, with its lower end being funnel-shaped. (d) An open expansion chamber (30) surrounded the upper side (11) of the carrier substrate (10), which is equipped with a gas outlet (23), (e) comprising a cooling tank (300) for cooling the liquid (2), To make the supply line (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10). The outlet valve (22) is a pneumatically switchable valve comprising a lifting cylinder (200), a piston (501), a piston tip (510), and a piston guide (520). The outlet opening (21) has a nozzle shape, and the diameter of the outlet opening (21) is smaller than the diameter of the carrier substrate (10). [Permanent cooling] According to another advantageous modification of the present invention, after a temperature change of the sample, particularly after cooling, the sample has a ki Permanent tempering of the carrier substrate (10) can be performed. For example, during the examination of a sample using inverted microscopy, a low temperature can be maintained by passing the upper side (11) of the carrier substrate (10) through a gaseous or liquid constant temperature medium. The gaseous or liquid constant temperature medium is supplied at a low pressure between 5 and 10 bar. Furthermore, freeze-fixation of rapidly frozen samples is also possible. Freeze-fixation can be carried out using one of the methods known to the extent that the carrier substrate (10) with the sample is transferred from the apparatus to a freeze-fixation apparatus, such as a cryotank containing liquid nitrogen, as needed.

[0294] The tempering fluid for permanent cooling may be supplied from a pressure tank (400) or via a separate line connected to the supply line. Preferably, the tempering fluid for permanent cooling is a cooling medium stored in a cooling tank (300).

[0295] Therefore, one embodiment of the present invention relates to an apparatus (100) for lowering the temperature of a sample, (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) The outlet valve (22) is connected to the upper side (11) of the carrier substrate (10) and configured to pass the liquid (2) through under a pressure higher than atmospheric pressure, and the supply line (20) has an inlet for tempering liquid for permanent cooling, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), To shorten the supply line (20) as much as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0296] In one embodiment of the present invention, the apparatus (100) for lowering the temperature of the sample is (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) An expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), (e) comprising a cooling tank (300) for cooling the liquid (2), To make the supply line (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10). The cooling tank (300) is connected to a pressure tank (400) for supplying tempering fluid to the supply line for permanent cooling.

[0297] It is preferable that the tempering solution be supplied quickly or instantaneously after cooling in order to keep the sample temperature consistently low. In this regard, in an embodiment of the apparatus of the present invention in which the tempering solution is supplied from a pressure tank (400), the supply of the tempering solution is controlled by a valve. The valve may be a check valve that opens as soon as the pressure in the tank drops due to the opening of the outlet valve (22). Alternatively, the valve may be a valve that opens after a predetermined time has elapsed.

[0298] Therefore, in one embodiment of the present invention, the apparatus (100) for lowering the temperature of the sample is, (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) An expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), (e) comprising a cooling tank (300) for cooling the liquid (2), To make the supply line (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10). The cooling tank (300) is connected via a valve to a pressure tank (400) for supplying tempering fluid to the supply line for permanent cooling.

[0299] The valve is preferably a check valve configured to open when the pressure inside the pressure tank reaches atmospheric pressure. Alternatively, the valve is preferably a check valve configured to open when the pressure inside the pressure tank falls below a predetermined pressure higher than atmospheric pressure.

[0300] Another embodiment of the present invention relates to an apparatus (100) for lowering the temperature of a sample, (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10) and further provided with an inlet for tempering liquid for permanent cooling, To shorten the supply line (20) as much as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10). [Fluorescence microscopy] The methods and apparatus disclosed herein are particularly useful for fluorescence microscopy of freeze-stopped samples (1). Ultra-rapid freeze-stopping of a sample (1), preferably a biological sample such as living cells, overcomes the fundamental resolution barriers imposed by motion blur and photochemical reactivity, thereby enabling the observation of the intrinsic molecular distribution and reaction patterns that cannot be resolved at physiological temperatures.

[0301] Therefore, further aspects of the present invention relate to a fluorescence microscopy method comprising the following steps.

[0302] (a) To provide a sample (1) containing fluorescent molecules on the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly. (d) Irradiating sample (1) (e) Detect the fluorescence generated by sample (1).

[0303] The sample (1) can be irradiated with any suitable light source, such as a laser, LED, xenon, or mercury gas lamp.

[0304] Preferably, the fluorescence microscopy method includes the following steps.

[0305] (a) To provide a sample (1) containing fluorescent molecules on the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen having a temperature below the critical temperature and helium having a pressure between 5 MPa and 100 MPa into a pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly. (d) Irradiating sample (1) (e) Detect the fluorescence generated by sample (1).

[0306] Preferably, the fluorescence microscopy method includes the following steps.

[0307] (a) To provide a sample (1) containing fluorescent molecules on the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly, so as to vitrify the sample (1) without crystallization, preferably without forming ice crystals with a diameter of 80 nm or more, preferably without forming ice crystals with a diameter of 10 nm or more, more preferably without forming ice crystals, (d) Irradiating sample (1) (e) Detect the fluorescence generated by sample (1).

[0308] In a preferred embodiment, the fluorescence microscopy method includes the following steps:

[0309] (a) To provide a sample (1) containing fluorescent molecules on the underside (12) of the carrier substrate (10) at atmospheric pressure, (a') Irradiating sample (1) and (a'') Detecting fluorescence generated by sample (1), (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly. (d') Repeat step (a') and step (a'').

[0310] [Optical microscopy using stimulated emission suppression (STED)] A further aspect of the present invention relates to a method for performing optical microscopy using the suppression of stimulated emission from a freeze-stopped sample (1), (a) To provide a sample (1) containing fluorescent molecules on the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly. (d1) Irradiating the sample (1) with a first pulsed laser beam focused to at least one focal region to excite fluorescent molecules in the sample (1) and cause them to emit fluorescence, (d2) Irradiate the sample (1) with a second torus-shaped laser beam containing an intensity zero point within at least one focal region to de-excite the fluorescent molecules in the sample (1), (e) Detect the fluorescence generated by sample (1), (f) optionally, the process includes repeating steps d1, d2, and e at various positions in the focal region of the first pulsed laser beam and at various positions in the intensity zero point of the second torus-shaped laser beam.

[0311] Preferably, the method for performing optical microscopy by suppressing the induced emission of the freeze-stopped sample (1) is as follows: (a) To provide a sample (1) containing fluorescent molecules on the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen having a temperature below the critical temperature and helium having a pressure between 5 MPa and 100 MPa into a pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly. (d1) Irradiating the sample (1) with a first pulsed laser beam focused to at least one focal region to excite fluorescent molecules in the sample (1) and cause them to emit fluorescence, (d2) Irradiate the sample (1) with a second torus-shaped laser beam containing an intensity zero point within at least one focal region to de-excite the fluorescent molecules in the sample (1), (e) Detect the fluorescence generated by sample (1), (f) optionally, the process includes repeating steps d1, d2, and e at various positions in the focal region of the first pulsed laser beam and at various positions in the intensity zero point of the second torus-shaped laser beam.

[0312] Preferably, the method for performing optical microscopy by suppressing the induced emission of the freeze-stopped sample (1) is as follows: (a) To provide a sample (1) containing fluorescent molecules on the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly, so as to vitrify the sample (1) without crystallization, preferably without forming ice crystals with a diameter of 80 nm or more, preferably without forming ice crystals with a diameter of 10 nm or more, more preferably without forming ice crystals, (d1) Irradiating the sample (1) with a first pulsed laser beam focused to at least one focal region to excite fluorescent molecules in the sample (1) and cause them to emit fluorescence, (d2) Irradiate the sample (1) with a second torus-shaped laser beam containing an intensity zero point within at least one focal region to de-excite the fluorescent molecules in the sample (1), (e) Detect the fluorescence generated by sample (1), (f) optionally, the process includes repeating steps d1, d2, and e at various positions in the focal region of the first pulsed laser beam and at various positions in the intensity zero point of the second torus-shaped laser beam. [Fluorescence Lifetime Imaging Microscopy (FLIM)] A further aspect of the present invention relates to a method for performing fluorescence lifetime imaging microscopy using time-correlated single-photon counting on a frozen-stopped sample (1), (a) To provide a sample (1) containing fluorescent molecules on the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly. (d) By irradiating the sample with a pulsed laser beam, fluorescent molecules are periodically excited and fluorescent photons are emitted, (e) Detecting the fluorescent photons generated by the sample along with the arrival time of the photons relative to the laser pulse (1), (f) Determining the fluorescence lifetime from the detected fluorescent photons and the arrival times of the photons.

