Method and apparatus for ultra-rapid freezing and fixing samples for microscopic research

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 a method for ultrarapid freezing and fixing a sample for time- and space-resolved microscopy, and an apparatus for ultrarapid freezing and fixing a sample at a specific time point of interest on a microscope. 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. [Background of the invention] Fluorescence microscopy is a powerful tool for studying the structure and dynamics of molecular systems.

[0002] Recent developments in fluorescence microscopy allow tracking well-separated molecules with nanoscale and millisecond localization precision (Balzarotti et al. 2017, Science, 355: 606-12). Super-resolution methods such as single molecule localization microscopy (SMLM) (Betzig et al. 2006, Science, 313: 1642-46), coordinate targeted nanoscopy (e.g. stimulated emission depletion (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 minimum photon flux (MINFLUX) nanoscopy (Balzarotti et al. 2017, Science, 355: 606-12) allow resolution beyond the diffraction limit of light even in crowded environments. Adding a spectroscopic dimension to the 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) or excited state lifetimes (Gadella, Jovin, and Clegg 1993, Biophysical Chemistry, 48:221-39; Bastiaens and Squire 1999, Trends in Cell Biology, 9:48-52), allows the extraction of information about molecular reaction states within spatially resolvable volume elements.

[0003] However, the spatial and spectroscopic resolution is 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 cycling of fluorophores between ground and excited states, determined by molecular cross-section, fluorescence quantum yield, and excited state lifetime, sets a limit on the fluorescence photon flux. This results in a minimum of achievable exposure times, leading to motion blur due to Brownian motion during photon acquisition. Motion blur is inversely proportional to the size of the observed structures and fundamentally limits localization precision and resolution (Figure 1). This fundamental limit on exposure times is further stretched by technical constraints of the optical system, such as laser scanning in coordinate targeted nanoscopy or switching of fluorophores in coordinate stochastic nanoscopy. Active motions, such as molecular motor-driven transport or cytoskeleton reorganization, further increase motion blur. Maximizing the saturable flux of fluorophores by increasing the illumination intensity increases the time that fluorophores spend in their chemically reactive excited states, leading to strong bleaching. This not only limits the amount of useful photons reaching the detector, reducing resolution (Figure 1), but also increases phototoxicity and perturbs reactions within the sample.

[0004] A specific example is fluorescence microscopy of biological systems such as living cells, where cellular functions appear on the micrometer scale and emerge from the non-equilibrium patterning dynamics of nanometer-sized molecules (Bastiaens et al. 2006, Trends Cell 2010). l 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 methods could in principle allow imaging of molecular patterns within cells and also measure the underlying reaction kinetics. However, the ultimate goal of linking collective molecular behavior 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 elusive due to fundamental problems: motion blur, as well as the photochemical reactivity of fluorescent probes.

[0005] To prevent molecular movement, such samples are usually chemically fixed. However, fixation by crosslinking or denaturation essentially alters the sample, making this method unsuitable for observing native non-equilibrium states, especially non-equilibrium molecular patterns in cells. The fixation process also takes at least a few minutes and is incomplete. Thus, it is not possible to fix a defined state (Huebinger et al. 2018, Sci Rep, 8:17756). Post-treatment to increase photostability is another source of artifacts.

[0006] Motion blur can be reduced by cooling the sample to a temperature where it becomes solid or reaches a very high viscosity, which also reduces the photochemical reactivity of the fluorophores. However, this needs to be done very quickly to avoid altering the sample through phase changes and loss of non-equilibrium states. For example, living cells need to be immobilized very quickly below their glass transition temperature of about -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 inhibited from reaching thermodynamic equilibrium at this low temperature and are trapped in liquid-like structures. Thus, even out-of-equilibrium polymer patterns of cells can be preserved by ultra-rapid cooling.

[0007] However, when applying liquids to the sample to cool it, a limiting factor is the limited wettability of the initially warm surface. The Leidenfrost phenomenon leads to the formation of an insulating vapor layer at the surface with a significantly reduced thermal conductivity compared to liquids, limiting the cooling rate at the surface. Furthermore, there are limitations to the technically possible flow rates and therefore the cooling capacity. However, when using gaseous cooling media, due to their low density, the heat capacity related to the volume is too low to achieve sufficient cooling capacity for practical investigations.

[0008] Ultrarapid cooling directly on the microscope is particularly desirable as it allows the combination of precise analysis in the absence of molecular motion and dynamic measurements at positive temperatures combined with the photochemical reactivity of the same samples that is greatly reduced under freeze-stop.

[0009] Methods for rapid cooling of samples known in the state of the art are for example high pressure freezing and propane jet freezing. In high pressure freezing, a pressure of 2000 bar is required to lower the melting point of the sample and to rapidly pass through the temperature range where ice crystals can form. Pressurizing the sample requires encapsulation. As for propane jet freezing, the sample needs to be jetted from two sides, above and below the sample, so the method and the device are not suitable to allow observation of the sample under a microscope, especially an inverted microscope, before, during and after the rapid cooling process. In an inverted microscope, the sample is fed above the microscope objective.

[0010] European patent EP 2877828 B1 discloses a method and a device for controlling the temperature of a sample. There, a supercritical medium is used to cool a sample provided on the front side of a carrier substrate. The supercritical medium is supplied to the rear side of the carrier substrate from below, i.e. against the direction of the Earth's gravity. Although it is possible to cool the sample by the aforementioned method, the rate of temperature change is too small for continuous observation of the sample without destroying the biological sample by ice crystals. Moreover, supplying the cooling medium against the Earth's gravity does not allow observation of the sample with 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 through an insulator. Despite the presence of a cryoprotectant, the cooling rate achieved was too low to prevent the formation of ice crystals (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 high water content, very poor glass formation, and relatively low thermal conductivity (<0.6 Wm -1 K -1 ), making arrest by freeze fixation particularly difficult.

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

[0014] EP3418710A1 relates to a method and apparatus for preparing a sample for inverted microscopy studies, where a sample is irradiated with light in a first area at time t1, and at a time t2 after time t1, a second area of ​​the sample is irradiated with light, and at a time t3 after time t2, the sample is fixed. The sample can be fixed by freezing (such as with liquid nitrogen) or by chemical fixation using a glutaraldehyde solution and a photocurable resin. WO2013 / 152239A1 relates to a freeze preparation system for almost instantaneous vitrification of biological samples. The system includes a capsule structure configured to hold the sample in a recess while the sample is ultra-rapidly frozen by exposure to a cryogenic cooling jet. Pulsed microwave energy is applied during the rapid cooling to avoid the formation of ice crystals within the sample. These methods do not disclose a method for generating a cooling jet that effectively avoids the formation of an insulating gas layer to cool the sample fast enough. 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 flow is generated by alternatingly opening and closing two valves. However, the pressure is limited to 1.379 MPa by a pump, limiting the cooling rate.

[0015] A common drawback of conventional methods is due to their specific design for freezing samples. Other tempering processes, such as rapid sample thawing, cannot usually be carried out with conventional methods. However, in the investigation of dynamic systems, it is important that after taking a still image and terminating the heat fixation, the processes in the system can continue as undisturbed as possible. In the case of biological samples, for example, it is important that the heating is carried out in such a way 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 to rapidly heat and / or The aim of the present invention is to provide an improved method and device for performing temperature adjustment and reducing the temperature of a sample. More precisely, the invention comprises a method for ultra-rapid freeze-fixing of a sample for spatially resolved microscopic measurements without the use of cryoprotectants, and a device for ultra-rapid freeze-fixing of a sample on a microscope. In particular, the invention aims to allow a temperature change of a sample with a higher rate of temperature adjustment, to realize a temperature increase or decrease, to set the final temperature of the temperature change and / or to monitor the sample, compared to the conventional techniques. The rapid temperature adjustment must be performed at any time, in particular during the observation of the sample. In particular, the invention aims to provide an improved technique for vitrifying biological samples and controlling their heating so that no ice crystals form or overheating occurs, which allows microscopic measurements of the sample before, during and after vitrification without altering the sample (reversible). In other words, the invention aims to provide a technique for fixing ("freeze-stop") short-lived states of a sample in a targeted and unaffected ("native") way, and to investigate them using fluorescence microscopy, whose recording speeds would otherwise be too slow to study these states. It is a further object of the present invention to provide an apparatus and method for cooling a sample on an inverted microscope sufficiently fast to allow continuous observation of the sample before and after cryofixation.

[0017] The object of the invention is solved by the teaching of the independent claims. Further advantageous features, aspects and details of the invention are evident from the dependent claims, the description, the drawings and the examples of the present application. [Brief Description of the Invention] This object has been solved by a method for reducing the temperature of a sample (1) for inverted microscopy, comprising the following steps:

[0018] (a) providing a sample (1) on an underside (12) of a carrier substrate (10) at atmospheric pressure; (b) providing at least one liquid (2) having a temperature below a critical temperature and a gas (500) having a pressure higher than atmospheric pressure in a pressure tank (400); (c) supplying at least one liquid (2) under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and bringing the at least one liquid (2) into contact with the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1), 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 reducing the temperature of a sample (1) for inverted microscopy, comprising the following steps:

[0020] (a) providing a sample (1) on an underside (12) of a 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 a pressure tank (400); (c) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and allowing the liquid nitrogen to contact the upper side (11) of the carrier substrate (10) and reduce, preferably rapidly reduce, the temperature of the sample (1).

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

[0022] (a) providing a sample (1) on an underside (12) of a carrier substrate (10) at atmospheric pressure; (b) Liquid nitrogen having a temperature below the critical temperature and a pressure of at least 2.5 MPa. supplying helium into a pressure tank (400); (c) applying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400), causing the liquid nitrogen to contact the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1).

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

[0024] (a) providing a sample (1) on an underside (12) of a 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 a pressure tank (400); (c) applying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400), causing the liquid nitrogen to contact the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1).

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

[0026] (a) providing a sample (1) on an underside (12) of a 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 a pressure tank (400); (c) passing liquid nitrogen under pressure from the pressure tank (400) to the top side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400), causing the liquid nitrogen to contact the top side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1).

[0027] Thus, rapid cooling of the sample (1) accommodated on the underside (12) of the carrier substrate (10) is achieved by supplying liquid nitrogen, having a temperature below the critical temperature, under pressure from a pressure tank (400) to the upper side (11) of the carrier substrate (10), which is pressurized by a gas (500) (helium) 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] Thus, in a preferred embodiment, a method for reducing the temperature of a sample (1) for inverted microscopy comprises the following steps:

[0030] (a) providing a sample (1) on an underside (12) of a 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 5 MPa into a pressure tank (400); (c) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and allowing the liquid nitrogen to contact the upper side (11) of the carrier substrate (10) and reduce, preferably rapidly reduce, the temperature of the sample (1).

[0031] In a further embodiment, 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 reducing the temperature of a sample (1) for inverted microscopy comprises the following steps:

[0033] (a) providing a sample (1) on an underside (12) of a 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) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and allowing the liquid nitrogen to contact the upper side (11) of the carrier substrate (10) and reduce, preferably rapidly reduce, the temperature of the sample (1).

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

[0035] The carrier substrate (10) separating the sample (1) from the liquid nitrogen acts here 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 liquid. Preferably, the carrier substrate (10) comprises or consists of a diamond disc.

[0036] In one embodiment, in step (c), the liquid nitrogen is completely released from the pressure tank (400) to allow maximum nitrogen to contact the topside of the carrier substrate, thereby maximizing cooling of the sample. Thus, a method for reducing the temperature of a sample (1) for inverted microscopy comprises the following steps:

[0037] (a) providing a sample (1) on an underside (12) of a 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 a pressure tank (400); (c) completely supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening the outlet valve (22) located at the bottom of the pressure tank (400), causing the liquid nitrogen to contact the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1).

[0038] According to the invention, a sudden or rapid or explosive temperature drop is achieved, which means that the sample temperature is reduced from an initial temperature (start temperature) to an end temperature (target temperature) at a rate (cooling rate, tempering rate) at which the molecular processes (such as ice crystal formation) in the sample are slower or negligible.

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

[0040] (a) providing a sample (1) on an underside (12) of a 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 a pressure tank (400); (c) completely supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening the outlet valve (22) located at the bottom of the pressure tank (400), causing the liquid nitrogen to contact the upper side (11) of the carrier substrate (10) and explosively reducing the temperature of the sample (1).

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

[0042] (a) providing a sample (1) on an underside (12) of a 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 a pressure tank (400); (c) supplying liquid nitrogen and gas (500) under pressure from the pressure tank (400) to the top side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400), causing the liquid nitrogen to contact the top side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1).

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

[0044] (a) providing a sample (1) on an underside (12) of a 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 a pressure tank (400); (c) supplying liquid nitrogen and gas (500) under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and allowing the liquid nitrogen to contact the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1); In step (c), liquid nitrogen is fed first and helium is fed second.

[0045] Liquid nitrogen as a cooling medium is added suddenly 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 at which the sample vitrifies depends heavily on the properties of the sample, primarily on the molecular components of the sample. For example, vitrification occurs in dilute aqueous buffers when the molecular components are cooled at rates of about 100,000 K / s to 1,000,000 K / s.

[0046] Thus, in one embodiment, a method for reducing the temperature of a sample (1) for inverted microscopy comprises the following steps.

[0047] (a) providing a sample (1) on an underside (12) of a 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 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) so that the liquid nitrogen comes into contact with the upper side (11) of the carrier substrate (10) and the temperature of the sample (1) is slightly reduced. The temperature must be reduced by at least -100,000K / sec.