[0313] Preferably, the method for performing fluorescence lifetime imaging microscopy by time-correlated single-photon counting on a freeze-stopped sample (1) is as follows: (a) To provide a sample (1) containing fluorescent molecules on the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen having a temperature below the critical temperature and helium having a pressure between 5 MPa and 100 MPa into a pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1), preferably rapidly. (d) By irradiating the sample with a pulsed laser beam, fluorescent molecules are periodically excited and fluorescent photons are emitted, (e) Detecting the fluorescent photons produced by sample (1), (f) This includes determining the fluorescence lifetime from the detected fluorescent photons.

[0314] Preferably, a method for performing fluorescence lifetime imaging microscopy by time-correlated single-photon counting on a freeze-stopped sample (1) is: (a) To provide a sample (1) containing fluorescent molecules on the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen at a temperature below critical temperature and helium at a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) to lower the temperature of the sample (1), preferably rapidly, and without forming ice crystals within the sample (1), or without forming ice crystals with a diameter of 80 nm or more, or without forming ice crystals with a diameter of 10 nm or more. (d) By irradiating the sample with a pulsed laser beam, fluorescent molecules are periodically excited and fluorescent photons are emitted, (e) Detecting the fluorescent photons produced by sample (1), (f) This includes determining the fluorescence lifetime from the detected fluorescent photons.

[0315] This disclosure is also related to the following items.

[0316] Item 1. A method for lowering the temperature of a sample (1) for inverted microscopy, including the following steps: (a) To provide the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure; (b) supplying at least one liquid (2) having a temperature below critical temperature and a gas (500) having a pressure higher than atmospheric pressure into a pressure tank (400); (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), at least one liquid (2) is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, bringing the at least one liquid (2) into contact with the upper side (11) of the carrier substrate (10) and lowering, preferably rapidly lowering, the temperature of the sample (1).

[0317] Item 2. The method described in Item 1, wherein the pressure of the gas (500) is between 50 bar and 1000 bar.

[0318] Item 3. The method according to Item 1 or Item 2, wherein at least one liquid (2) in step (b) is liquid nitrogen.

[0319] Item 4. The gas (500) is helium, as described in any of items 1 through 3.

[0320] Item 5. The method of any of items 1 through 4, further including step (d): (d) Temper the sample (1) on the lower side (12) of the carrier substrate (10) by passing it over the upper side (11) of the carrier substrate (10) through a further liquid.

[0321] Item 6. The method according to any one of items 1 to 5, wherein the sample (1) is a living sample of biological material, preferably living cells.

[0322] Item 7. The method according to any one of items 1 to 7, wherein in step (c), the temperature of sample (1) is reduced, preferably rapidly, without forming ice crystals in sample (1) or without forming ice crystals with a diameter greater than 10 nm in sample (1).

[0323] Item 8. Apparatus (100) for lowering the temperature of sample (1), (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) having an upper end connected to an outlet valve (22) and an outlet opening (21) at its lower end facing the upper side (11) of the carrier substrate (10), and configured to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) An apparatus (100) comprising an expansion chamber (30) surrounding an outlet opening (21).

[0324] Item 9. Apparatus (100) as described in Item 8, which lowers the temperature of sample (1) so that ice crystals do not form within sample (1), or so that ice crystals with a diameter greater than 10 nm do not form within sample (1).

[0325] Item 10. The apparatus (100) according to item 8 or 9, wherein the outlet valve (22) is a switchable valve, preferably a quick-opening switchable valve, preferably an electrically or pneumatically switchable valve, preferably a switchable piston valve.

[0326] Item 11. The apparatus described in any of items 8 through 10, wherein the bottom of the pressure tank (400) is funnel-shaped and the outlet valve (22) is located in the center of the bottom.

[0327] Item 12. An apparatus (100) according to any of items 8 to 11, wherein the carrier substrate (10) is diamond, in particular CVD diamond.

[0328] Item 13. The apparatus (100) described in any of items 8 to 12, wherein the distance between the exit opening (21) and the upper side (11) of the carrier substrate (10) is in the range of 0.5 mm to 1.5 mm.

[0329] Item 14. The dimensions of the expansion chamber (30) are 50 cm 3 From 100cm 3 A device (100) described in any of items 8 through 13, which falls within the range of the above.

[0330] Item 15. The apparatus (100) according to any one of items 8 to 14, wherein a pressure tank (400) is surrounded by a cooling tank (300) for cooling a liquid (2), and / or the supply line is funnel-shaped, preferably funnel-shaped at the outlet opening (21). [Brief explanation of the drawing]

[0331] [Figure 1] This figure shows the localization accuracy calculated relative to the actual structure size (left) and resolution (right), plotted against the structure size of various amounts of photons (legend on the right) emitted at room temperature (solid line) or under freeze-stopped conditions (dotted line) simulating Brownian motion. Horizontal black dotted line: localization accuracy of 10% of the actual structure size. This figure illustrates the limit of the spatial resolution ("fundamental barrier") of the observed system. PSF represents the point spreading function of the microscope. This limit is inversely proportional to the size of the observed structure and is caused by Brownian motion or active motion ("room temperature" vs. "freeze-stopped"), and by the number of photons collected, which is limited by the exposure time, which is limited by the maximum achievable fluorescence photon flux determined by molecular cross-section, fluorescence quantum yield, excited state lifetime, and photochemical stability. [Figure 2] A schematic diagram of one embodiment of the apparatus (100) of the present invention applied to an inverted microscope (1000) is shown. The following means are shown: sample (1), liquid (2), gas (500), carrier substrate (10), upper (11) and lower (12) sides of the carrier substrate (10), cooling tank (300), pressure tank (400), supply line (20), outlet opening (21), outlet valve (22), expansion chamber (30), and inverted microscope (100). [Figure 3A] A detailed diagram of the apparatus (100) according to the present invention is shown: The carrier substrate (10) functions as a heat exchanger, receiving the sample (1) on its underside, and has a supply line (20) for the liquid (2) and an outlet opening (21). When the outlet valve (22) opens, the liquid (2) and gas (500) flow out from the pressure tank (400), pass through the supply line to the upper side (11) of the carrier substrate (10), and flow into the expansion chamber (30). [Figure 3B] A detail drawing of one embodiment of the apparatus (100) of the present invention having an expansion chamber (30) equipped with a gas outlet (23) is shown, the gas outlet (23) may further be equipped with a quick coupling for an exhaust hose. [Figure 3C]A detailed diagram of one embodiment of the apparatus (100) of the present invention, which has a closed expansion chamber (30) and a supply line (20) having a funnel-shaped outlet opening (21), is shown. The outlet opening may be in the shape of a nozzle. [Figure 3D] A detailed diagram of an embodiment of the apparatus (100) of the present invention, which has a closed expansion chamber (30) and a supply line (20) having a funnel-shaped outlet opening (21), is shown. The outlet opening may be in the shape of a nozzle. [Figure 3E] A detailed diagram of one embodiment of the apparatus (100) of the present invention is shown, in which an observation window (15) is positioned below the sample (1), thereby forming a sample chamber (14). [Figure 4A] An exemplary embodiment of a pressure tank (400) of the apparatus according to the present invention, having a round or semicircular bottom, is shown. [Figure 4B] An exemplary embodiment of a pressure tank (400) of the apparatus according to the present invention, having a flat bottom, is shown. [Figure 5] An exemplary embodiment of a pressure tank (400) of the apparatus according to the present invention, equipped with a switchable outlet valve (22), is shown. The following means are shown: a lifting cylinder (200), a piston guide (520), a piston tip (510), and a piston (501). The piston tip is preferably made of high-density polyethylene (HDPE). This switchable valve allows for quick opening of a large cross-section. [Figure 6] The temperature profile obtained using the apparatus according to the present invention is shown. A cylindrical sample with a diameter of 4 mm and a thickness of 100 μm was used for the cooling experiment. The temperature was measured using a 50 μm non-adiabatic wire thermocouple (T-type, Omega Engineering Inc.) in an aqueous imaging medium (DMEM containing HEPES buffer from PAN-Biotech GmbH). As can be seen, the temperature can initially be maintained at the desired temperature. When the switching valve is opened, the temperature drops sharply to below -130°C. Subsequently, by passing the upper side (11) of the carrier substrate (10) through liquid nitrogen, the temperature is always maintained below -130°C. [Figure 7A]Figures 7A and 7C show a comparison of the temperature and cooling rate achieved by the apparatus according to the present invention (Figure 7A) and the apparatus according to EP2877828B1 (Figure 7B). Temperature was measured using a 50 μm non-adiabatic wire thermocouple (T-type, Omega Engineering Inc.) in an aqueous imaging medium (DMEM containing HEPES buffer from PAN-Biotech GmbH). The bottom curve in Figure 7A shows the cooling rate in °C / sec for cooling experiments using the apparatus or method according to the present invention. The thick line represents the average value for individual cooling experiments and shows the standard deviation. The other curves show the temperature profile for individual cooling experiments. Here the cooling process is clearly visible, and it can be seen that the cooling rate (bottom curve) exceeds 10,000 K / sec in the temperature range between 0°C and -130°C. Ice can normally form in that temperature range. [Figure 7B] The bottom curve shows the cooling rate in °C / second for cooling experiments using the apparatus or method described in EP2877828B1. [Figure 7C] This shows a comparison of the cooling rates achieved by the apparatus according to the present invention operating with a mixture of liquid nitrogen (LN2) and helium (He) at a pressure of 50 bar (solid line) and lower pressures of 25 bar and 15 bar (dashed lines), and by the apparatus according to the present invention operating with helium at 50 bar without LN2. Temperature was measured using a 50 μm non-adiabatic wire thermocouple (T-type, Omega Engineering Inc.) in an aqueous imaging medium (DMEM containing HEPES buffer from PAN-35 Biotech GmbH). It is clearly evident that the cooling rate slows down with lower helium pressure. The lowest cooling rate was observed when liquid nitrogen was omitted. [Figure 8]This document compares the temperature and temperature change rate achieved by the apparatus according to the present invention with the apparatus according to EP2877828B1 ("prior art apparatus"), which is adapted to an inverted microscope by extending the supply line to the upper side of the carrier substrate. The temperature change rate was measured using a 50 μm non-adiabatic wire thermocouple (T-type, Omega Engineering Inc.) in an aqueous imaging medium (DMEM containing HEPES buffer from PAN-Biotech GmbH). [Figure 9] Electron microscope images (input: electron diffraction pattern) of ice crystals in dilute aqueous solution (PBS) at various cooling rates are shown. All cooling rates (relative measurements) were recorded using a 50 μm thermocouple. Actual cooling rates are much faster. (A) A sample frozen at a measured cooling rate of approximately 1000 K / s, obtained with a conventional apparatus adapted to an inverted microscope (Figure 8). The electron diffraction pattern shows that only one hexagonal ice crystal is found. The actual size of the ice crystal cannot be determined. However, the sample consists only of hexagonal ice, and at this size, the molecular components within the cell would be displaced, leading to cell destruction. (B) and (C) show samples frozen at measured cooling rates of approximately 2500 K / s and 3500 K / s, respectively. Individual ice crystals were observed. At a cooling rate of approximately 3500 K / s, the diameter of the ice crystals is smaller than 50 nm. (D) shows a cell sample containing 30% dextran (cryoprotectant) frozen at a measured cooling rate of approximately 3500 K / second. Cubic ice crystals less than 10 nm in diameter were formed. The arrows point in the direction of the cell membrane. [Figure 10] A cross-sectional view of one embodiment of the apparatus (100) according to the present invention is shown. The following means are shown: a lifting cylinder (200), a cooling tank (300), a pressure tank (400), a piston tip (510), a heat exchanger (600), a measuring device (1000), such as an inverted microscope, and a gas outlet (23). A sealed expansion chamber (30) together with a carrier substrate (10) forms a heat exchanger (600). The gas outlet (23) further includes a quick coupling for an exhaust hose. [Figure 11]Figure 10 shows an external view of an embodiment of the apparatus (100) according to the present invention. Means (800) for adjusting and locking the height are shown. [Figure 12] Figure 10 shows the lower part of an embodiment of the apparatus (100) according to the present invention. In particular, the piston tip (510), heat exchanger (600), and diamond window (700) are shown. [Figure 13] Figure 10 shows the lower part of the device (100) according to the present invention, specifically the gas outlet (23), along with the quick coupling for the exhaust hose shown in Figure 10. [Figure 14] Figure 10 shows a detailed view of the upper part of a pressure tank (400) as part of the apparatus (100) of the present invention. Screw holes (450) for assembly components such as a pressure gauge, rupture disc, and gas supply pipe are shown. [Figure 15] Figure 10 shows a top view of a screw hole (450) in one embodiment of the apparatus (100) according to the present invention. Means (800) for adjusting and locking the height, a screw hole (450), and a hole (210) for a lifting cylinder are shown. [Figure 16] Figure 10 shows a more detailed view of the lifting cylinder (200) of one embodiment of the apparatus (100) according to the present invention. In particular, the piston guide (520) is shown. [Figure 17] The images show the results of wide-field fluorescence microscopy of EGFP-transfected HeLa cells before cryopreservation (first three images) and during cryopreservation (rightmost image). Scale bar: 10 μm [Figure 18] The leftmost image shows a wide-field fluorescence image of the fluorescent dye rhodamine 6G in cell culture medium after ultra-rapid freeze-stopping using a microscope. The sample was almost homogeneous, except for highly fluorescent structures already present before freeze-stopping. Arrows: Expansion cracks. The three images on the far right: The same sample during reheating. The cracks completely disappeared, and then, after removing the fluorescent dye, ice crystals became visible (arrows). Subsequently, the ice crystals grew first, and then melted at higher temperatures, causing the liquid phase between the ice crystals to expand (illustrated by the arrows in the far right image). Scale bar: 10 μm [Figure 19]Examples of MCF7 cells expressing TC-PTP-mCitrine, imaged by confocal scanning microscopy at a specified time before freeze-stopping (Images 1 to 3 from left). Right half: The same cells are imaged by confocal microscopy (total of 10 frames) and wide-field microscopy (top: single frame, bottom: total of 100 frames). Bottom right image: SRRF (super resolution radial variation) reconstruction of 100 frames. Scale bar: 10 μm [Figure 20A] This image shows MCF7 cells expressing R-PTP-γ-mCitrine and Alexa647-labeled Snap-EGFR, previously imaged by confocal laser scanning microscopy at specified times (3.75-second scan time) after application of 100 ng / mL EGF (single frame, images 1-3) and during freeze-stop (total of 10 frames, 37.5 seconds, image 4). Right, top: Confocal scan of a wider area including several cells. Second column: SRRF reconstruction of 100 frames of wide-field recording of the same cells (left) and an example of cells fixed without prior EGF stimulation. Third column: Magnified view of a box region with a mask for the plasma membrane (white dotted line). Scale bar: 10 μm [Figure 20B] Motion blur measured by CLSM of Alexa647-Snap-EGFR labeled endosomes at room temperature (top) and under freeze-stop conditions (bottom). Left image: individual frames, right image: total of 10 frames. Graph: corresponding cumulative background correction line profiles, color-coded by frame number. [Figure 21A] CLSM and STED nanoscopy of Alexa647-Snap-EGFR in live MCF7 cells (top) and freeze-stopped MCF7 cells (bottom). CLSM of cells (first row, pixel size: 100 nm). Detail of box region (pixel size: 40 nm) CLSM scan (second row) and STED scan (third row) at specified laser irradiation intensity. Live cells: single scan. Freeze-stopped cells: total of 10 scans. [Figure 21B]Alexa647 photobleaching during STED scans at room temperature (rt) or under cryo-stopped conditions at specified depletion laser power. λ: attenuation rate (mean + / - standard deviation, N=5) determined by exponential fitting. [Figure 21C] STED spatial frequency spectra normalized by corresponding CLSM for various regions containing vesicles (cryo: n=11; chemically fixed: n=8; rt raw: n=6). Horizontal line: frequency range where STED information content is significantly higher than that of CLSM (Student's t-test); Vertical line: digital resolution. [Figure 21D] Top row: 3D-CLSM of freeze-arrested MCF7 cells. Bottom row: Detailed high-resolution scan of the specified region in the top row (pixel size: 40nm): CLSM (left column), STED (center column), and merged (right column). [Figure 22] Representative intensity-weighted mean fluorescence lifetime (τ) images obtained by confocal FLIM from Cos7 cells stably expressing LIFEA2. Images are shown for and without clustered ephrin A1 stimulation (left column) and with clustered ephrin A1 stimulation (right column) at room temperature (rt, top) or under freeze-stop conditions (cryo, bottom). Bar: τ (ns). [Figure 23] Confocal FLIM of LIFEA2-expressing Cos7 cells after clustered ephrin A1 stimulation at specified times before and during ultrarapid freeze-stop. Top panel: number of fluorescence photons, bottom panel: τ (ns), graph: corresponding τ histograms at room temperature and freeze-stop. [Figure 24A] Normalized cumulative fluorescence decay histograms of LIFEA2 at room temperature and freeze-stopped, with and without clustered ephrin A1 stimulation (rt: B, N=6; n=9; cryo: D, N=7; n=16). [Figure 24B] The imaginary (Im) and real (Re) parts of the phasor (R) derived from the first harmonic of the fluorescence decay data in Figure 24A. Dashed line: Linear regression of the phasor. [Figure 25]The number of fluorescent photons at various z-heights (rows) (upper panel) and the fluorescence intensity weighting ratio of the active structure of LIFEA2 (lower panel) were obtained by confocal FLIM during freeze-stop after 14 minutes of stimulation with clustered ephrin. Bar: Percentage of LIFEA2 in the active structure (α). [Figure 26] This is a representative series consisting of 10 consecutive STED scans of a 5×5 μm region using Alexa647-Snap-EGFR in EGF-stimulated (100 ng / ml) MCF7 cells under freeze-stop. The image is displayed as a sum of two scans and has been gamma-corrected (γ: 0.24) to highlight faint fluorescence. The protruding plasma membrane appears as a continuous fluorescence region separating cytoplasmic fluorescence with endocytosis structures from extracellular autofluorescence (extracellular). This series was acquired at maximum laser intensity (0.42 W / μm²). Ice crystals above 80 nm (digital resolution), which exclude fluorescent dye molecules, scatter light, and appear as dark spots, were not detected in the integrated image and were not formed during scanning due to heating that may occur with the STED beam. [Modes for carrying out the invention]