[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 subjecting the sample to high pressures of about 200 MPa, however, the use of cryoprotectants can also affect the properties and activity of the sample.

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

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

[0051] In other embodiments, the sample inherently or additionally contains cryoprotective compounds such as salts, sugars (e.g., trehalose, dextran), organic solvents (e.g., dimethylsulfoxide, ethylene glycol), proteins, or antifreeze proteins in certain cells. In these embodiments, cooling rates for vitrification slower than those mentioned above may be applied.

[0052] Thus, in one embodiment, a method for reducing the temperature of a sample (1) for inverted microscopy comprises the following steps.

[0053] (a) providing a sample (1) on an underside (12) of a 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 a pressure tank (400); (c) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400), 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 a rate of at least -10,000 K / s.

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

[0055] (a) providing a sample (1) on an underside (12) of a 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 a pressure tank (400); (c) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and contacting the liquid nitrogen with the upper side (11) of the carrier substrate (10) to reduce the temperature of the sample (1) at a rate of at least -10,000 K / s; The sample includes a cryoprotectant compound.

[0056] In these embodiments, the temperature of the sample (1) is preferably reduced at a rate of 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 sample's properties such as thermal conductivity and thickness within the sample. A person skilled in the art can easily select an appropriate sample thickness to ensure complete vitrification of the sample using the method according to the invention. Thus, in one embodiment, a method for reducing the temperature of a sample (1) for inverted microscopy comprises the following steps:

[0058] (a) providing a sample (1) on an underside (12) of a 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 a pressure tank (400); (c) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and allowing the liquid nitrogen to contact the upper side (11) of the carrier substrate (10) and reduce, preferably rapidly reduce, the temperature of the sample (1); 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] Thus, in one embodiment, a method for reducing the temperature of a sample (1) for inverted microscopy comprises the following steps.

[0061] (a) providing a sample (1) on an underside (12) of a 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 a pressure tank (400); (c) supplying at least one liquid (2) under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and bringing the at least one liquid (2) into contact with the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1), The thickness of sample (1) is less than 20 μm.

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

[0063] (a) providing a sample (1) on an underside (12) of a 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 a pressure tank (400); (c) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400), causing the liquid nitrogen to contact the upper side (11) of the carrier substrate (10) and reducing the temperature of the sample (1), preferably rapidly reducing it from room temperature to at least -120°C, preferably -130°C.

[0064] In a preferred embodiment of the method of the present invention for reducing the temperature of a sample (1) for inverted microscopy, the sample (1) is vitrified (i.e., no ice crystals with a diameter of more than 10 nm are formed). Nanometer-sized ice crystals do not usually 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] Thus, a method for reducing the temperature of a sample (1) for inverted microscopy comprises the following steps.

[0066] (a) providing a sample (1) on an underside (12) of a 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 a pressure tank (400); (c) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400), causing the liquid nitrogen to contact the upper side (11) of the carrier substrate (10), reducing, preferably rapidly reducing, the temperature of the sample (1) and vitrifying the sample (1) without crystallizing, preferably without forming ice crystals having a diameter of more than 80 nm, preferably without forming ice crystals having a diameter of more than 10 nm, more preferably without forming ice crystals having a diameter of 1 nm or more, and even more preferably without forming ice crystals.

[0067] All of the methods for reducing the temperature of sample (1) disclosed herein are capable of reducing the temperature of sample (1) without forming ice crystals or without forming ice crystals greater than 10 nm in diameter.

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

[0069] Thus, the temperature of the sample (1) drops explosively, i.e. very rapidly. It is difficult to define the minimum cooling temperature to avoid the formation of ice crystals with a diameter of more than 10 nm, since the required cooling rate depends on the thickness and size of the sample (1), the material or mixture of materials of the sample (1), the presence of cryoprotective compounds (i.e. cryoprotectants) and other parameters disclosed herein. However, as an example, the method for lowering the temperature of the sample (1) disclosed herein is capable of lowering the temperature of a 100 μm thick aqueous sample from room temperature to a temperature between −120° C. and −130° C. in a time frame of less than 10 milliseconds using a 50 μm thermocouple at a cooling rate of −15,000 K / s or less (see FIG. 7A). Said cooling rate approaches the theoretical heat diffusion limit of the aqueous sample and thus corresponds to a cooling rate of up to −200,000 K / s for a 10 μm to 15 μm thick aqueous sample containing adherent mammalian cells.

[0070] Another aspect of the invention relates to an apparatus (100) for controlling the temperature of a sample (1), the apparatus (100) comprising: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) located at the bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to keep the supply lines (20) as short as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0071] The cooling liquid is forced under pressure from a pressure tank (400) onto the upper side (11) of the carrier substrate (10). By supplying the liquid naturally, the formation of an insulating gas layer on the carrier substrate (Leidenfrost phenomenon) is prevented. Using only liquid at low pressure does not prevent the formation of an insulating gas layer, which results in a slower cooling rate. This problem can be solved by additionally pressurizing the cooling liquid, especially with a gas such as helium (500), which does not liquefy under these conditions. If this liquid-gas mixture is rapidly released onto the carrier substrate, the flow of the coolant in the carrier substrate is very high, allowing rapid convection cooling and suppressing film boiling. Moreover, the device is upside down compared to the device according to EP 2 877 828 B1, so that the tank storing the cooling medium is located above the sample and the cooling medium is applied from above onto the carrier substrate.

[0072] In order to minimize the temperature rise of the cooling liquid (2) in the supply lines (20), the pressure tank (400) is placed on the upper side (11) of the carrier substrate so that the supply lines (20) can be as short as possible. By shortening the lines from the outlet valve (22) of the pressure tank (400) to the upper side (11) of the carrier substrate (10), the heat exchange between the cooling liquid (2) and the surroundings can be reduced. This can be achieved by mounting or connecting the pressure tank (400) with the outlet valve (22) at its bottom to the top (11) of the carrier substrate (10).

[0073] In a preferred embodiment of the device (100), the temperature rise of the cooling liquid (2) is further minimized by insulating the supply line (20). Thus, a preferred embodiment of the device (100) for controlling the temperature of the sample (1) comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) located at the bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to make the supply lines (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 internally insulated, preferably with high density polyethylene.

[0074] The device (100) according to the present invention allows for cryoprotectant-free fixation and observation of native, non-equilibrium molecular states, such as cytoplasmic molecular organization and reaction patterns, in living cells. When the device (100) is coupled to an inverted microscope setup, it is possible to continuously observe dynamic cellular processes at physiological temperatures, followed by rapid freezing and measuring the molecular patterns of the same cells in a cryopreserved state.

[0075] In other words, the device (100) according to the invention is a device for reducing, preferably rapidly reducing, the temperature of a sample (1), (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having 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 a lower end facing the upper side (11) of the carrier substrate (10), the supply line (20) being configured to pass liquid (2) under pressure higher than atmospheric pressure; (d) an expansion chamber (30) surrounding the outlet opening (21).

[0076] Surprisingly, the inventors have found that higher cooling rates can be achieved using the device according to the invention compared to the device according to EP 2877828 B1. We show that when a 100 μm aqueous sample was cooled from the top side with the thermocouple element placed on the underside of the carrier substrate, a cooling rate of approximately -15,000 K / s was measured (theoretically, the vitrification cooling rate of water is estimated to reach up to -200,000 K / s for aqueous samples of 10 μm to 15 μm thickness such as adherent cells). When the apparatus of EP2877828B1 was configured so that the sample was placed on the underside of the carrier substrate and cooled from the top side, the observed rate of temperature change was only -1,000 K / s (Figure 7B).

[0077] Moreover, no ice crystals, including microcrystals, could be observed in a solution of fluorescent dye Rhodamine 6G in a 15 μm-thick cell culture medium under an inverted optical microscope after cooling (see FIG. 18). No dehydration of the sample was observed.

[0078] More importantly, we were able to demonstrate by wide-field fluorescence imaging that the morphology of EGFP expressing adherent HeLa cells was preserved during cryofixation 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 showed that the morphology of large organelles such as the endoplasmic reticulum (ER) remained intact during freeze fixation. In this regard, the ER membrane was labeled by using MCF7 cells expressing T-cell protein tyrosine phosphatase (TC-PTP) fused to fluorescent mCitrine (see Figure 19). The high photostability of mCitrine at cryogenic temperatures also allowed us to subsequently acquire 100 wide-field fluorescence images of the same arrested cells to generate a super resolution radial fluctuation (SRRF) reconstruction of the ER, thereby showing that the spatial localization of the fluorescent protein is fully preserved after freeze fixation using the device according to the invention.

[0080] Advantageously, the present invention makes it possible to freeze ("freeze-arrest") short-lived states in a targeted and unaffected manner, and to investigate short-lived states with methods whose recording speeds are too slow to study these states otherwise. These include, for example, many optical microscopy techniques with improved spatial and spectral resolution, such as CLSM and stimulated emission depletion microscopy (STED).

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

[0082] The vesicle dynamics of both fluorescent fusion proteins at room temperature could be followed by CLSM upon stimulation with epidermal growth factor (EGF, 100 ng / ml), after which freeze-stop allowed us to precisely capture the spatial patterns of both proteins in relation to each other (Figure 20A). The absence of motion blur (Figure 20B) and the increased photostability of the fluorescent markers allowed colocalization analysis using SRRF reconstruction of 100 wide-field fluorescent images from each channel. Thus, this freeze-stop analysis revealed a previously unknown coorganization of EGFR and R-PTP-γ into nanoscale clusters along the plasma membrane and within endosomes, as well as their dissociation upon EGF stimulation. This growth factor-induced dissociation of EGFR nanoclusters from inhibitory phosphatases is consistent with a switch of the system from a dynamically silent to a transiently active signaling state.

[0083] STED nanoscopy constitutes a special case among super-resolution techniques, since the fluorescence intensity is directly related to the density of the fluorescent marker, allowing quantitative molecular patterns to be obtained. In laser scanning nanoscopy, a second high-intensity donut-shaped laser beam is used to stimulate the emission of excited fluorophores, achieving resolution beyond the diffraction limit. As a result, high requirements for photostability, fluorescence quantum yield, and density of fluorescent labels hinder STED imaging of sparse fluorescently tagged molecules in live cells.

[0084] In fact, low depletion laser power (<42mW / μm 2 ) preserved enough fluorescent marker to acquire noisy STED images of Alexa647-Snap-EGFR in MCF7 cells at room temperature (see Figures 21A and 21B), with no statistically significant increase in information content above the diffraction limit over regular CLSM (Figures 21A and 21C). Resolution could be improved slightly by reducing motion blur by chemical fixation of the cells, allowing the accumulation of photons from five consecutive STED frames before bleaching the sample (Figure 21C). However, after ultrarapid freeze arrest of live cells, the bleaching rate decreased by about 45-fold (Figure 21B), which was consistent with the depletion laser power (420 mW / μm 2 ) and exposure time (radiation energy density: 35 mJ / μm 2 ) by 10-fold, resulting in clear resolution of endosomal structures containing Alexa647-Snap-EGFR (Figure 21D). The amount of information increased dramatically from a scale of <125 nm up to a digital (pixel-limited) resolution of 80 nm (Figure 21C).

[0085] Moreover, due to the absence of motion blur and the significantly reduced photochemistry, it is possible to 3D scan the whole cell by CLSM and then record super-resolution STED images of Alexa647-Snap-EGFR in different regions at different z-levels (Figure 21D). This allowed the identification of vesicle clusters beyond the diffraction limit that cannot be resolved at room temperature. Here, the total acquisition time was over 15 min and the average irradiation energy density over the whole cell region was 20 mJ / μm 2 The lethal light dose of mammalian cell lines at positive temperatures exceeds approximately 1 mJ / μm2 Thus, we were able to demonstrate that cryofixation with the device according to the invention allows imaging of whole living cells in 3D with improved spatial and spectroscopic resolution, in the absence of molecular motion, and with significantly extended exposure times due to reduced motion blur and greatly improved photostability.

[0086] As a result, the inventive method described herein allows for freeze-fixation of dilute aqueous media and cells without ice crystals detectable at this resolution as non-fluorescent regions in the cytoplasm of cells, even though cellular structures imaged beyond the diffraction limit by STED or SRRF (Figure 26). Furthermore, after repeated STED scans at maximum depletion laser intensity, the structure and arrangement of endocytic structures was preserved, but no ice crystals were observed, demonstrating that high-intensity STED illumination does not warm biological samples enough to produce ice crystals. Furthermore, the distribution of molecular conformations of the LIFEA2 sensor measured by FRET-FLIM was preserved, indicating that the protein structure remains intact after freeze-stop. Thus, ultrafast freeze-fixation by the method described herein faithfully freezes the transient states 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 pulling it out of its natural state, moving it, or manipulating it with additives or, for example, excessive pressure buildup.