[0332] [Example 1: Design and Operation of an Exemplary Embodiment of the Device of the Invention] The main components of the cooling system (100) are a pressure tank (400) that holds N2 and helium, and a carrier substrate (10) that functions as a heat exchanger unit. The pressure tank (400) is made of stainless steel and its joints are sealed with copper gaskets. The pressure tank (400) is cooled from the outside by liquid N2 filling a cooling tank (300) insulated with Styrodur® made from steel plate. The pressure tank (400) included a custom-made pneumatically controlled piston that functions as a valve (22). The piston is sealed on a shaft with a spring-loaded PTFE seal. The tip of the piston is made of high-density polyethylene (HDPE) that withstands pressure while forming a seal against the stainless steel of the tank when closed. As the piston rises, the pressurized fluid is released onto the upper side (11) of the carrier substrate (10). The supply line (20), which functions as a connecting component, was kept as short as possible and insulated from the inside with HDPE to minimize heating of the coolant from the metal components. Within the heat exchanger unit, the mixture is guided directly to the upper side (11) of the carrier substrate (10), which includes a diamond window, through a nozzle at the outlet opening (21). The mixture then spreads out and is guided to the exhaust port.

[0333] Using a mixture of helium and N2 yielded significantly better cooling performance than using each alone. N2 alone rapidly liquefies at -196°C and 50 bar, causing a drop in pressure within the container and slowing release. As a result, the cooling rate was much slower. Helium cannot liquefy under these conditions, but using only the gas results in incomplete and slow freeze-fixation due to its low heat capacity. N2 was first filled into the apparatus for 1 minute by introducing nitrogen from an internal source (approximately 7-8 bar) through a copper spiral immersed in liquid nitrogen. The pressure was then increased with helium from a pressurized bottle (200 bar, Westfalen AG, Münster, Germany). The pressure used to fill the pressure tank was limited to 50 bar by a pressure reducing valve.

[0334] All valves were controlled via a graphical user interface created using LabVIEW software (National Instruments, Austin, Texas, USA). A check valve suitable for cryogenic use (CGS250SS, Fluessiggas-Anlagen GmbH, Salzgitter, Germany) was installed between the pressure vessel and the copper spiral for nitrogen supply to prevent high pressure from entering the copper spiral. The entire system was controlled by an electronically controlled lifting cylinder (1000N, Drive-System Europe). It is mounted on the frame above the epifluorescence microscope in a manner that allows it to be raised for mounting the sample (using a device called Ltd., Werther, Germany).

[0335] The heat exchanger unit was always heated to 37°C before each shot by two 50W heating bands (Friedr.Freek GmbH, Menden, Germany) mounted on the heat exchanger unit and controlled by a PID controller (KT4, Panasonic Corporation, Kadoma, Japan).

[0336] To maintain the temperature at -196°C after rapid cooling, N2 from an internal supply (approximately 8 bar) was used, introduced through a copper spiral immersed in liquid nitrogen. A check valve between the copper coil and the pressure vessel opens as soon as the pressure inside the vessel falls below the pressure inside the copper coil. This allows liquid nitrogen to flow through the vessel on the diamond, keeping the vessel at a temperature close to -196°C.

[0337] A transparent, flexible foil was attached to the stage around the objective lens by custom 3D-printed connectors. The chamber inside this foil was filled with a constant low flow rate of room-temperature dry helium throughout the experiment. The temperature of the objective lens was controlled by an objective lens heater (Bioptechs Inc., Butler, Pennsylvania, USA). The objective lens heater was always set to 25°C. Imaging was performed for 30 to 60 minutes. During the experiment, the temperature of the objective lens was typically between 7°C and 10°C. [Example 2: Temperature measurement using the apparatus according to the present invention] A 50 μm non-adiabatic wire thermocouple was attached to the underside (12) of the carrier substrate (10) of the apparatus (100) according to the present invention. The cooling tank (300) and the permanent cooling device were cooled with liquid nitrogen until the vigorous boiling of the liquid nitrogen stopped and the components reached a temperature close to 77 K. Next, the pressure tank (400) was filled with 50 bar liquid nitrogen and helium. A droplet of HEPES-buffered DMEM on the microscope coverslide was allowed to adhere to the underside (12) of the carrier substrate (10) by water adhesion, so that the droplet surrounded the thermocouple. Since the wire thermocouple was not flat, a gap remained between the carrier substrate and the wire, resulting in a cylindrical sample with a diameter of 4 mm and a thickness of approximately 100 μm. Temperature measurement was started, and cooling was initiated by opening the outlet valve (22) during the measurement. The results of this measurement are shown in Figures 6, 7A, and 8. [Example 3: Temperature measurement using the apparatus according to the present invention at pressures of 15 bar, 25 bar, and 50 bar] A 50 μm non-adiabatic wire thermocouple was attached to the underside (12) of the carrier substrate (10) of the apparatus (100) according to the present invention. The cooling tank (300) and permanent cooling device were cooled with liquid nitrogen until the vigorous boiling of the liquid nitrogen stopped and the components reached a temperature close to 77 K. Then, the pressure tank (400) was filled with 15 bar, 25 bar, or 50 bar of liquid nitrogen and helium, or filled with 50 bar of helium without liquid nitrogen. A droplet of HEPES-buffered DMEM on a microscope coverslide was allowed to adhere to the underside (12) of the carrier substrate (10) by water adhesion, so that the droplet surrounded the thermocouple. Since the wire thermocouple was not flat, a gap remained between the carrier substrate and the wire, resulting in a cylindrical sample with a diameter of 4 mm and a thickness of approximately 100 μm. Temperature measurement was started, and cooling was initiated by opening the outlet valve (22) during the measurement. The cooling rate decreased significantly as the pressure decreased, and the lowest cooling rate was observed in the absence of liquid nitrogen (Figure 7C). [Example 4: Temperature measurement using a conventional device adapted for an inverted microscope] The apparatus disclosed in EP2877828B1 was set up and adapted to an inverted microscope by extending the supply line to the upper side of the carrier substrate. A thermocouple was attached to the lower side of the carrier substrate. Temperature measurements were performed as described in Example 2. The results of these measurements are shown in Figures 7B and 8. [Example 5: Microscopic view of adherent and proliferated mammalian cells] An inverted microscope equipped with the temperature control device of the present invention was set up. First, the cooling tank (300) and the permanent cooling device were cooled with liquid nitrogen until the vigorous boiling of the liquid nitrogen stopped and the components reached a temperature close to 77K. Next, the pressure tank (400) was filled with nitrogen and helium at 50 bar. The coverslide, to which the cells had attached and grown, was attached to the underside (12) of the carrier substrate (10) by the adhesion of water. As a result, most of the cell culture medium was removed, leaving the cells in a thin layer of about 15 μm. When the stage was lowered onto the objective lens of the microscope, living cells could be observed. At the target time, the valve was opened to rapidly freeze-stop the cells. After this, detailed microscopic measurements of the freeze-stopped cells could be performed. [Example 6: Spatial-resolved spectroscopy of living cells] ·method [Calculation of resolution and positioning accuracy] Resolution and localization accuracy were calculated considering the effects of the imaging system, molecular motion, quantum yield of fluorescent dye molecules, and photostability. Simply put, since motion was modeled as molecular diffusion through a low-Reynolds number liquid, the diffusion coefficient D of the object was obtained as a function of temperature (T) and radius (r) through the following Stokes-Einstein equation.

[0338]

number

[0339] Here, η is the viscosity of the liquid, k Bis the Boltzmann constant. The viscosity of water is 0.6922 mPas at 37°C. Upon freeze-stop (CA), the viscosity increases by more than 15 orders of magnitude, so the object was considered to be at rest. The photophysical properties (quantum yield and photostability) of fluorescent proteins were obtained from the literature, and their changes upon freeze-stop were obtained from the data shown here (QY). CA / QY RT = 1.2, (emitted photons) CA (Emitted photon) RT >10).

[0340] Using a Gaussian approximation for the point spread function (PSF), and knowing that the variance of a convolution of multiple Gaussian distributions is the sum of their individual variances, the variance of the PSF (EPSF) including motion blur can be calculated.

[0341]

number

[0342] It will be as follows:

[0343]

number

[0344] Here, ν MB corresponds to the mean square displacement (4DT) of the diffusion process. The calculations and procedures described are appropriate when a single point light source is used as a label for the imaged structure. In contrast, if the structure of interest is fully labeled, additional procedures are required. Assuming uniform labeling, there is one light source at each point of the structure, so the observed object becomes a convolution of the EPSF and the object. In the Gaussian approximation, the output variance is again the sum of the two input variances. The calculations were performed as a function of the number of photons emitted by the molecule at a rate of (1 / 500 ns) at room temperature. This value was found to be consistent with other values ​​reported in the literature, ensuring that the fluorescent dye molecule is not re-excited when active. Furthermore, it was assumed that only 10% of the emitted photons reached the detector due to reduced collection and transfer efficiency.

[0345] The resolution, as a function of the number of photons, was calculated using Monte Carlo simulations. Simply put,

[0346]

number

[0347] N photons are extracted from a distribution consisting of two Gaussian distributions defined by and at a predetermined distance The data was separated at a distance d. The results were binned (size a) to simulate pixelation. A Gaussian mixture model was used to reconstruct these Gaussian distributions, and contrast was defined as follows:

[0348]

number

[0349] Here, I min This corresponds to the number of counts in the valley between two Gaussian distributions, and I max This corresponds to the maximum height of the two peaks. According to Abbe's criteria, the structure was considered to have decomposed when C > 1.7%. This process is

[0350]

number

[0351] The process was repeated 1000 times for each combination of d and N.

[0352]

number

[0353] If it was greater than the specified value, the structure was considered to have disintegrated.

[0354]

number

[0355] The resolution of N was defined as the minimum d at which 95% of the realized values ​​are resolved. Localization accuracy was calculated as a function of N, a, and detector noise. Importantly, PSF was replaced with EPSF to account for motion blur. [Measuring the cooling rate] Cooling performance was measured using a 50 μm constantan copper thermocouple (Omega Engineering Inc., Norwalk, Connecticut, USA; response time: 3 ms) attached to a μL aqueous buffer droplet. The total sample thickness was approximately 100 μm, as inferred from the droplet covering the entire area of ​​the attached coverslide, based on the thermocouple thickness. The thermocouple was connected to a computer via a USB data acquisition module (Omega Engineering Inc., Norwalk, Connecticut, USA), and L The readings were taken at a frequency of 1000 Hz (digital resolution: 2 milliseconds) using abview software (National Instruments, Austin, Texas, USA). [Calculation of cooling rate limited by thermal diffusivity] To relate the measured cooling rate to a cooling rate limited by the thermal diffusivity of the aqueous sample, heat flux-limited cooling from one surface (i.e., cooling of an aqueous solution from diamond) is based on the following heat flow equation.

[0356]

number

[0357] Area (A) is the area of ​​the cover slide (2 × 10 -5 m 2 The thermal conductivity of water (κ) is 0.5562 Wm². -2The initial temperature difference (ΔT) is approximately 233°C, between the 37°C sample and liquid nitrogen (-196°C). The thickness (L) was calculated from the volume collected with a pipette covering the entire coverslide. The freezing rate of samples containing a 50 μm thermocouple was calculated assuming the thermocouple was measured at a depth of 50 μm (L=50 μm) within a 100 μm thick sample. For aqueous samples without a thermocouple, the thickness was measured between 10 and 15 μm, assuming a typical L of approximately 13 μm for attached mammalian cell samples.

[0358] The heat flow Q can also be explained as follows:

[0359]

number

[0360] Here, c is the heat capacity (water: 4 J cm²). -3 K -1 ), where m is the mass of the sample (0.25 mg).

[0361] When combined, the following result is obtained.

[0362]

number

[0363] The estimated temperature progression of a sample with thickness L was calculated using MATLAB, starting from an initial temperature of 37°C.

[0364]

number

[0365] Despite neglecting heat transport along the thermocouple and the different thermal conductivity and capacity of the thermocouple and aqueous solution, the resulting diffusion-limited rate of temperature change was remarkably similar to the measured rate of temperature change. [Generation of stable transgenic cell lines] A polyclonal stable cell line of MCF7 expressing TC-PTP fused to mCitrine (TC-PTP-mCitrine) was generated in the background of MCF7 cells modified by CRISPR gene editing to suppress the expression of endogenous TC-PTP. TC-PTP-mCitrine (Stanoev et al. 2018, Cell Systems 7, 295-309.e11) was first cloned into a PiggyBac vector (System Bioscience, Palo Alto, California). MCF7 cells were then transfected with this construct and PiggyBac transposase (System Bioscience) in a 1:1 ratio. Transfection was performed 24 hours after seeding using FuGENE® 6 (Roche Diagnostics) as the transfection reagent. Transfection was performed according to the manufacturer's protocol. One day later, transfected cells were selected with 2 μg / mL puromycin for 30 hours. After being left in standard cell culture medium for another week, cells were sorted by FACS based on whether they expressed high levels of R-PTP-γ-mCitrine.

[0366] A polyclonal Cos7 cell line stably expressing LIFEA2 (Sabet et al. 2015, Nature Communications 6, 1-13) was generated by cloning LIFEA2 into a PiggyBac vector (System Bioscience, Palo Alto, California). Subsequently, MCF7 cells were transfected with this construct and PiggyBac transposase (System Bioscience) in a 1:1 ratio. Transfection was performed 24 hours after seeding using FuGENE® 6 (Roche Diagnostics) as the transfection reagent. Transfection was performed according to the manufacturer's protocol. One day later, transfected cells were selected with 3 μg / mL puromycin for 14 days.