[0088] Furthermore, the inventors evaluated functional imaging of molecular reactions in cells after ultrarapid freeze-arrest. FLIM adds the spectroscopic dimension of nanosecond fluorescence decay kinetics to fluorescence microscopy. Hereby, excited-state reactions between fluorophores (e.g., FRET) triggered by molecular proximity can be imaged to spatially resolve macromolecular reactions such as interactions and conformational changes. The genetically encoded biosensor LIFEA2 is able to determine molecular conformation and its Here we report on the kinase activity of the cell guidance ephrin receptor type A (EphA2) via FRET between the fluorescent proteins mCitrine and mCherry. At room temperature, receptor activation by ligand-clustered ephrinA1 could be observed at the plasma membrane of Cos7 cells by a slight (~0.2 ns) decrease in the mean fluorescence lifetime of the FRET donor mCitrine in LIFEA2 (Figure 22). However, motion blur and poor separation of the fluorescence decay profile and derived phasors significantly limited the spatial and fluorescence lifetime resolution of membrane structures in cells (Figure 22). However, at cryogenic temperatures, the increased quantum yield of mCitrine significantly improved the separation of the decay profile, mean fluorescence lifetime (τ), and associated phasors of LIFEA2 in stimulated and unstimulated cells (Figure 22). Clustered receptor activity could be resolved in plasma membrane patches and endocytic structures (Figure S2), and basal activity in the absence of exogenous stimulation was likely driven by endogenous ephrins in adjacent cells. This contrast enhancement in molecular activity imaging was particularly evident when comparing the same ephrinA1-stimulated cells before and during freeze arrest (Figure S22).

[0089] Under freeze arrest, the large separation of the cumulative phasors (Figure 24C) allows the derivation of spatially invariant fluorescence lifetimes of active (τ = 0.9 ± 0.2 ns) and inactive (τ = 3.5 ± 0.1 ns) conformations by global analysis, as well as the fraction of LIFEA2 containing non-absorbable mCherry acceptors (~0.2). Using these constraints, the spatially varying molecular fraction of active LIFEA2 (α) can be derived from the phasors in each voxel of the image. Thereby, freeze arrest allowed the acquisition (times >10 min) and reconstruction of 3D molecular activity maps of LIFEA2 in stimulated Cos7 cells (Figure 25), allowing the distinction of individual vesicles with activated receptors emerging from the plasma membrane from gradually inactivated receptors in perinuclear endosomal compartments.

[0090] Thus, we were able to show that cryofixation with our device allows the spatial distribution of fluorescence decay kinetics to be measured with greatly improved resolution by fluorescence lifetime imaging microscopy (FLIM), thereby enabling unprecedented mapping of the reaction state of fluorescently labeled molecules, preferably genetically encoded one or two component fluorescent proteins containing indicators. An example is given of the genetically encoded conformational FRET sensor LIFEA2, which can be used to precisely measure the activity state of EphA2 receptors in numerous individual endosomes and endomembrane structures within cells (Figure 25).

[0091] Thus, a further aspect of the invention is directed to a method of fluorescence microscopy comprising the steps of:

[0092] (a) providing a sample (1) comprising fluorescent molecules on an underside (12) of a 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 a pressure tank (400); (c) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1); (d) irradiating the sample (1) with laser light; (e) detecting fluorescence produced by the sample (1).

[0093] Thus, a further aspect of the invention is directed to a method for performing optical microscopy by stimulated emission suppression of a freeze-stopped sample (1), comprising the steps of:

[0094] (a) providing a sample (1) comprising fluorescent molecules on an underside (12) of a 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 a pressure tank (400); (c) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1); (d1) irradiating a sample (1) with a first pulsed laser beam focused at at least one focal region to excite fluorescent molecules in the sample (1) to emit fluorescence; (d2) irradiating the sample (1) with a second torus-shaped laser beam having an intensity zero point in at least one focal region to de-excite fluorescent molecules in the sample (1); (e) detecting fluorescence produced by the sample (1); and (f) Optionally, repeating steps d1, d2, and e at different positions of the focal region of the first pulsed laser beam and different positions of the intensity zero point of the second torus-shaped laser beam.

[0095] Thus, a further aspect of the invention is directed to a method for performing time-correlated single-photon counting fluorescence lifetime imaging microscopy of a freeze-stopped sample (1), comprising the steps of:

[0096] (a) providing a sample (1) comprising fluorescent molecules on an underside (12) of a 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 a pressure tank (400); (c) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1); (d) periodically exciting the fluorescent molecules to emit fluorescent photons by irradiating the sample with a pulsed laser beam; (e) detecting fluorescent photons produced by the sample (1); and (f) Determining the fluorescence lifetime from the detected fluorescence photons.

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

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

[0099] The present application therefore relates to a method for reducing the temperature of a living sample (1), preferably a living cell (1) for inverted microscopy, comprising the following steps:

[0100] (a) providing a sample (1) on an underside (12) of a carrier substrate (10) at atmospheric pressure; (b) Liquid nitrogen having a temperature below the critical temperature and a pressure of at least 2.5 MPa. supplying helium into a pressure tank (400); (c) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and allowing the liquid nitrogen to contact the upper side (11) of the carrier substrate (10) and reduce, preferably rapidly reduce, the temperature of the sample (1).

[0101] The present application therefore relates to a device (100) for controlling the temperature of a living sample (1), preferably a living cell (1), (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) located at the bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10), and in order to keep the supply lines (20) as short as possible, a pressure tank (400) is attached to the upper side (11) of said carrier substrate (10). [Description of the Invention] The term "reducing the temperature" of a sample is typically used to describe a temperature change in response to a temperature difference between the tempering liquid and the carrier substrate containing the sample, including a decrease in temperature (cooling).

[0102] The tempering liquid is therefore called a cooling medium or cryogen. If the carrier substrate containing the sample is placed at a temperature above the freezing point of water, the use of a cooling medium causes the sample to change into a solid state. The transition to the solid state is also referred to here as freezing of the sample.

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

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

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

[0106] The term "freeze fixation" as used herein refers to the (almost) instantaneous (rapid or explosive) freezing of a sample, whereby molecular motion within the sample stops and the water molecules contained in the sample are unable to form hydrogen-bonded networks of ice crystals. The resulting amorphous ice is called "glass ice" and remains in a disordered liquid water-like arrangement without disturbing the native structure of the sample. Thus, freeze fixation avoids ice crystal growth and / or only leads to the formation of nano-dispersed ice (vitrification), without affecting the structure of the sample. Nano-dispersed ice comprises ice particles smaller than 100 nm, in particular smaller than 10 nm, e.g. smaller than 5 nm.

[0107] For living cells, cryofixation can in principle preserve them so well that many cells will remain viable if thawed at a suitable rate at a later time, and the process can therefore be considered reversible.

[0108] "Freeze arrest" is used synonymously herein with "cryofixation."

[0109] A tempering liquid is usually a substance that contains a chemical element or compound or composition that can be provided in a liquid state in the temperature range of interest to cool the sample. Preferably, the liquid contains nitrogen. Further examples of liquids as temperature control media are hydrogen, argon, helium or methane.

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

[0111] As used herein, the "top side" (11) of the carrier substrate (10) refers to the side of the carrier substrate (10) that faces the outlet opening (21) of the supply line (20) and faces upward, i.e., away from the direction of the Earth's gravity (10).

[0112] As used herein, "underside" (12) refers to the side of the carrier substrate (10) opposite the exit openings (21) of the supply lines (20) and faces downward, i.e., towards the direction of the Earth's gravity.

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

[0114] As used herein, "room temperature" refers to temperatures above 4° C., preferably from 15 to 40° C., from 15 to 30° C., and from 15 to 24° C., and from 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 degrees Celsius" refers to temperatures above the melting point of water, i.e., 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 objective lens of the microscope is below the stage and points upwards, so that the sample can be observed from below. The sample can be placed at the bottom of a container, which has the best optical characteristics, or attached to the underside of a carrier substrate. Inverted microscopy thus 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 depletion microscopy (STED), fluorescence lifetime imaging microscopy with time-correlated single-photon counting, and electron microscopy.

[0117] As used herein, "critical temperature" refers to the temperature below which pressure can cause a gas to liquefy, and above which this is no longer possible. In embodiments involving more than one liquid (2), the critical temperature refers to the lowest critical temperature of the individual liquids.

[0118] According to the invention, the supply line (20) is configured to conduct the tempering liquid to the carrier substrate at a pressure higher than atmospheric pressure, and the underside (12) of the carrier substrate is configured to receive, accommodate or hold the sample at atmospheric pressure.

[0119] It has been 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 is no longer limited by the Leidenfrost phenomenon. Thus, one embodiment of the present invention is directed to a method for reducing the temperature of a sample, where the pressure of the gas (500) is between 50 bar and 1000 bar. Thus, an embodiment according to the invention relates to a method for reducing the temperature of a sample (1), preferably a living sample (1), more preferably a living cell (1), for inverted microscopy, comprising the following steps:

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

[0121] Thus, one embodiment of the present invention is directed to a method for reducing the temperature of a sample, wherein the helium pressure is between 5 MPa and 100 MPa. Thus, an embodiment according to the invention relates to a method for reducing 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) providing a sample (1) on an underside (12) of a 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) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening the outlet valve (22) located at the bottom of the pressure tank (400) and bringing the liquid nitrogen (2) into contact with the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1).

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

[0124] In step b) of the inventive method described herein, preferably, at least one liquid (2) is first charged 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 inventive method described herein, the at least one liquid (2) can be provided in the pressure tank (400) by filling it with a liquid cooled to a temperature below its critical temperature, i.e. by filling the pressure tank with a pre-cooled liquid. Alternatively, the at least one liquid (2) can be filled in the pressure tank (400) as a gas and then cooled to a temperature below its critical temperature such that the gas condenses to 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, the at least one liquid (2) is liquid nitrogen. In some embodiments, the at least one liquid (2) is a further cooling liquid, such as liquid argon or liquid methane. Accordingly, an embodiment according to the present invention is directed to a method for reducing the temperature of a sample (1), preferably a living sample (1), more preferably a living cell (1), for inverted microscopy, comprising the steps of:

[0127] (a) providing a sample (1) on an underside (12) of a carrier substrate (10) at atmospheric pressure; (b) providing at least one liquid (2) having a temperature below a critical temperature and helium having a pressure of at least 2.5 MPa in a pressure tank (400); (c) supplying at least one liquid (2) under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and bringing liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1), The at least one liquid (2) comprises liquid nitrogen.

[0128] In another embodiment, a method for reducing the temperature of a sample (1), preferably a living sample (1), more preferably a living cell (1), for inverted microscopy comprises the following steps.

[0129] (a) providing a sample (1) on an underside (12) of a carrier substrate (10) at atmospheric pressure; (b) providing at least one liquid (2) having a temperature below a critical temperature and helium having a pressure of at least 2.5 MPa in a pressure tank (400); (c) supplying at least one liquid (2) under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and bringing liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1), The 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 reducing the temperature of a sample (1), preferably a living sample (1), more preferably a living cell (1), for inverted microscopy comprises the following steps.

[0131] (a) providing a sample (1) on an underside (12) of a carrier substrate (10) at atmospheric pressure; (b) providing at least one liquid (2) having a temperature below a critical temperature and helium having a pressure between 5 MPa and 100 MPa in a pressure tank (400); (c) supplying at least one liquid (2) under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and bringing liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1), The at least one liquid (2) comprises a mixture of (i) liquid nitrogen and liquid argon, or (ii) liquid nitrogen and liquid methane, or (iii) liquid nitrogen, liquid argon and liquid methane.

[0132] In a preferred embodiment, a method for reducing the temperature of a sample (1) for inverted microscopy comprises the following steps.

[0133] (a) providing a sample (1) on an underside (12) of a 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 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), causing the liquid nitrogen to come into contact with the upper side (11) of the carrier substrate (10) and lowering the temperature of the sample (1). and preferably rapidly and without the formation of ice crystals in the sample (1) or without the formation of ice crystals having a diameter of more than 80 nm or without the formation of ice crystals having a diameter of more than 10 nm in the sample (1).

[0134] In a preferred embodiment, a method for reducing the temperature of a sample (1) for inverted microscopy comprises the following steps.

[0135] (a) providing a sample (1) on an underside (12) of a 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) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400), causing the liquid nitrogen to contact the upper side (11) of the carrier substrate (10) and reducing the temperature of the sample (1), preferably rapidly and without the formation of ice crystals within the sample (1) or without the formation of ice crystals greater than 80 nm in diameter or greater than 10 nm in diameter within the sample (1).

[0136] The temperature of the at least one liquid (2) used in the method of the invention is at least 5° C., preferably at least 10° C. below the critical temperature of the liquid. Thus, an embodiment according to the invention relates to a method for reducing the temperature of a sample, wherein the temperature of the at least one liquid (2) is at least 10° C. below its critical temperature.

[0137] Accordingly, an embodiment according to the present invention is directed to a method for reducing the temperature of a sample (1), preferably a living sample (1), more preferably a living cell (1), for inverted microscopy, comprising the following steps:

[0138] (a) providing a sample (1) on an underside (12) of a carrier substrate (10) at atmospheric pressure; (b) supplying into a pressure tank (400) at least one liquid (2) having a temperature at least 10° C. lower than the critical temperature and a gas (500) having a pressure of at least 2.5 MPa; (c) supplying at least one liquid (2) under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and bringing the at least one liquid (2) into contact with the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1), At least one of the liquids (2) is liquid nitrogen and the gas (500) is helium.

[0139] In a further embodiment, a method for reducing the temperature of a sample (1), preferably a living sample (1), more preferably a living cell (1), for inverted microscopy comprises the following steps.

[0140] (a) providing a sample (1) on an underside (12) of a carrier substrate (10) at atmospheric pressure; (b) providing 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) supplying at least one liquid (2) under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and bringing the at least one liquid (2) into contact with the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1).

[0141] In principle, any gas (500), such as helium or hydrogen, can be used in the inventive method described herein. This gas is a compressible gas and can be present in a pressure tank. It does not liquefy under the conditions and does not react with or dissolve in liquids. However, the desired cooling rates were achieved using helium as the gas (500).

[0142] In one embodiment, a mixture of gases (500) is used in the method of the invention. A mixture of gases may be advantageous for adjusting the pressure in the pressure tank (500). Thus, one embodiment of the present invention is directed to a method for reducing the temperature of a sample (1), preferably a living sample (1), more preferably a living cell (1), for inverted microscopy, comprising the following steps:

[0143] (a) providing a sample (1) on an underside (12) of a carrier substrate (10) at atmospheric pressure; (b) supplying into a pressure tank (400) a mixture of liquid nitrogen having a temperature below the critical temperature and a gas (500) having a pressure of at least 2.5 MPa; (c) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and allowing the liquid nitrogen to contact the upper side (11) of the carrier substrate (10) and reduce, preferably rapidly reduce, the temperature of the sample (1); The mixture of gases (500) includes helium.

[0144] A further embodiment of the present invention is directed to a method for reducing the temperature of a sample (1), preferably a living sample (1), more preferably a living cell (1), for inverted microscopy, comprising the following steps:

[0145] (a) providing a sample (1) on an underside (12) of a carrier substrate (10) at atmospheric pressure; (b) supplying into a pressure tank (400) a mixture of liquid nitrogen having a temperature below the critical temperature and a gas (500) having a pressure of at least 2.5 MPa; (c) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and allowing the liquid nitrogen to contact the upper side (11) of the carrier substrate (10) and reduce, preferably rapidly reduce, the temperature of the sample (1); The mixture of gases (500) includes helium and hydrogen.

[0146] One embodiment of the present invention is also directed to a method for reducing the temperature of a sample (1), preferably a living sample (1), more preferably a living cell (1), for inverted microscopy, comprising the steps of:

[0147] (a) providing a sample (1) on an underside (12) of a carrier substrate (10) at atmospheric pressure; (b) supplying into a pressure tank (400) a mixture of liquid nitrogen having a temperature below the critical temperature and a gas (500) having a pressure between 2.5 MPa and 1000 MPa; (c) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and allowing the liquid nitrogen to contact the upper side (11) of the carrier substrate (10) and reduce, preferably rapidly reduce, the temperature of the sample (1); The mixture of gases (500) includes helium.

[0148] One embodiment of the present invention is also directed to a method for reducing the temperature of a sample (1), preferably a living sample (1), more preferably a living cell (1), for inverted microscopy, comprising the steps of:

[0149] (a) providing a sample (1) on an underside (12) of a carrier substrate (10) at atmospheric pressure; (b) supplying into a pressure tank (400) a mixture of liquid nitrogen having a temperature below the critical temperature and a gas (500) having a pressure of at least 2.5 MPa; (c) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400), causing the liquid nitrogen to contact the upper side (11) of the carrier substrate (10) and reducing the temperature of the sample (1), preferably rapidly and without the formation of ice crystals within the sample (1) or without the formation of ice crystals greater than 80 nm in diameter or greater than 10 nm in diameter within the sample (1).

[0150] As used herein, "sample" refers to any specimen that is cryofixed or cryostopped and analyzed using the methods and devices of the present invention. In general, each type of sample can be cooled. Samples usually contain a variable liquid composition of matter, such as soft matter, materials, chemical reaction partners, or, particularly preferred, biological materials.

[0151] The sample may comprise (polymeric) 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 an unaffected original state, modified, excited, non-equilibrium or equilibrium state. The sample may be a biological sample such as (mammalian) cells, tissues, body fluids (blood, serum, plasma, etc.). Preferably, the sample comprises living biological material, more preferably living cells, living biological 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. If the sample consists of a very thin layer between the carrier substrate and the observation window, adhesion is not absolutely necessary.

[0152] According to the invention, a sample is prepared at normal (atmospheric) pressure and tempered (frozen or thawed). Thus, an embodiment according to the invention relates to a method for reducing the temperature of a sample, wherein the sample (1) comprises biological cells, preferably live biological cells. Thus, an embodiment according to the invention is directed to a method for reducing the temperature of a sample (1), preferably a live sample (1), more preferably a live cell (1), for inverted microscopy, comprising the steps of:

[0153] (a) providing biological cells (1), preferably live biological cells (1), on the underside (12) of a carrier substrate (10) at atmospheric pressure; (b) providing in a pressure tank (400) at least one liquid (2) having a temperature below a critical temperature and a gas (500) having a pressure of at least 2.5 MPa; (c) supplying at least one liquid (2) under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and bringing the at least one liquid (2) into contact with the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the biological cells (1), preferably live biological cells (1), At least one of the liquids (2) is nitrogen and the gas (500) is helium.

[0154] A further embodiment according to the present invention is directed to a method for reducing the temperature of a sample (1), preferably a living sample (1), more preferably a living cell (1), for inverted microscopy, comprising the following steps:

[0155] (a) providing biological cells (1), preferably live biological cells (1), on the underside (12) of a carrier substrate (10) at atmospheric pressure; (b) providing liquid nitrogen having a temperature below a critical temperature and helium having a pressure between 2.5 MPa and 1000 MPa in a pressure tank (400); (c) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and allowing the liquid nitrogen to contact the upper side (11) of the carrier substrate (10) and absorbing the biological cells (1), preferably or reducing, preferably rapidly reducing, the temperature of a living biological cell (1).

[0156] A further embodiment according to the present invention is directed to a method for reducing the temperature of a sample (1), preferably a living sample (1), more preferably a living cell (1), for inverted microscopy, comprising the following steps:

[0157] (a) providing biological cells (1), preferably live biological cells (1), on the underside (12) of a carrier substrate (10) at atmospheric pressure; (b) supplying liquid nitrogen having a temperature below the critical temperature and helium having a pressure between 2.5 MPa and 1000 MPa into a pressure tank (400); (c) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400), causing the liquid nitrogen to contact the upper side (11) of the carrier substrate (10), and reducing the temperature of the biological cells (1), preferably live biological cells (1), preferably rapidly, without the formation of ice crystals within the biological cells (1) or without the formation of ice crystals having a diameter of more than 80 nm or more than 10 nm within the biological cells (1).

[0158] According to the invention, a liquid (2) is brought into contact with a carrier substrate (10). To achieve a rapid temperature change, the liquid is released under pressure from a pressure tank (400). A flow of liquid towards the upper side of the carrier substrate is formed. The flow can be achieved with a defined start time and a predefined flow rate. The flow rate is selected depending on the liquid properties (e.g. viscosity) and the desired temperature.

[0159] Advantageously, there are various ways of directing the flow of the tempering liquid against the upper side of the carrier substrate. The flow can be directed by the design of the supply line (20) and its outlet opening (21). According to a first variant, a flow direction parallel to the surface normal of the upper side of the carrier substrate is provided. The tempering liquid flows vertically to the upper side and then laterally away from the carrier substrate. In this case, it may have the advantage that a particularly efficient heat transfer is obtained. Alternatively, in a second variant, the flow direction is perpendicular to the surface normal of the upper side of the carrier substrate. The tempering liquid flows parallel over the upper side so as to pass through the carrier substrate. In this case, it has the advantage that the 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, so that the liquid flows in the direction of gravity, thereby increasing the flow velocity to some extent.

[0161] According to the present invention, a rapid temperature drop is achieved. The term "rapid temperature drop" (or abrupt, immediate, instantaneous, rapid or burst temperature drop) is used to describe a rate at which the temperature of a sample is reduced from an initial (start) temperature to an end (target) temperature (cooling rate, tempering rate) in comparison to a slower or negligible rate of molecular processes within the sample (such as ice crystal formation or denaturation of biomolecules or biological systems).

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

[0163] For the investigation of samples, especially biological samples, it may be desirable to keep the sample at a constant low temperature after the rapid (or explosive) cooling of step (c) in order to avoid the formation of ice crystals. In this respect, it is possible to permanently immerse the upper side (11) of the carrier substrate (10) in at least one liquid (2), e.g. liquid nitrogen. Thus, an embodiment according to the invention relates to a method for reducing the temperature of a sample, comprising the additional step (d).

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

[0165] Accordingly, one embodiment of the present invention is directed to a method for reducing the temperature of a sample (1), preferably a living sample (1), more preferably a living cell (1), for inverted microscopy, comprising the following steps:

[0166] (a) providing a sample (1) on an underside (12) of a carrier substrate (10) at atmospheric pressure; (b) supplying at least one liquid (2) having a temperature below a critical temperature and a gas (500) having a pressure of at least 2.5 MPa into a pressure tank (400); (c) supplying at least one liquid (2) under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and bringing the at least one liquid (2) into contact with the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1); (d) tempering the sample (1) on the underside (12) of the carrier substrate (10) by passing at least one liquid over the upper side (11) of the carrier substrate (10), At least one of the liquids (2) is liquid nitrogen and the gas (500) is helium.

[0167] A further embodiment of the present invention is directed to a method for reducing the temperature of a sample (1), preferably a living sample (1), more preferably a living cell (1), for inverted microscopy, comprising the following steps:

[0168] (a) providing a sample (1) on an underside (12) of a 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) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1); (d) Tempering the sample (1) on the underside (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 is directed to a method for reducing the temperature of a sample (1), preferably a living sample (1), more preferably a living cell (1), for inverted microscopy, comprising the following steps:

[0170] (a) providing a sample (1) on an underside (12) of a 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 a pressure tank (400); (c) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and contacting the liquid nitrogen with the upper side (11) of the carrier substrate (10) and reducing the temperature of the sample (1), preferably rapidly and without forming ice crystals in the sample (1) or without forming ice crystals in the sample (1) having a diameter of more than 10 nm or without forming ice crystals in the sample (1) having a diameter of more than 80 nm; (d) Liquid nitrogen is passed over the top side (11) of the carrier substrate (10) to remove the Tempering the sample (1) on the underside (12) of the carrier substrate (10).

[0171] A preferred embodiment of the present invention is directed to a method for reducing the temperature of a sample (1), preferably a living sample (1), more preferably a living cell (1), for inverted microscopy, comprising the following steps:

[0172] (a) providing a sample (1) on an underside (12) of a 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 a pressure tank (400); (c) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and contacting the liquid nitrogen with the upper side (11) of the carrier substrate (10) and reducing the temperature of the sample (1), preferably rapidly and without forming ice crystals in the sample (1) or without forming ice crystals in the sample (1) having a diameter of more than 10 nm or without forming ice crystals in the sample (1) having a diameter of more than 80 nm; (d) tempering the sample (1) without forming ice crystals within the sample (1) on the underside (12) of the carrier substrate (10) by passing liquid nitrogen over the upper side (11) of the carrier substrate (10).

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

[0174] Accordingly, one embodiment of the present invention is directed to a method for reducing the temperature of a sample (1), preferably a living sample (1), more preferably a living cell (1), for inverted microscopy, comprising the following steps:

[0175] (a) providing a sample (1) on an underside (12) of a carrier substrate (10) at atmospheric pressure; (b) supplying into a pressure tank (400) a first liquid (2) having a temperature below a critical temperature and a gas (500) having a pressure of at least 2.5 MPa; (c) supplying a first liquid (2) under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) so that at least one liquid (2) contacts the upper side (11) of the carrier substrate (10) and reduces, preferably rapidly reduces, the temperature of the sample (1); (d) tempering the sample (1) on the underside (12) of the carrier substrate (10) by passing a second liquid over the upper side (11) of the carrier substrate (10), The first liquid (2) is liquid nitrogen and the gas (500) is helium.

[0176] In other words, one embodiment of the present invention is directed to a method for reducing the temperature of a sample (1), preferably a living sample (1), more preferably a living cell (1), for inverted microscopy, comprising the following steps:

[0177] (a) providing a sample (1) on an underside (12) of a 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 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), so that the liquid nitrogen contacts the upper side (11) of the carrier substrate (10) and the temperature of the sample (1) is reduced. and decreasing, preferably rapidly, (d) tempering the sample (1) on the underside (12) of the carrier substrate (10) by passing a further liquid over the upper side (11) of the carrier substrate (10).

[0178] In other words, one embodiment of the present invention is directed to a method for reducing the temperature of a sample (1), preferably a living sample (1), more preferably a living cell (1), for inverted microscopy, comprising the following steps:

[0179] (a) providing a sample (1) on an underside (12) of a 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) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1); (d) tempering the sample (1) on the underside (12) of the carrier substrate (10) by passing a further liquid over the upper side (11) of the carrier substrate (10).

[0180] In other words, one embodiment of the present invention is directed to a method for reducing the temperature of a sample (1), preferably a living sample (1), more preferably a living cell (1), for inverted microscopy, comprising the following steps:

[0181] (a) providing a sample (1) on an underside (12) of a 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 a pressure tank (400); (c) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and contacting the liquid nitrogen with the upper side (11) of the carrier substrate (10) and reducing the temperature of the sample (1), preferably rapidly and without forming ice crystals in the sample (1) or without forming ice crystals in the sample (1) having a diameter of more than 10 nm or without forming ice crystals in the sample (1) having a diameter of more than 80 nm; (d) tempering the sample (1) on the underside (12) of the carrier substrate (10) by passing a further liquid over the upper side (11) of the carrier substrate (10).