[0367] To generate a polyclonal stable cell line of MCF7 expressing EGFR with an extracellular SNAP tag (SNAP-EGFR) (Ibach et al. 2015, Plos One 10, e0143162) and R-PTP-γ fused to mCitrine (R-PTP-γ-mCitrine), both constructs were first cloned into a PiggyBac vector (System Bioscience, Palo Alto, California). Subsequently, MCF7 cells were transfected with the constructs and PiggyBac transposase (System Bioscience) in a 1:1:2 ratio. Transfection was performed 24 hours after seeding using FuGENE® 6 (Roche Diagnostics) as the transfection reagent. Transfection was performed according to the manufacturer's protocol. After 1 day, transfected cells were selected with 2 μg / mL puromycin for 30 hours. After being left in standard cell culture medium for another week, cells were sorted by FACS based on whether they expressed high levels of R-PTP-γ-mCitrine. We found that cells express endogenous R-PTP-γ-mCitrine, and endogenous We observed that MCF7 cells that do not express sex EGFR cannot survive for more than a few days. Snap-EGFR labeling was performed by incubating cells with 0.5 μM Snap-Surface Alexa 647 (New England Biolabs GmbH, Frankfurt, Germany) for at least 60 minutes. Due to a certain degree of EGFR recycling, EGFR was labeled not only in intracellular compartments but also in the plasma membrane. Cells labeled with Snap-Surface Alexa 647 were microscopically confirmed to also express SNAP-EGFR in over 99% of these cells. [Cell culture, transfection, and preparation] HeLa (ATCC number CCL-185), Cos7 (ATCC number CRL-1651), and MCF7 (ATCC number HTB-22) cells were obtained from ATCC. Cells were authenticated by short tandem repeat (STR) analysis and did not contain DNA sequences from mice, rats, or hamsters (Leibniz Institute DSMZ). Cells were regularly tested for mycoplasma infection using the MycoAlert mycoplasma detection kit (Lonza, Basel, Switzerland). They were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 200 mM L-glutamine, and 1% non-essential amino acids, and cultured at 37°C, 95% air, and 5% CO2.

[0368] In the ultra-rapid freeze-stop experiment, cells were placed in 4 mm coverslides on 3.5 cm plastic petri dishes, with 1 × 10⁶ cells per well. 5 From 2 x 10 5 Seed at cell concentration, or 4 x 10 mm coverslides. 4 The cover slide was mounted in a biocompatible silicone (4-well microinsert; ibidi GmbH, Grafelfing, Germany) chamber filled with 20 μL of cell culture medium at a cell / mL concentration. The latter approach had the advantage of not leaving any medium on the bottom of the cover slide. Cells were seeded at least one day before the experiment. To facilitate mounting, the cover slide was placed on a custom 3D printed holder, removing excess liquid before the experiment and allowing the remaining aqueous solution to adhere directly to the diamond heat exchanger. Experiments were conducted at 37°C in glass-bottom cell culture dishes (MatTek Corporation, Ashland, Massachusetts, USA), with 1 × 10⁶ cells per dish. 5 From 4x10 5 The procedure was performed on cells.

[0369] Transient transfection of HeLa cells with EGFP (GFP-N1 vector, Clontech Laboratories Inc., Mountain View, California, USA) was performed 6 to 24 hours after cell seeding, using 1 μg of DNA per well and FuGENE® 6 (Roche Diagnostics) as the transfection reagent. Transfection was performed according to the manufacturer's protocol.

[0370] Chemical fixation for STED imaging was performed by incubating cells in 2% glutaraldehyde in 4% formaldehyde in phosphate-buffered saline at 37°C for 20 minutes. This fixes cells faster and with fewer artifacts than other aldehyde fixations (Huebinger et al. 2018, Sci Rep 8, 17756). [Fluorescence microscopy] Cryofluorescence microscopy and corresponding control experiments were performed using a commercially available confocal laser scanning STED microscope (Expert Line, Abberier Instruments, Göttingen, Germany) equipped with 488 nm, 561 nm, and 640 nm excitation lasers, STED lasers with wavelengths of 595 nm (nominal 1 W) and 775 nm (1.25 W), and FLIM hardware and software integration. This system was further equipped with an MT20 illumination system for wide-field imaging (Olympus Deutschland GmbH, Hamburg, Germany) and an Orca-R2 camera (Hamamatsu Photonics, Hamamatsu, Japan).

[0371] Wide-field fluorescence imaging of EGFP-expressing HeLa cells, rhodamine 6G solution, and a control consisting only of FLIM donors was performed using a 40x 0.6NA objective lens (LucPlanFLN; Olympus Deutschland GmbH, Hamburg, Germany). All other imaging was acquired using a 40x 0.95NA objective lens (UPlanApo, Olympus Deutschland GmbH, Hamburg, Germany).

[0372] Confocal imaging, including FLIM measurements, was performed with 485 nm laser excitation (mCitrine) or 640 nm laser excitation (Alexa647, measurement irradiation power at the sample surface for photobleaching: 0.2 mW) and a pixel length of 100 nm or 200 nm (FLIM). Wide-field fluorescence imaging was performed using filter sets for EGFP and mCitrine (excitation: 470 / 40 nm, dichroic mirror 495 nm, emission 520 / 35 nm, measurement power at the sample surface: 10 mW) or Alexa647 (excitation: 620 / 60 nm, dichroic mirror 640 nm, emission 537 / 700 / 75 nm) and a pixel length of 163 nm. Sequential wide-field fluorescence images for SRRF reconstruction were acquired at a frame rate of 1 frame / second. This relatively low frame rate allowed for successful reconstruction of super-resolution images from reduced fluorescence variability under cryogenic conditions.

[0373] STED imaging was performed using a 640 nm laser for excitation and a 775 nm laser for stimulated emission suppression. The sample was scanned with a pixel length of 40 nm and a residence time of 15 us. Each frame was aligned using a rigid registration algorithm with the open-source program Fiji (Schindelin et al. 2012, Nature Methods 9, 676-682) to correct for sample drift. To achieve a highly sensitive donut-shaped depression beam under cryogenic conditions, spherical aberration had to be corrected using a correction ring on the objective lens. [Image Analysis] General image processing, including fluorescence intensity quantification, line profile extraction, and Manders coefficient calculation, was performed using the open-source program Fiji (Schindelin et al. 2012 Nature Methods, 9:676-82). Bleaching quantification was performed by fitting fluorescence intensity with a simple exponential fit using Microsoft Excel (Microsoft Corporation, Redmond, Washington, USA). If fluorescence intensity could not be fitted with a simple exponential fit because fluorescence converges to 0, the first simple exponential portion of the data was fitted. Super-Resolution Radial Fluctuation (SRRF) analysis was performed using the NanoJ plugin for Fiji (Laine et al. 2019 Journal of Physics D:Applied Physics, 52:163001). The coefficient of determination was calculated using the Pearson function implemented in SciPy (version 1.5.2) (Virtanen et al. 2020, Nature Methods 17, 261-272). [Analysis of the FLIM dataset] To compensate for stage drift that may occur when applying a transformation that only applies rigid body movement, each photon count dataset was registered. After computing the photon count image stack, the X and Y shifts of each frame relative to the first frame were found using a discrete Fourier transform-based algorithm implemented in Python (imreg_dft 2.0) (Reddy et al. 1996, IEEE Transactions on Image Processing 5, 1266-1271). These values ​​were rounded to the nearest integer. The new, registered photon count dataset was assembled by shifting each photon within each frame by a predetermined number of pixels. Photons outside the image defined by Rheh were removed.

[0374] The registered photon count dataset was corrected directly from the data for background and instrument response function (IRF), as previously described (Grecco et al. 2009, Opt. Express 17, 6493-6508). Briefly, the background was estimated in two ways. First, a photon count image was generated by summing all frames in the image stack. After denoising using a 9-pixel median filter implemented in Python (SciPy 1.5.2), the position of the minimum value (P) was found. Next, the total background was calculated as the mean value around P pixels from the original image using a 21- to 51-pixel window (depending on the image). The background was also estimated from the photon count histogram. Again, after median denoising (21-bin window), the background per bin was estimated as the mean value of the original histogram around the minimum value of the denoised value (51-bin window). Next, this value was extrapolated to the entire image by multiplying it by the number of bins. The consistency of both background values ​​was cross-checked. IRF was estimated from the harmonics of the histogram.

[0375] The resulting corrected dataset was denoised using pawFLIM (Silberberg et al. 2017, Methods Appl Fluoresc 5, 024016). In short, this is a weighted translation-invariant Haar wavelet transform denoising algorithm for phasor images that significantly reduces bias and mean squared error.