[0182] Accordingly, one embodiment of the present invention is directed to a method for reducing the temperature of a sample (1), preferably a living sample (1), more preferably a living cell (1), for inverted microscopy, comprising the following steps:

[0183] (a) providing a sample (1) on an underside (12) of a carrier substrate (10) at atmospheric pressure; (b) supplying liquid nitrogen having a temperature T1 and helium having a pressure of at least 2.5 MPa into a pressure tank (400); (c) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1); (d) tempering the sample (1) on the underside (12) of the carrier substrate (10) by passing a further liquid at temperature T2 over the upper side (11) of the carrier substrate (10), The temperature T1 is below the critical temperature of liquid nitrogen and optionally the temperature T2 is below the critical temperature of a further liquid.

[0184] In a preferred embodiment, to avoid rapid temperature changes of the sample, the difference between T1 and T2 is not more than 10 K. Preferably, T1 and T2 are identical or nearly identical, i.e., the difference between T1 and T2 is not more than 5 K.

[0185] Accordingly, one embodiment of the present invention is directed to a method for reducing the temperature of a sample (1), preferably a living sample (1), more preferably a living cell (1), for inverted microscopy, comprising the following steps:

[0186] (a) providing a sample (1) on an underside (12) of a 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 a pressure tank (400); (c) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1); (d) Tempering the sample (1) on the underside (12) of the carrier substrate (10) by passing liquid nitrogen over the upper side (11) of the carrier substrate (10).

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

[0188] In order to maintain sufficient cooling of the sample over an extended period of time, in step (d) of the method of the invention, a tempering liquid is passed over the top side of 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, most preferably between 0.5 MPa and 1 MPa. Thus, one embodiment of the present invention is directed to a method for reducing 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) providing a sample (1) on an underside (12) of a 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 a pressure tank (400); (c) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1); (d) Tempering the sample (1) on the underside (12) of the carrier substrate (10) by passing liquid nitrogen over the upper side (11) of the carrier substrate (10) at a pressure greater than atmospheric pressure.

[0190] One embodiment of the present invention is also directed to a method for reducing the temperature of a sample (1), preferably a living sample (1), more preferably a living cell (1), for inverted microscopy, comprising the steps of:

[0191] (a) providing a sample (1) on an underside (12) of a 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 delivered under pressure from the pressure tank (400) to the top side (11) of the carrier substrate (10) by opening the outlet valve (22) located at the bottom of the pressure tank (400). and contacting the liquid nitrogen with the upper side (11) of the carrier substrate (10) to reduce, preferably rapidly reduce, the temperature of the sample (1); (d) Tempering the sample (1) on the underside (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, a further embodiment of the present invention is directed to a method for reducing the temperature of a sample (1), preferably a living sample (1), more preferably a living cell (1), for inverted microscopy, comprising the following steps:

[0193] (a) providing a sample (1) on an underside (12) of a 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 a pressure tank (400); (c) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and contacting the liquid nitrogen with the upper side (11) of the carrier substrate (10) and reducing the temperature of the sample (1), preferably rapidly and without forming ice crystals in the sample (1) or without forming ice crystals in the sample (1) having a diameter of more than 10 nm or without forming ice crystals in the sample (1) having a diameter of more than 80 nm; d) Tempering the sample (1) on the underside (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.

[0194] One embodiment of the present invention is also directed to a method for reducing the temperature of a sample (1), preferably a living sample (1), more preferably a living cell (1), for inverted microscopy, comprising the steps of:

[0195] (a) providing a sample (1) on an underside (12) of a 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 a pressure tank (400); (c) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1); (d) Tempering the sample (1) on the underside (12) of the carrier substrate (10) by passing liquid nitrogen over the upper side (11) of the carrier substrate (10) at a pressure greater than atmospheric pressure. [Device] The present invention is directed to an apparatus (100) for reducing the temperature of a sample (1) in a manner (i.e. very rapidly or explosively) such that ice crystals are not formed in the sample (1), or that ice crystals having a diameter of more than 10 nm are not formed in the sample (1), or that ice crystals having a diameter of more than 80 nm are not formed in the sample (1), or that biological molecules or biological systems, such as cells, are not denatured, the apparatus (100) comprising: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); , In order to keep the supply lines (20) as short 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 a device (100) for controlling the temperature of a sample (1), (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to keep the supply lines (20) as short as possible, a pressure tank (400) is connected to the upper side (11) of the carrier substrate (10).

[0197] The present invention also relates to a device (100) for controlling the temperature of a sample (1), (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to keep the supply lines (20) as short as possible, the pressure tank (400) is mounted directly on the upper side (11) of the carrier substrate (10).

[0198] The present invention also relates to a device (100) for controlling the temperature of a sample (1), (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to keep the supply lines (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 invention also relates to a device (100) for controlling the temperature of a sample (1), the device (100) comprising: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); , In order to keep the supply lines (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 a device (100) for controlling the temperature of a sample (1), (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to keep the supply lines (20) as short as possible, the bottom of the pressure tank (400) is attached directly to the upper side (11) of the carrier substrate (10). [Supply Line] The supply line (20) is connected at its upper end to an outlet valve (22) of the pressure tank (400). At its lower end, the supply line is provided with an outlet opening (21) for releasing at least one liquid (2), as shown in Figure 2. The supply line (20) is arranged between the pressure tank (400) and the upper side (11) of the carrier substrate, thereby allowing the supply of at least one liquid (2) (and optionally a gas (500)) to the upper side (11) of the carrier substrate.

[0201] The supply line (20) is preferably as short as possible in order to reduce undesired temperature changes of the at least one liquid (2), which may result in a slower cooling rate of the sample. On the other hand, the supply line must be long enough so that the outlet opening (21) is optimally positioned for the supply of liquid to the upper side of the carrier substrate, i.e. the discharged liquid (2) must come into direct contact with the upper side of the carrier substrate and not with other components of the device, as shown in Fig. 3.

[0202] To minimize the temperature rise of the cooling liquid (2) in 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 polymeric 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 at low temperature and high pressure. Therefore, the supply line is most preferably insulated from the inside with high density polyethylene.

[0203] The supply line (20) can have any shape, such as cylindrical or funnel-shaped. Funnel-shaped in this specification means that the diameter of the top of the supply line is larger than the diameter of the bottom of the supply line, specifically the diameter of the top of the supply line is larger than the diameter of the outlet opening (21) of the supply line. The supply line (20) is preferably funnel-shaped at its lower end (outlet opening (21)) as shown in Figures 3C and 3D.

[0204] Accordingly, an embodiment according to the invention is directed to an apparatus (100) for reducing the temperature of a sample for inverted microscopy, in which the supply line (20) is funnel-shaped.

[0205] Accordingly, an embodiment of the present invention provides an apparatus (100) for reducing the temperature of a specimen. In regard to (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a funnel-shaped supply line (20) connecting an outlet valve (22) to the upper side (11) of the carrier substrate (10) and configured to pass the liquid (2) under pressure higher than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to keep the supply lines (20) as short as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0206] A further embodiment according to the invention relates to an apparatus (100) for reducing the temperature of a sample, comprising: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a funnel-shaped supply line (20) connecting an outlet valve (22) to the upper side (11) of the carrier substrate (10) and configured to pass the liquid (2) under pressure higher than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to keep the supply lines (20) as short as possible, the pressure tank (400) is mounted directly on the upper side (11) of the carrier substrate (10).

[0207] A further embodiment according to the invention relates to an apparatus (100) for reducing the temperature of a sample, comprising: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a funnel-shaped supply line (20) connecting an outlet valve (22) to the upper side (11) of the carrier substrate (10) and configured to pass the liquid (2) under pressure higher than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to keep the supply lines (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10) and the supply lines (20) are insulated from the inside, preferably with high density polyethylene.

[0208] Preferably, the device (100) of the present invention for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a funnel-shaped supply line (20) at its lower end, the funnel-shaped supply line (20) being configured to connect an outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass a liquid (2) at a pressure higher than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to keep the supply lines (20) as short as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0209] Preferably, the device (100) of the present invention for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a funnel-shaped supply line (20) at its lower end, the funnel-shaped supply line (20) being configured to connect an outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass a liquid (2) at a pressure higher than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to keep the supply lines (20) as short as possible, the pressure tank (400) is mounted directly on the upper side (11) of the carrier substrate (10).

[0210] Preferably, the device (100) of the present invention for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a funnel-shaped supply line (20) at its lower end, the funnel-shaped supply line (20) being configured to connect an outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass a liquid (2) at a pressure higher than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to keep the supply lines (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10) and the supply lines (20) are insulated from the inside, preferably with high density polyethylene.

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

[0212] Also, the supply line (20) is preferably straight (as shown in FIG. 3) and does not contain corners, edges, or turns in order to supply the at least one liquid (2) at a higher flow rate. [Pressure tank (400)] The device (100) of the present invention comprises at least one pressure tank (400). The pressure tank (400) of the device (100) is preferably a pressure vessel configured to contain the tempering liquid (2) and the gas (500) and / or an insulated storage vessel configured to contain the tempering liquid (2) and the gas (500). Since the term "fluid" includes liquids and gases, the pressure tank (400) of the device (100) is also preferably a pressure vessel configured to contain a fluid and / or an insulated storage vessel configured to contain a fluid. More preferably, the pressure tank (400) is an insulated pressure vessel configured to contain the tempering liquid (2) and the 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] Thus, in one embodiment, the device (100) of the present invention for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having a volume of 1 L to 5 L and equipped with an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to keep the supply lines (20) as short as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0215] Preferably, the device (100) of the present invention for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having a volume of 1 L to 5 L and equipped with an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to keep the supply lines (20) as short as possible, the pressure tank (400) is mounted directly on the upper side (11) of the carrier substrate (10).

[0216] Preferably, the device (100) of the present invention for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having a volume of 1 L to 5 L and equipped with an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to keep the supply lines (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10) and the supply lines (20) are 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 FIG. 4A. Thus, in one embodiment, the apparatus (100) of the present invention for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid, the pressure tank having a rounded bottom and an outlet valve (22) at the bottom; (c) an outlet valve (22) connected to the upper side (11) of the carrier substrate (10) and adapted to provide a pressure above atmospheric pressure; a supply line (20) configured to pass a liquid (2) under high pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to keep the supply lines (20) as short as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0218] Preferably, the device (100) of the present invention for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid, the pressure tank having a rounded bottom and an outlet valve (22) at the bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to keep the supply lines (20) as short as possible, the pressure tank (400) is mounted directly on the upper side (11) of the carrier substrate (10).

[0219] Preferably, the device (100) of the present invention for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid, the pressure tank having a rounded bottom and an outlet valve (22) at the bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to keep the supply lines (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10) and the supply lines (20) are 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 outlet valve (22) and the connection with the supply line (20). Thus, in one embodiment, the device (100) of the present invention for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid, the pressure tank having a funnel-shaped bottom and an outlet valve (22) at the bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to keep the supply lines (20) as short as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0221] Preferably, the device (100) of the present invention for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid, the pressure tank having a funnel-shaped bottom and an outlet valve (22) at the bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to keep the supply lines (20) as short as possible, the pressure tank (400) is mounted directly on the upper side (11) of the carrier substrate (10).

[0222] Preferably, the device (100) of the present invention for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid, the pressure tank having a funnel-shaped bottom and an outlet valve (22) at the bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to keep the supply lines (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10) and the supply lines (20) are insulated from the inside, preferably with high density polyethylene.

[0223] In a preferred embodiment, the device (100) of the present invention for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid, the pressure tank having a funnel-shaped bottom and an outlet valve (22) at the bottom; (c) a funnel-shaped supply line (20) configured to connect an outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass liquid (2) at a pressure higher than atmospheric pressure, the supply line (20) having a lower end thereof; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to keep the supply lines (20) as short 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 the at least one liquid (2) in the pressure tank (400). The cooling tank (300) of the apparatus (100) of the present invention is preferably a tank made of stainless steel, but may be made of other materials. The cooling tank (300) preferably has an opening at the top for convenient supply of a cooling medium. The cooling tank (300) thus surrounds the pressure tank (400) for cooling the liquid.

[0224] Thus, in one embodiment, the device (100) of the present invention for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (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); In order to keep the supply lines (20) as short as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0225] In a preferred embodiment, the device (100) of the present invention for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (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); In order to keep the supply lines (20) as short as possible, the pressure tank (400) is mounted directly on the upper side (11) of the carrier substrate (10).

[0226] In a further embodiment, the device (100) of the present invention for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (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); In order to keep the supply lines (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10) and the supply lines are insulated from the inside, preferably with high density polyethylene.

[0227] In a preferred embodiment, the device (100) of the present invention for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); (e) A cooling tank (300) surrounding the pressure tank (400) for cooling the liquid (2).

[0228] In a preferred embodiment, the device (100) of the present invention for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); (e) a cooling tank (300) surrounding the pressure tank (400) for cooling the liquid (2); In order to make the supply line (20) as short as possible, the pressure tank (400) is mounted on a carrier base. It is attached directly to the upper side (11) of the plate (10).