[0376] The mean fluorescence lifetime of FRET donor mCitrine fused to TC-PTP or R-PTP-γ expressed in measured MCF7 cells increased from 3.04+ / -0.01ns at room temperature to 3.71+ / -0.02ns (mean + / - standard deviation) under freeze-stop (p<0.0001 using Student's t-test), but remained close to a simple exponential function. Measurements using LIFEA2 showed a clearly degraded non-exponential fluorescence decay profile under freeze-stop, indicating a mixture of active and inactive structures, with a clear mean FRET efficiency and a proportion of LIFEA2 containing non-absorbable mCherry acceptors. The resulting denoised cumulative phasor plots were analyzed by global analysis using a three-component model derived as an extension of the previously described two-component model (Verveer et al. 2000, Science 290, 1567-1570). These three spatially invariant states are (1) a fixed proportion of the donor alone (sensor with a non-absorbing acceptor; proportion under freeze-stop: 0.2) and fluorescence lifetime (3.71+ / -0.02 ns), (2) a closed (active) structure sensor with fluorescence lifetime (0.9+ / -0.2 ns) and corresponding FRET efficiency (0.75+ / -0.25), and (3) an extended (inactive) structure sensor with fluorescence lifetime (3.5+ / -0.2 ns) and corresponding FRET efficiency (0.05+ / -0.01), each represented as a phasor. Thus, each observed phasor is the sum of three phasors weighted by the corresponding normalized number of photons from each state. The complete phasor data for the image was then fitted with a linear combination of the three phasors and the corresponding photon proportions using a custom-made Python program based on Imfit 1.0.2. As previously described (Verveer et al. 2000, Biophys J 78, 2127-2137), the conversion from the photon ratio of each state to the molecular ratio was performed by dividing each by its corresponding fluorescence lifetime (a measure of quantum yield) and normalizing the sum to 1. The ratio of the active structure was normalized by dividing by (1 - ratio of donors only), taking into account the ratio of sensors including non-absorbing acceptors. • Fluorescence microscopy of cryopreserved HeLa cells expressing EGFP HeLa cells transfected with EGFP were prepared for cryomicroscopy as outlined above. Wide-field fluorescence imaging of these EGFP-expressing HeLa cells was performed. It was shown that after cryopreservation using the apparatus of the present invention, EGFP fluorescence and cell morphology were preserved, and no detectable ice crystals that could create non-fluorescent regions were observed (Figure 17). • Fluorescence microscopy of rhodamine 6G solution A rhodamine 6G solution was prepared in a 15 μm thick cell culture medium and cryopreserved and thawed using the apparatus described herein. Wide-field fluorescence imaging of the fluorescent solution showed that no detectable ice crystals were formed during or after cryopreservation. Upon thawing the solution, ice crystals formed (Figure 18). • Confocal imaging of MCF7 cells expressing TC-PTP-mCitrine To investigate whether complex intracellular tissues are preserved after ultrarapid freeze-stopping, extensively and dynamically maintained endoplasmic reticulum (ER) (Guo et al. 2018, Cell, 175:1430-42.e17) were imaged before and after freeze-stopping using multiple fluorescence microscopy techniques. For this purpose, MCF7 cells expressing TC-PTP fused to the fluorescent protein mCitrine (TC-PTP-mCitrine) (Stanoev et al. 2018, Cell Systems, 7:295-309.e11) were used. Confocal laser scanning microscopy (CLSM) before and after freeze-stopping demonstrated that the ER is highly preserved even after ultrarapid freeze-fixation.

[0377] Due to the high photostability of fluorescent dye molecules at extremely low temperatures, 100 wide-field fluorescence images of the same stopped cell could be acquired, enabling SRRF (super resolution radial fluctuation) reconstruction with improved resolution and contrast (Gustafsson et al. 2016, Nature Communications, 7:12471) (Figure 19), demonstrating that delicate ER cell organelles are preserved after cryopreservation. • Confocal imaging of MCF7 cells co-expressing Alexa647-SNAP-EGFR and R-PTP-γ-mCitrine To evaluate how ultrarapid freeze-stopping can improve the study of molecular tissue in dynamic cellular systems, the spatial distribution of oncoprotoproteins and functionally related candidate tumor suppressor proteins was imaged using multimodal fluorescence imaging. For this purpose, MCF7 cells co-expressing a fluorescence fusion construct of the receptor tyrosine kinase epidermal growth factor receptor (EGFR, Alexa647 labeled with Snap-EGFR: Alexa647-SNAP-EGFR) and the receptor-like protein tyrosine phosphatase γ (R-PTP-γ) (R-PTP-γ-mCitrine) fused to mCitrine (Stanoev et al. 2018, Cell Systems, 7:295-309.e11) were mounted on a cryomicroscope according to the present invention. Stimulation with epidermal growth factor (EGF, 100 ng / ml) allowed tracking of vesicular transport dynamics of both proteins at room temperature, and subsequent freeze-stopping enabled accurate capture of the spatial pattern of the interactions between the two proteins (Figure 20A).

[0378] SRRF reconstruction of 100 wide-field fluorescence images of the same arrest cell became possible (Figure 20A). From these reconstructions, the heterogeneous distribution of EGFR along the sides of the plasma membrane (Masip The cluster was identified as a patch of approximately 400 nm in size (Baumdick et al. 2016, Nature Methods, 13). Dual-color SRRF revealed high co-localization of R-PTP-γ and Alexa647-SNAP-EGFR in these patches within quiescent cells. Both proteins also showed co-localization in endosomal structures, but were lost after 15 minutes of EGF stimulation, indicating that both proteins had already separated at the plasma membrane (Figure 19). This cryogenic co-localization analysis revealed nanoscale organization of the EGFR / R-PTP-γ complex co-migrating within the endosomal system. These results are consistent with the recycling of the EGFR / R-PTP-γ complex, which maintains the silent signaling state of EGFR at the plasma membrane in the absence of stimulation (Baumdick et al. 2015, eLife, 4). Observation of EGFR / R-PTP-γ clusters upon EGF stimulation. The resulting separation indicates that the EGFR-EGF dimer complex can no longer interact with R-PTP-γ. The EGFR-EGF complex is then rerouted to late endosomes, generating a finite endosomal signaling response (Stanoev et al. 2018, Cell Systems, 7:295-309.e11). STED microscopy of MCF7 cells co-expressing Alexa647-SNAP-EGFR and R-PTP-γ-mCitrine MCF7 cells co-expressing Alexa647-SNAP-EGFR and R-PTP-γ-mCitrine were obtained and prepared for STED imaging as outlined above.

[0379] STED nanoscopy represents a special case among super-resolution techniques because fluorescence intensity is directly related to the density of fluorescent markers, and quantitative molecular patterns can be obtained. In this laser-scanning nanoscopy method, resolution beyond the diffraction limit is achieved by stimulated emission of excitation fluorescent dye molecules using a second high-intensity donut-shaped laser beam. As a result, high demands on photostability, fluorescence quantum yield, and fluorescence label density hinder STED imaging of sparsely distributed fluorescently tagged molecules in living cells.

[0380] In fact, low depletion laser power (<42mW / μm 2 Only ) preserved enough fluorescent markers to obtain noisy STED images of Alexa647-Snap-EGFR in MCF7 cells at room temperature (see Figures 21A and 21B), and there was no statistically significant increase in information beyond the diffraction limit compared to normal CLSM (Figures 21A and 21C). Resolution could be slightly improved by reducing motion blur through chemical fixation of cells, allowing for the accumulation of photons from five consecutive STED frames before bleaching the sample (Figure 21C). However, after ultrarapid freeze-stopping of living cells, the bleaching rate decreased to approximately 1 / 45th (Figure 21b), resulting in a depletion laser output (420 mW / μm). 2 ) and exposure time (radiation energy density: 35 mJ / μm 2 Both of these could be increased tenfold, and endosomal structures containing Alexa647-Snap-EGFR were clearly resolved (Figure 21D). The amount of information increased significantly from a scale of less than 125 nm to a digital (pixel-limited) resolution of 80 nm (Figure 21C). At this resolution, no ice crystals were detected as non-fluorescent regions within the cytoplasm of these cells (Figure 26). This was also true when the structure and arrangement of endocytotic structures were preserved after repeating STED scans at the maximum depletion laser intensity. This indicates that high-intensity STED illumination did not heat the sample enough to generate ice crystals.