[0229] In a further embodiment, the device (100) of the present invention for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); (e) a cooling tank (300) surrounding the pressure tank (400) for cooling the liquid (2); In order to keep the supply lines (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10) and the supply lines are 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 a cooling tank (300). At this point, the temperature of the cooling medium is below the critical temperature of the liquid (2). The cooling medium may be the same substance as the liquid (2), for example liquid nitrogen. Thus, in one embodiment, the device (100) of the present invention for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater 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 contain a cooling medium for cooling the liquid (2); In order to keep the supply lines (20) as short as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0231] In a preferred embodiment, the device (100) of the present invention for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (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); The cooling tank (300) and the pressure tank (400) are not insulated and in order to keep the supply lines (20) as short as possible, the pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0232] In a preferred embodiment, the device (100) of the present invention for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a funnel-shaped bottom configured to hold a fluid and having an outlet valve (22) at its bottom; a pressure tank (400) having (c) a funnel-shaped supply line (20) configured to connect an outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass liquid (2) at a pressure higher than atmospheric pressure, the supply line (20) having a lower end thereof; (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); In order to keep the supply lines (20) as short as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0233] In a preferred embodiment, the device (100) of the present invention for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid, the pressure tank having a funnel-shaped bottom and an outlet valve (22) at the bottom; (c) a funnel-shaped supply line (20) configured to connect an outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass liquid (2) at a pressure higher than atmospheric pressure, the supply line (20) having a lower end thereof; (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); In order to keep the supply lines (20) as short as possible, the pressure tank (400) is mounted directly on the upper side (11) of the carrier substrate (10).

[0234] In a preferred embodiment, the device (100) of the present invention for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid, the pressure tank having a funnel-shaped bottom and an outlet valve (22) at the bottom; (c) a funnel-shaped supply line (20) configured to connect an outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass liquid (2) at a pressure higher than atmospheric pressure, the supply line (20) having a lower end thereof; (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); In order to keep the supply lines (20) as short as possible, the pressure tank (400) is mounted directly on the upper side (11) of the carrier substrate (10) and the supply lines are insulated from the inside, preferably with high density polyethylene. [Outlet valve] According to the invention, the supply of the tempering liquid to the upper side of the carrier substrate is activated (started) by an outlet valve, for example an electric or pneumatic valve, in the supply line (20) of the tempering liquid. A control element for opening and closing the outlet valve is arranged separately from the heat exchanger (carrier substrate). By activating the final control element, a defined temperature control can be started. When the outlet valve is opened, the tempering liquid hits the carrier substrate (10) with a certain defined delay. This allows fixing a selected state of the sample that has been prespecified during observation with an inverted microscope. Therefore, the device (100) is equipped with an outlet valve (22), preferably a switchable outlet valve, more preferably a fast-opening switchable outlet valve. The switchable outlet valve (22) can be a pneumatically or electrically switchable valve and / or a fluidic rotary valve.

[0235] Accordingly, embodiments of the present invention are directed to an apparatus (100) for reducing the temperature of a sample, in which the pressure tank (400) is provided with a switchable outlet valve, more preferably a fast-opening switchable outlet valve. ... The present invention relates to an apparatus (100) for (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to keep the supply lines (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 fast-opening switchable outlet valve.

[0236] Preferably, the device (100) for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to keep the supply lines (20) as short as possible, the pressure tank (400) is mounted directly on the upper side (11) of the carrier substrate (10) and the outlet valve is a switchable outlet valve, more preferably a fast-opening switchable outlet valve.

[0237] Preferably, the device (100) for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to make the supply lines (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 preferably a fast opening switchable outlet valve; The supply lines are internally insulated, preferably with high density polyethylene.

[0238] In another embodiment according to the invention, an apparatus (100) for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater 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 mounted on a carrier base. Attached to the upper side (11) of the plate (10), The outlet valve may be a pneumatically or electrically switchable valve and / or a fluid rotary valve.

[0239] Any valve is suitable for the inventive device (100) and method 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) can be an electrically or pneumatically switchable valve. Preferably, the outlet valve (22) is a pneumatically switchable valve. The switchable outlet valve (22) can be composed of a lift cylinder (200), a piston (501) and a piston tip (510) as shown in Figure 5. The piston tip is preferably composed of high density polyethylene (HDPE).

[0240] Furthermore, the switchable outlet valve (22) may further comprise a piston guide (520). Preferably, the switchable outlet valve (22) is a pneumatically switchable valve consisting of a lift cylinder (200), a piston (501) and a piston tip (510). Furthermore, the pneumatically switchable valve may further comprise 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 lift cylinder (200), thereby acting as an outlet valve for the pressure tank (400). The pressure tank (400) can be filled with liquids and gases through tubes threaded into the threaded holes. Cooling of the pressure tank (400) by the surrounding cooling tank (300) allows the gases to cool and condense.

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

[0243] A preferred embodiment of the present invention is therefore directed to an apparatus (100) for reducing the temperature of a sample, in which a pressure tank (400) is provided with a pneumatically switchable valve with a lift cylinder (200), a piston (501) and a piston tip (510). A preferred embodiment of the present invention therefore relates to an apparatus (100) for reducing the temperature of a sample, comprising: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to make the supply lines (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 including a lift cylinder (200), a piston (501) and a piston tip (510).

[0244] Preferably, the device (100) for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (4) configured to hold a fluid and having an outlet valve (22) at its bottom; 00) and (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to keep the supply lines (20) as short as possible, the pressure tank (400) is mounted directly on the upper side (11) of the carrier substrate (10); The outlet valve (22) is a pneumatically switchable valve including a lift cylinder (200), a piston (501) and a piston tip (510).

[0245] Preferably, the device (100) for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to make the supply lines (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 with a lift cylinder (200), a piston (501) and a piston tip (510), and the supply line is internally insulated, preferably with high density polyethylene.

[0246] Another preferred embodiment according to the present invention is directed to an apparatus (100) for reducing the temperature of a sample, wherein the pressure tank (400) comprises a pneumatically switchable valve with a lift cylinder (200), a piston (501), a piston tip (510) and a piston guide (520). Thus, a preferred embodiment according to the present invention relates to an apparatus (100) for reducing the temperature of a sample, comprising: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to make the supply lines (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 including a lift cylinder (200), a piston (501), a piston tip (510), and a piston guide (520).

[0247] In a preferred embodiment, the device (100) of the present invention for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid, the pressure tank having a funnel-shaped bottom and an outlet valve (22) at the bottom; (c) an outlet valve (22) connected to the upper side (11) of the carrier substrate (10) and configured to pass liquid (2) under pressure higher than atmospheric pressure, the outlet valve (22) being a funnel-shaped supply at its lower end; Line (20), (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); In order to make the supply lines (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 including a lift cylinder (200), a piston (501), a piston tip (510), and a piston guide (520).

[0248] Preferably, the device (100) for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid, the pressure tank having a funnel-shaped bottom and an outlet valve (22) at the bottom; (c) a funnel-shaped supply line (20) configured to connect an outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass liquid (2) at a pressure higher than atmospheric pressure, the supply line (20) having a lower end thereof; (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); In order to keep the supply lines (20) as short as possible, the pressure tank (400) is mounted directly on the upper side (11) of the carrier substrate (10); The outlet valve is a pneumatically switchable valve that includes a lift cylinder (200), a piston (501), a piston tip (510) and a piston guide (520).

[0249] Preferably, the device (100) for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid, the pressure tank having a funnel-shaped bottom and an outlet valve (22) at the bottom; (c) a funnel-shaped supply line (20) configured to connect an outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass liquid (2) at a pressure higher than atmospheric pressure, the supply line (20) having a lower end thereof; (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); In order to make the supply lines (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 including a lift cylinder (200), a piston (501), a piston tip (510), and a piston guide (520); The supply line (20) is internally insulated, preferably with high density polyethylene. [Exit opening] Further advantages of the invention arise, especially in designs with expansion of the tempering medium, if the lower end of the supply line (20), which forms the outlet opening (21), has at least one of the following features: According to a first variant, the end can be arranged 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 can also be designed such that the diameter of the outlet opening is variable. The mentioned distances and / or diameters can be selected depending on the concrete application of the invention for optimal heat transfer and / or a given temperature setting of the tempering medium. According to another particularly preferred variant, the outlet opening (21) has the shape of a nozzle. 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 according to the invention, the lower end is arranged 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 can also be designed such that the diameter of the outlet opening is variable. The mentioned distances and / or diameters can be selected depending on the concrete application of the invention for optimal heat transfer and / or a given temperature setting of the tempering medium. According to another particularly preferred variant, the outlet opening (21) has the shape of a nozzle. The nozzle forms the outlet opening (21) of the supply line (20) and is shaped such that the outlet opening (21) has a smaller diameter (funnel-shaped) than the supply line (20). In another embodiment according to the invention, the lower end is arranged such that the distance of the outlet opening (21) from the upper side (11) of the carrier substrate is variable. The nozzle (24) is arranged so that it can be separated from the line (20). For example, an exchangeable nozzle (24) can be provided. If the end, and in particular the nozzle, is coupled to the supply line via a threaded connection, both exchangeability (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) has the form of a nozzle. Accordingly, an embodiment according to the invention is directed to a device (100) for reducing the temperature of a sample, in which the outlet opening (21) has the form of a nozzle. Accordingly, an embodiment according to the invention is directed to a device (100) for reducing the temperature of a sample, in which (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to make the supply lines (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 device (100) for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to keep the supply lines (20) as short as possible, the pressure tank (400) is mounted directly on the upper side (11) of the carrier substrate (10); The outlet opening (21) has the shape of a nozzle.

[0252] Preferably, the device (100) for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater 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), the supply line (20) is insulated from the inside, preferably with high density polyethylene, and the outlet opening (21) has the shape of a nozzle.

[0253] In a preferred embodiment, the device (100) of the present invention for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid, the pressure tank having a funnel-shaped bottom and an outlet valve (22) at the bottom; (c) a funnel-shaped supply line (20) configured to connect an outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass liquid (2) at a pressure higher than atmospheric pressure, the supply line (20) having a lower end thereof; (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); In order to make the supply lines (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 including a lift 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 has a diameter 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, most preferably between 1.5 mm and 5 mm. Preferably, the nozzle has a diameter of 3 mm. Thus, an embodiment according to the invention relates to an apparatus (100) for reducing the temperature of a sample, comprising: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to make the supply lines (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 with a diameter between 1.5 mm and 5 mm. [Exit opening and carrier board] The temperature change rate can be further improved by adjusting the distance between the exit 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 the distance is 1 mm.

[0255] Accordingly, an embodiment according to the invention is directed to an apparatus (100) for reducing the temperature of a sample, in which 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. Accordingly, an embodiment according to the invention relates to an apparatus (100) for reducing the temperature of a sample, (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to make the supply lines (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 outlet opening (21) and the upper side (11) of the carrier substrate (10) is 0.5 mm. to 1.5 mm.

[0256] It has also been found that the ratio of the diameter of the outlet opening (21) to the diameter of the carrier substrate (10) can have a positive effect on the rate of temperature change. Therefore, an embodiment according to the invention relates to an apparatus (100) for reducing the temperature of a sample, (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to make the supply lines (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 outlet opening (21) is smaller than the diameter of the carrier substrate (10).

[0257] In a preferred embodiment, the device (100) of the present invention for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid, the pressure tank having a funnel-shaped bottom and an outlet valve (22) at the bottom; (c) a funnel-shaped supply line (20) configured to connect an outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass liquid (2) at a pressure higher than atmospheric pressure, the supply line (20) having a lower end thereof; (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); In order to make the supply lines (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 lift cylinder (200), a piston (501), a piston tip (510) and a piston guide (520), and the outlet opening (21) has the shape of a nozzle, and the diameter of the outlet opening (21) is smaller than the diameter of the carrier substrate (10). [Carrier board] The carrier substrate (10) (or cooling substrate) forms the sample holder and the heat exchanger. The carrier substrate (10) is usually a plate-like flat or curved component, one surface of which (herein called the underside) forms the receptacle for the sample and the other surface (herein called the upper side) forms the surface of the heat exchanger. In the case of self-adhesive samples, such as adherent cell cultures, the exposed surface of the underside, which may carry a modification layer, provides the sample holder. In other cases, for example in the case of non-adhesive samples, a receiving structure may be provided on the underside, for example to form a receptacle for receiving a liquid reaction partner sample. The upper side is preferably an exposed flat surface for wetting with the cooling medium. However, the upper side can also have surface structures, such as protrusions or recesses, with which the tempering fluid can come into contact.

[0258] The tempering rate (cooling or heating rate) is preferably greater than 5,000 K / s, in particular greater than 20,000 K / s, for example more than 50,000 K / s or even 100,000 K / s or more. 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 comprises a material whose thermal conductivity value is selected such that the temperature of the sample changes with a tempering rate (temperature gradient) exceeding the mentioned limits. The thermal conductivity of the carrier substrate (10) It is particularly preferred that the conductivity is greater than 1,000 W / Km, especially 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, more preferably less than 500 μm. Rapid heat transfer combined with adequate stability required for the high pressures applied to the carrier substrate can be achieved when the thickness is in the range of 0.1 mm to 10 mm, more preferably in the range of 0.1 mm to 2 mm, and most preferably in the range of 0.5 mm to 1.5 mm.

[0260] Accordingly, an embodiment according to the invention is directed to an apparatus (100) for reducing the temperature of a sample, in which the carrier substrate (10) has a thickness in the range of 0.1 mm to 10 mm. Accordingly, an embodiment according to the invention relates to an apparatus (100) for reducing the temperature of a sample, (a) a carrier substrate (10) having a thickness in the range of 0.1 mm to 10 mm, the carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to keep the supply lines (20) as short 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 that has the desired thermal conductivity and sufficient strength under the pressure of the tempering fluid. The carrier substrate (10) therefore acts 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, it is important to ensure that the carrier substrate (10) has a sufficient thickness, although the thickness of the carrier substrate required to obtain sufficient strength may have a negative effect on the thermal conductivity value and the achievable rate of temperature change (tempering rate). However, this is not decisive if the requirements for the tempering rate are low and / or if the samples are placed even under high pressure with respect to atmospheric pressure.