[0381] In this experiment, it was confirmed that freeze-fixation significantly prevented motion blur and bleaching, enabling 3D scanning of the entire cell using confocal microscopy, and subsequently allowing recording of STED images of various regions at different z-levels (Figure 21D). The total acquisition time exceeded 15 minutes.

[0382] As a result, freeze-fixation significantly improves the signal, facilitating STED microscopy of multiple sub-areas within larger volumes. Confocal FRET-FLIM microscopy of Cos7 cells expressing LIFEA2 Cos7 cells expressing LIFEA2 were obtained and prepared for cryomicroscopy as outlined above. A FRET-FLIM study was performed to investigate the activity state distribution of tyrosine kinase EphA2 after freeze-fixation using the apparatus of the present invention.

[0383] At room temperature, receptor activation by ligand-clustered ephrin A1 was observed in the plasma membrane of Cos7 cells by a slight (approximately 0.2 ns) decrease in the mean fluorescence lifetime of FRET donor mCitrine in LIFEA2 (Figure 22). However, motion blur and insufficient separation of fluorescence decay profiles and derived phasers severely limited the spatial resolution and fluorescence lifetime resolution of intracellular membrane structures (Figure 22). However, at cryogenic temperatures, m Increased citrine quantum yield significantly improved the decay profile, mean fluorescence lifetime (τ), and separation of associated phasors of LIFEA2 in stimulated and unstimulated cells (Figure 22). Clustered receptor activity was degradable in plasma membrane patches and endocytosis structures (Figure 22), and basal activity in the absence of exogenous stimulation was likely induced by endogenous ephrin in adjacent cells. This improvement in contrast in molecular activity imaging was particularly evident when comparing the same ephrin A1-stimulated cells before and during freeze-stop (Figure 22).

[0384] Under freeze-stop conditions, the cumulative phasors are significantly separated (Figure 24C), allowing for the derivation of spatially invariant fluorescence lifetimes of active (τ=0.9±0.2ns) and inactive (τ=3.5±0.1ns) structures, as well as the proportion of LIFEA2 containing non-absorbable mCherry acceptors (approximately 0.2), through global analysis. Using these constraints, the spatially variable molecular proportion (α) of active LIFEA2 can be derived from the phasors within each voxel of the image. This enables the acquisition (time > 10 min) and reconstruction of a 3D molecular activity map of LIFEA2 in stimulated Cos7 cells (Figure 25), allowing for the differentiation of individual vesicles with activated receptors emerging from the plasma membrane from gradually inactivated receptors in the perinuclear endosomal compartment.

[0385] Therefore, under freeze-fixation conditions using the apparatus of the present invention, the spatial distribution of fluorescence lifetime can be measured with significantly improved resolution, enabling unprecedented mapping of the three-dimensional structure of receptors against numerous individual endosomes and inner membrane structures within cells (Figure 25).

Claims

1. A method for lowering the temperature of a sample (1) for inverted microscopy, (a) A step of providing the sample (1) to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) A step of supplying liquid nitrogen having a temperature below the critical temperature and helium having a pressure of at least 2.5 MPa into a pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, the liquid nitrogen is brought into contact with the upper side (11) of the carrier substrate (10), and the temperature of the sample (1) is lowered. A method that includes this.

2. The method according to claim 1, further, (d) A step of tempering the sample (1) on the lower side (12) of the carrier substrate (10) by passing it through the upper side (11) of the carrier substrate (10), A method that includes this.

3. The method according to claim 1 or 2, wherein the sample (1) is a living sample of a biological material.

4. The method according to claim 1 or 2, wherein the pressure of the helium is between 5 MPa and 100 MPa.

5. The method according to claim 1 or 2, wherein in step (c), the temperature of the sample (1) is lowered without forming ice crystals in the sample (1) or without forming ice crystals with a diameter greater than 10 nm in the sample (1).

6. A device (100) for lowering the temperature of a sample (1), (a) A carrier substrate (10) having a lower side (12) for housing a sample (1) and an upper side (11) exposed for supplying liquid (2), (b) A pressure tank (400) configured to hold fluid and having an outlet valve (22) at the bottom, (c) A supply line (20) is configured to connect the outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass the liquid (2) through under a pressure higher than atmospheric pressure, (d) comprising an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), In order to shorten the supply line (20) as much as possible, the device (100) is configured such that the pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

7. The apparatus (100) according to claim 6, wherein the supply line (20) is insulated from the inside.

8. The apparatus (100) according to claim 6, wherein the temperature of the sample (1) is lowered so that ice crystals do not form in the sample (1), or so that ice crystals with a diameter greater than 10 nm do not form in the sample (1).

9. The apparatus (100) according to claim 6, wherein the outlet valve (22) is a switchable valve, a switchable valve that opens quickly, a valve that can be switched electrically or pneumatically, or a switchable piston valve.

10. The apparatus according to claim 6, wherein the bottom of the pressure tank (400) is funnel-shaped, and the outlet valve (22) is located in the center of the bottom.

11. The apparatus (100) according to claim 6, wherein the carrier substrate (10) is a diamond.

12. The apparatus (100) according to claim 6, wherein the distance between the outlet opening (21) of the supply line (20) and the upper side (11) of the carrier substrate (10) is in the range of 0.5 mm to 1.5 mm.

13. The dimensions of the expansion chamber (30) are 50 cm ３ From 100cm ３ The apparatus (100) according to claim 6, which is within the range of [specified range].

14. The apparatus (100) according to claim 6, wherein the pressure tank (400) is surrounded by a cooling tank (300) for cooling the liquid (2), and / or the supply line (20) is funnel-shaped at its outlet opening (21).

15. A method of fluorescence microscopy, (a) A step of providing a sample (1) containing fluorescent molecules to the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) A step of supplying liquid nitrogen having a temperature below the critical temperature and helium having a pressure of at least 2.5 MPa into a pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, the liquid nitrogen is brought into contact with the upper side (11) of the carrier substrate (10), and the temperature of the sample (1) is lowered. (d) The step of irradiating the sample (1), (e) A step of detecting fluorescence generated by the sample (1), A method that includes this.

16. A method for performing optical microscopy by suppressing stimulated emission from a freeze-stopped sample (1), (a) To provide a sample (1) containing fluorescent molecules on the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen having a temperature below the critical temperature and helium having a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, the liquid nitrogen is brought into contact with the upper side (11) of the carrier substrate (10), and the temperature of the sample (1) is lowered. (d1) Irradiating the sample (1) with a first pulsed laser beam focused to at least one focal region to excite the fluorescent molecules in the sample (1) and cause them to emit fluorescence, (d2) Irradiating the sample (1) with a second torus-shaped laser beam that includes an intensity zero point within at least one focal region to de-excite the fluorescent molecules in the sample (1), (e) Detecting the fluorescence generated by the sample (1), (f) Optionally, repeating steps (d1), (d2), and (e) at various positions in the focal region of the first pulsed laser beam and at various positions in the intensity zero point of the second torus-shaped laser beam. A method that includes this.

17. A method for performing fluorescence lifetime imaging microscopy using time-correlated single-photon counting on a frozen-stopped sample (1), (a) To provide a sample (1) containing fluorescent molecules on the underside (12) of the carrier substrate (10) at atmospheric pressure, (b) Supplying liquid nitrogen having a temperature below the critical temperature and helium having a pressure of at least 2.5 MPa into the pressure tank (400), (c) By opening the outlet valve (22) located at the bottom of the pressure tank (400), liquid nitrogen is supplied from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressurized conditions, the liquid nitrogen is brought into contact with the upper side (11) of the carrier substrate (10), and the temperature of the sample (1) is lowered. (d) By irradiating the sample with a pulsed laser beam, the fluorescent molecules are periodically excited and emit fluorescent photons, (e) Detecting the fluorescent photons generated by the sample (1) along with the arrival time of the photons to the laser pulse (1), (f) Determining the fluorescence lifetime from the detected fluorescent photon and the arrival time of the photon, A method that includes this.

18. The method according to claim 3, wherein the living sample of the biological material is a living cell.

19. The apparatus (100) according to claim 11, wherein the diamond is a CVD diamond.

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