[0262] The carrier substrate comprises a diamond disk, for example a synthetic diamond disk having a thickness of less than 2 mm, in particular less than 500 μm. The use of diamond offers particular advantages with regard to optimum values ​​of thermal conductivity (product dependent, e.g. 1,500 W / Km) and strength at low substrate thicknesses.

[0263] Thus, an embodiment according to the invention is directed to an apparatus (100) for reducing the temperature of a sample, in which the carrier substrate (10) is diamond, in particular CVD diamond. Preferably, the CVD diamond has impurities of less than 6 ppb. The carrier substrate may comprise a diamond disk, for example a synthetic diamond disk with a thickness of less than 2 mm, preferably less than 1 mm, more preferably less than 500 μm. The use of diamond has particular advantages in terms of optimum values ​​of thermal conductivity (product dependent, e.g. 1,500 W / Km) and strength at low substrate thicknesses. Particularly preferred is single crystal CVD diamond, which exhibits a thermal conductivity of more than 2200 W / Km.

[0264] Therefore, an embodiment according to the invention provides a temperature control device (10) for reducing the temperature of a sample, the carrier substrate (10) being made of CVD diamond with impurities of less than 6 ppb. 00). The use of such CVD diamond increases thermal conductivity and the rate of temperature change.

[0265] Accordingly, an embodiment according to the invention relates to an apparatus (100) for reducing the temperature of a specimen, comprising: (a) a carrier substrate (10) made of CVD diamond with less than 6 ppb impurities, having a lower side (12) for receiving a sample (1) and an exposed upper side (11) for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to keep the supply lines (20) as short as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0266] Preferably, the device (100) for reducing the temperature of a sample comprises: (a) a carrier substrate (10) made of CVD diamond with less than 6 ppb impurities, having a lower side (12) for receiving a sample (1) and an exposed upper side (11) for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to keep the supply lines (20) as short as possible, the pressure tank (400) is mounted directly on the upper side (11) of the carrier substrate (10).

[0267] Preferably, the device (100) for reducing the temperature of a sample comprises: (a) a carrier substrate (10) made of CVD diamond with less than 6 ppb impurities, having a lower side (12) for receiving a sample (1) and an exposed upper side (11) for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to make the supply lines (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 internally insulated, preferably with high density polyethylene.

[0268] Another embodiment of the present invention relates to an apparatus (100) for reducing the temperature of a specimen, the apparatus (100) comprising: (a) a carrier substrate (10) made of single crystal CVD diamond having a lower side (12) for accommodating a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (4) configured to hold a fluid and having an outlet valve (22) at its bottom; 00) and (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to keep the supply lines (20) as short as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0269] A further advantage of the invention is provided by the temperature reduction from the top side of the carrier substrate (10), which allows the sample to be exposed on the underside. For example, it is possible to carry out optical and / or electrical measurements of the sample before, during and / or after the supply of the tempering medium. For example, an optical microscope, a confocal microscope, an inverted confocal microscope or a measuring device (1000) comprising observation optics and / or an impedance measuring device (1000) is arranged, in particular an inverted optical microscope, more particularly an inverted confocal microscope. The microscope allows the sample to be observed with high resolution while remaining in an unchanging position on the carrier substrate. The impedance measuring device may comprise a measuring electrode arranged on the front side of the carrier substrate.

[0270] Thus, the apparatus (100) may comprise a measurement device (1000), which preferably comprises a microscope, more preferably an inverted microscope.

[0271] Accordingly, one embodiment of the present invention is directed to an apparatus (100) for reducing the temperature of a specimen, comprising: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); (f) a measuring device (1000); In order to keep the supply lines (20) as short as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0272] Preferably, the device (100) for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); (f) a measuring device (1000); In order to keep the supply lines (20) as short as possible, the pressure tank (400) is mounted directly on the upper side (11) of the carrier substrate (10).

[0273] Preferably, the device (100) for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (4) configured to hold a fluid and having an outlet valve (22) at its bottom; 00) and (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); (f) a measuring device (1000); In order to make the supply lines (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 internally insulated, preferably with high density polyethylene.

[0274] In a preferred embodiment of the device (100) according to the invention, the measuring device (1000) is an inverted microscope. Therefore, another aspect of the invention is directed to an inverted cryo-microscope (2000), (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); (f) a measuring device (1000); In order to keep the supply lines (20) as short 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 cryo-microscope (2000) of the present invention comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid, the pressure tank having a funnel-shaped bottom and 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), the supply line being configured to pass the liquid (2) under a pressure higher than atmospheric pressure, the lower end of the supply line being funnel-shaped; (d) an expansion chamber (30) surrounding the outlet opening (21); (c) a funnel-shaped supply line (20) configured to connect an outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass liquid (2) at a pressure higher than atmospheric pressure, the supply line (20) having a lower end thereof; (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) a measuring device (1000); In order to make the supply lines (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 lift cylinder (200), a piston (501), a piston tip (510) and a piston guide (520), and the outlet opening (21) has the shape of a nozzle, and the diameter of the outlet opening (21) is smaller than the diameter of the carrier substrate (10). [Sample chamber (14)] The device may further comprise a sample chamber (14). Accordingly, an embodiment according to the invention is directed to a device (100) for reducing the temperature of a sample, further comprising a sample chamber (14). The sample chamber (14) may comprise a spacer, but preferably does not comprise a spacer. This allows the rate of temperature change to be further significantly improved, i.e. the rate of temperature change is increased.

[0276] Accordingly, an embodiment according to the invention relates to an apparatus (100) for reducing the temperature of a specimen, comprising: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); (g) a sample chamber (14); In order to keep the supply lines (20) as short as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0277] Preferably, the device (100) for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); (g) a sample chamber (14); In order to keep the supply lines (20) as short as possible, the pressure tank (400) is mounted directly on the upper side (11) of the carrier substrate (10).

[0278] Preferably, the device (100) for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); (g) a sample chamber (14); In order to make the supply lines (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 internally insulated, preferably with high density polyethylene.

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

[0280] A preferred embodiment according to the invention is directed to an apparatus (100) for reducing the temperature of a sample, comprising a spacer-free sample chamber (14). Accordingly, a preferred embodiment according to the invention relates to an apparatus (100) for reducing the temperature of a sample, comprising: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); (g) a sample chamber (14) that does not include a spacer; In order to keep the supply lines (20) as short as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0281] If an even higher rate of temperature change is required, it is even more preferred that the device according to the invention does not even include a sample chamber 14. Thus, an embodiment according to the invention is directed to an apparatus 100 for reducing the temperature of a sample that does not include a sample chamber 14 and does not include a spacer.

[0282] Accordingly, an embodiment according to the invention relates to an apparatus (100) for reducing the temperature of a specimen, comprising: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); Equipped with In order to make the supply lines (20) as short as possible, the pressure tank (400) is mounted on the upper side (11) of the carrier substrate (10); The device (100) does not include a sample chamber (14) and a spacer. [Expansion chamber] It has been found that a high pressure difference between the outlet opening and the expansion chamber allows for fast transport of the liquid (2) in the carrier substrate (10). The larger the expansion chamber (30), the greater the pressure difference between the outlet opening (21) and the expansion chamber (30), which allows for a high transport rate of the liquid in the carrier substrate (10).

[0283] Thus, an embodiment according to the present invention has an expansion chamber (30) dimension of 50 cm 3 From 100cm 3 The present invention relates to an apparatus (100) for reducing the temperature of a sample, the apparatus (100) being in the range of

[0284] Accordingly, an embodiment according to the invention relates to an apparatus (100) for reducing the temperature of a specimen, comprising: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) A 50 cm 2 diameter rectangular box (11) encircling the upper side (11) of the carrier substrate (10).3 From 100cm 3 and an expansion chamber (30) having a range of In order to keep the supply lines (20) as short as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0285] To achieve maximum cooling rates, the carrier substrate (10) is made of CVD diamond and the expansion chamber (30) has dimensions of 50 cm 3 From 100cm 3 The supply line is in the range The funnel (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] Thus, in a preferred embodiment according to the present invention, the carrier substrate (10) is made of CVD diamond and the expansion chamber (30) has dimensions of 50 cm 3 From 100cm 3 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] Thus, a preferred embodiment according to the invention relates to an apparatus (100) for reducing the temperature of a sample, comprising: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) a carrier substrate (10) having a top side (11) and a distance between the outlet opening (21) and the front surface (11) of the carrier substrate (10) in the range of 0.5 mm to 1.5 mm and a dimension of 50 cm3 From 100cm 3 and an expansion chamber (30) having a range of In order to keep the supply lines (20) as short 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 device (100) of the invention is preferably open so as to ensure a low pressure in the chamber when the tempering liquid is supplied. The expansion chamber may be in a housing with a gas outlet (23), as shown in Figures 3B, 3C and 3D. Thus, the device according to the invention may further comprise a gas outlet (23). The gas outlet (23) is preferably connected to an exhaust hose via a quick coupling for the exhaust hose. The exhaust hose is preferably connected to the housing of the expansion chamber (30).

[0289] Accordingly, an embodiment according to the invention relates to an apparatus (100) for reducing the temperature of a specimen, comprising: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an open expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to keep the supply lines (20) as short 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 reducing the temperature of a specimen, comprising: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) an outlet valve (22) connected to the upper side (11) of the carrier substrate (10) and adapted to provide a pressure above atmospheric pressure; a supply line (20) configured to pass a liquid (2) under high pressure; (d) an open expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10) and provided with a gas outlet (23); In order to keep the supply lines (20) as short as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0291] Preferably, the device (100) for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an open expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10) and provided with a gas outlet (23); In order to keep the supply lines (20) as short as possible, the pressure tank (400) is mounted directly on the upper side (11) of the carrier substrate (10).

[0292] Preferably, the device (100) for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an open expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10) and provided with a gas outlet (23); In order to make the supply lines (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 internally insulated, preferably with high density polyethylene.

[0293] In a preferred embodiment, the device (100) of the present invention comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid, the pressure tank having a funnel-shaped bottom and an outlet valve (22) at the bottom; (c) a funnel-shaped supply line (20) configured to connect an outlet valve (22) to the upper side (11) of the carrier substrate (10) and to pass liquid (2) at a pressure higher than atmospheric pressure, the supply line (20) having a lower end thereof; (d) an open expansion chamber (30) with a gas outlet (23) surrounding the upper side (11) of the carrier substrate (10); (e) a cooling tank (300) for cooling the liquid (2); In order to make the supply lines (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 lift cylinder (200), a piston (501), a piston tip (510) and a piston guide (520), and the outlet opening (21) has the shape of a nozzle, and the diameter of the outlet opening (21) is smaller than the diameter of the carrier substrate (10). [Permanent cooling] According to another advantageous variant of the invention, after a temperature change of the sample, in particular after cooling, the sample is subjected to a cooling process. A permanent tempering of the carrier substrate (10) can be performed. For example, during the examination of the sample by inverted microscopy, a low temperature can be maintained by passing a gaseous or liquid thermostatic medium over the upper side (11) of the carrier substrate (10). The gaseous or liquid thermostatic medium is supplied at a low pressure between 5 and 10 bar. Furthermore, cryofixation of snap-frozen samples is also possible. Cryofixation can be performed using one of the methods known per se, whereby the carrier substrate (10) with the sample is transferred as required from the device to a cryofixation device, for example a cryotank with liquid nitrogen.

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

[0295] Thus, one embodiment according to the invention relates to an apparatus (100) for reducing the temperature of a sample, comprising: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the upper side (11) of the carrier substrate (10) and configured to pass a liquid (2) at a pressure higher than atmospheric pressure and equipped with an inlet for a tempering liquid for permanent cooling; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10); In order to keep the supply lines (20) as short as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10).

[0296] In one embodiment according to the invention, an apparatus (100) for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (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); In order to make the supply lines (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 liquid to the supply line for permanent cooling.

[0297] The tempering liquid is preferably supplied quickly or instantaneously after cooling in order to keep the sample temperature constantly low. In this respect, in an embodiment of the device of the invention in which the tempering liquid is supplied from a pressure tank (400), the supply of the tempering liquid is controlled by a valve. The valve may be a check valve that opens as soon as the pressure in the tank is reduced by opening the outlet valve (22). Alternatively, the valve may be a valve that opens after a predefined time has elapsed.

[0298] Thus, in one embodiment according to the invention, an apparatus (100) for reducing the temperature of a sample comprises: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (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); In order to make the supply lines (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 liquid to the supply line for permanent cooling.

[0299] The valve is preferably a check valve configured to open when the pressure in the pressure tank reaches atmospheric pressure.The valve is preferably a check valve configured to open when the pressure in the pressure tank falls below a predetermined pressure above atmospheric pressure.

[0300] Another embodiment of the present invention relates to an apparatus (100) for reducing the temperature of a specimen, comprising: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting an outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass liquid (2) through the supply line (20) at a pressure greater than atmospheric pressure; (d) an expansion chamber (30) surrounding the upper side (11) of the carrier substrate (10) and further comprising an inlet for a tempering liquid for permanent cooling; In order to keep the supply lines (20) as short as possible, a pressure tank (400) is attached to the upper side (11) of the carrier substrate (10). [Fluorescence microscopy] The methods and devices disclosed herein are particularly useful for fluorescence microscopy of freeze-stopped samples 1. Ultra-rapid freeze-stop of samples 1, preferably biological samples such as living cells, overcomes fundamental resolution barriers imposed by motion blur and photochemical reactivity, thereby enabling the observation of native molecular distributions and reaction patterns that cannot be resolved at physiological temperatures.

[0301] Thus, a further aspect of the invention is directed to a method of fluorescence microscopy comprising the steps of:

[0302] (a) providing a sample (1) comprising fluorescent molecules on an underside (12) of a 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 a pressure tank (400); (c) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1); (d) irradiating the sample (1); and (e) detecting fluorescence produced by the sample (1).

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

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

[0305] (a) providing a sample (1) comprising fluorescent molecules on an underside (12) of a 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) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1); (d) irradiating the sample (1); and (e) detecting fluorescence produced by the sample (1).

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

[0307] (a) providing a sample (1) comprising fluorescent molecules on an underside (12) of a 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 a pressure tank (400); (c) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400), causing the liquid nitrogen to contact the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1) to vitrify the sample (1) without crystallization, preferably without forming ice crystals with a diameter of more than 80 nm, preferably without forming ice crystals with a diameter of more than 10 nm, more preferably without forming ice crystals with a diameter of more than 1 nm, and even more preferably without forming ice crystals; (d) irradiating the sample (1); and (e) detecting fluorescence produced by the sample (1).

[0308] In a preferred embodiment, the method of fluorescence microscopy comprises the following steps.

[0309] (a) providing a sample (1) comprising fluorescent molecules on an underside (12) of a carrier substrate (10) at atmospheric pressure; (a') irradiating a sample (1); (a'') detecting fluorescence produced by a sample (1); (b) 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) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1); (d') repeating step (a') and step (a'').

[0310] [Optical microscopy using stimulated emission depletion (STED)] A further aspect of the invention is directed to a method for performing optical microscopy using stimulated emission suppression of a freeze-stopped sample (1), comprising: (a) providing a sample (1) comprising fluorescent molecules on an underside (12) of a 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 a pressure tank (400); (c) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1); (d1) irradiating a sample (1) with a first pulsed laser beam focused at at least one focal region to excite fluorescent molecules in the sample (1) to emit fluorescence; (d2) irradiating the sample (1) with a second torus-shaped laser beam having an intensity zero point in at least one focal region to de-excite fluorescent molecules in the sample (1); (e) detecting fluorescence produced by the sample (1); and (f) optionally repeating steps d1, d2, and e at different positions of the focal region of the first pulsed laser beam and different positions of the intensity zero point of the second torus-shaped laser beam.

[0311] Preferably, the method for performing optical microscopy by stimulated emission suppression of a freeze-stopped sample (1) comprises the steps of: (a) providing a sample (1) comprising fluorescent molecules on an underside (12) of a 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) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1); (d1) irradiating a sample (1) with a first pulsed laser beam focused at at least one focal region to excite fluorescent molecules in the sample (1) to emit fluorescence; (d2) irradiating the sample (1) with a second torus-shaped laser beam having an intensity zero point in at least one focal region to de-excite fluorescent molecules in the sample (1); (e) detecting fluorescence produced by the sample (1); and (f) optionally repeating steps d1, d2, and e at different positions of the focal region of the first pulsed laser beam and different positions of the intensity zero point of the second torus-shaped laser beam.

[0312] Preferably, the method for performing optical microscopy by stimulated emission suppression of a freeze-stopped sample (1) comprises the steps of: (a) providing a sample (1) comprising fluorescent molecules on an underside (12) of a 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 a pressure tank (400); (c) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400), causing the liquid nitrogen to contact the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1) to vitrify the sample (1) without crystallization, preferably without forming ice crystals with a diameter of more than 80 nm, preferably without forming ice crystals with a diameter of more than 10 nm, more preferably without forming ice crystals with a diameter of more than 1 nm, and even more preferably without forming ice crystals; (d1) irradiating a sample (1) with a first pulsed laser beam focused at at least one focal region to excite fluorescent molecules in the sample (1) to emit fluorescence; (d2) irradiating the sample (1) with a second torus-shaped laser beam having an intensity zero point in at least one focal region to de-excite fluorescent molecules in the sample (1); (e) detecting fluorescence produced by the sample (1); and (f) optionally repeating steps d1, d2, and e at different positions of the focal region of the first pulsed laser beam and different positions of the intensity zero point of the second torus-shaped laser beam. [Fluorescence Lifetime Imaging Microscopy (FLIM)] A further aspect of the invention is directed to a method for performing time-correlated single-photon counting fluorescence lifetime imaging microscopy of a freeze-stopped sample (1), comprising: (a) providing a sample (1) comprising fluorescent molecules on an underside (12) of a 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 a pressure tank (400); (c) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1); (d) periodically exciting the fluorescent molecules to emit fluorescent photons by irradiating the sample with a pulsed laser beam; (e) detecting fluorescence photons produced by the sample along with the arrival time of the photons relative to the laser pulse (1); (f) determining a fluorescence lifetime from the detected fluorescent photons and the arrival times of the photons.

[0313] Preferably, the method for performing time-correlated single-photon counting fluorescence lifetime imaging microscopy of a freeze-stopped sample (1) comprises the steps of: (a) providing a sample (1) comprising fluorescent molecules on an underside (12) of a 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) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and bringing the liquid nitrogen into contact with the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1); (d) periodically exciting the fluorescent molecules to emit fluorescent photons by irradiating the sample with a pulsed laser beam; (e) detecting fluorescent photons produced by the sample (1); and (f) determining a fluorescence lifetime from the detected fluorescence photons.

[0314] Preferably, a method for performing time-correlated single-photon counting fluorescence lifetime imaging microscopy of a freeze-stopped sample (1) comprises the steps of: (a) providing a sample (1) comprising fluorescent molecules on an underside (12) of a 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 a pressure tank (400); (c) supplying liquid nitrogen under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and contacting the liquid nitrogen with the upper side (11) of the carrier substrate (10) and reducing 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 more than 80 nm or without forming ice crystals with a diameter of more than 10 nm in the sample (1); (d) periodically exciting the fluorescent molecules to emit fluorescent photons by irradiating the sample with a pulsed laser beam; (e) detecting fluorescent photons produced by the sample (1); and (f) determining a fluorescence lifetime from the detected fluorescence photons.

[0315] This disclosure also relates to the following:

[0316] Item 1. A method for reducing the temperature of a sample (1) for inverted microscopy, comprising the steps of: (a) providing a sample (1) on an underside (12) of a carrier substrate (10) at atmospheric pressure; (b) providing at least one liquid (2) having a temperature below a critical temperature and a gas (500) having a pressure higher than atmospheric pressure in a pressure tank (400); (c) supplying at least one liquid (2) under pressure from the pressure tank (400) to the upper side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400), bringing the at least one liquid (2) into contact with the upper side (11) of the carrier substrate (10) and reducing, preferably rapidly reducing, the temperature of the sample (1).

[0317] Item 2. The method according to 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 2, wherein the at least one liquid (2) in step (b) is liquid nitrogen.

[0319] Item 4. The method according to any one of Items 1 to 3, wherein the gas (500) is helium.

[0320] Item 5. The method according to any one of Items 1 to 4, further comprising step (d): (d) tempering the sample (1) on the underside (12) of the carrier substrate (10) by passing a further liquid over the upper side (11) of the carrier substrate (10).

[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 a living cell.

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

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

[0324] Item 9. The apparatus (100) according to item 8, wherein the temperature of the sample (1) is reduced so as to prevent ice crystals from forming in the sample (1) or to prevent ice crystals having a diameter of more than 10 nm from forming in the sample (1).

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

[0326] Item 11. The apparatus according to any one of items 8 to 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 one of items 8 to 11, wherein the carrier substrate (10) is diamond, in particular CVD diamond.

[0328] Item 13. The apparatus (100) according to any of items 8 to 12, 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.

[0329] Item 14. The expansion chamber (30) has a dimension of 50 cm 3 From 100cm 3 14. The apparatus (100) according to any one of items 8 to 13, wherein

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

[0331] [Figure 1] Localization precision calculated relative to the real structure size (left) and resolution (right) plotted against the structure size for different amounts of photons emitted (right legend) at room temperature (solid line) or under freeze-arrest (dotted line) simulating Brownian motion. Horizontal black dotted line: localization precision of 10% of the real structure size. The figure shows the spatial resolution limit ("fundamental barrier") of the observed system. PSF denotes the point spread function of the microscope. This limit is caused by the number of collected photons, which is inversely proportional to the size of the observed structure and determined by Brownian or active motion ("room temperature" vs. "freeze-arrest"), and by the exposure time, which is limited by the maximally achievable fluorescence photon flux, which is determined by the molecular cross-section, fluorescence quantum yield, excited state lifetime and photochemical stability. [Diagram 2] Shown is a schematic diagram of one embodiment of the device (100) of the present invention applied to an inverted microscope (1000). Shown are: sample (1), liquid (2), gas (500), carrier substrate (10), upper side (11) and lower side (12) 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 view of the device (100) according to the invention is shown: a carrier substrate (10) acts as a heat exchanger, receives the sample (1) on its underside, a supply line (20) for liquid (2), and an outlet opening (21). When the outlet valve (22) is opened, the liquid (2) and the gas (500) flow out of the pressure tank (400), through the supply line to the upper side (11) of the carrier substrate (10) and into the expansion chamber (30). [Figure 3B] A detailed view of one embodiment of the device (100) of the present invention is shown having an expansion chamber (30) with a gas outlet (23), which may further include a quick coupling for an exhaust hose. [Figure 3C]FIG. 1 shows a detailed view of an embodiment of the device (100) of the present invention having a closed expansion chamber (30) and a supply line (20) with a funnel-shaped outlet opening (21), which may be in the form of a nozzle. [Figure 3D] FIG. 1 shows a detailed view of an embodiment of the device (100) of the present invention having a closed expansion chamber (30) and a supply line (20) with a funnel-shaped outlet opening (21), which may be in the form of a nozzle. [Figure 3E] FIG. 1 shows a detailed view of one embodiment of the device (100) of the present invention, in which an observation window (15) is placed below the sample (1), thereby forming a sample chamber (14). [Figure 4A] An exemplary embodiment of a pressure tank (400) of a device according to the invention is shown, having a round or semicircular bottom. [Figure 4B] 4 shows an exemplary embodiment of a pressure tank (400) of a device according to the invention, having a flat bottom. [Diagram 5] An exemplary embodiment of the pressure tank (400) of the device according to the invention with a switchable outlet valve (22) is shown. The following means are shown: lift cylinder (200), piston guide (520), piston tip (510) and piston (501). The piston tip is preferably made of high density polyethylene (HDPE). This switchable valve allows for quick opening of large cross sections. [Figure 6] Figure 1 shows the temperature profile by using the device according to the invention. A cylindrical sample with a diameter of 4 mm and a thickness of 100 μm was used for the cooling experiments. The temperature was measured by using a 50 μm non-insulated wire thermocouple (T type, Omega Engineering Inc.) in an aqueous imaging medium (DMEM with HEPES buffer from PAN-Biotech GmbH). As can be seen, the temperature can be initially maintained at the desired temperature. Opening the switching valve causes the temperature to drop sharply to below -130°C. Afterwards, the temperature is constantly kept below -130°C by passing liquid nitrogen over the top side (11) of the carrier substrate (10). [Figure 7A]Figures 7A-7C show a comparison of the temperature and cooling rate achieved by the device according to the invention (Figure 7A) and the device according to EP 2877828 B1 (Figure 7B). The temperature was measured by using a 50 μm non-insulated wire thermocouple (T type, Omega Engineering Inc.) in an aqueous imaging medium (DMEM with HEPES buffer from PAN-Biotech GmbH). The bottom curve in Figure 7A shows the cooling rate in °C / s for a cooling experiment using the device or method according to the invention. The bold line is the average value of the individual cooling experiments, showing the standard deviation. The other curves show the temperature profile of the individual cooling experiments. Here the cooling process can be clearly seen, with a cooling rate (bottom curve) exceeding 10,000 K / s in the temperature range between 0 °C and -130 °C. In that temperature range ice can usually be formed. [Figure 7B] The bottom curve shows the cooling rate in °C / sec for cooling experiments using the apparatus or method according to EP2877828B1. [Figure 7C] Figure 1 shows a comparison of the cooling rates achieved by a device according to the invention operated with a mixture of liquid nitrogen (LN2) and helium (He) at a pressure of 50 bar (solid line) and at lower pressures of 25 bar and 15 bar (dashed lines), and with a device according to the invention operated without LN2 and with helium at 50 bar. Temperatures were measured using a 50 μm non-insulated wire thermocouple (T type, Omega Engineering Inc.) in an aqueous imaging medium (DMEM with HEPES buffer from PAN-35 Biotech GmbH). It can be clearly seen that the lower the helium pressure, the slower the cooling rate. The lowest cooling rate was observed when liquid nitrogen was omitted. [Figure 8]1 shows a comparison of the temperature and rate of temperature change achieved by a device according to the invention and a device according to EP 2877828 B1 ("prior art device") adapted to an inverted microscope by extending the supply lines to the top side of the carrier substrate. The rate of temperature change was measured using a 50 μm non-insulated wire thermocouple (T type, Omega Engineering Inc.) in an aqueous imaging medium (DMEM with HEPES buffer from PAN-Biotech GmbH). [Figure 9] Electron microscope images (inlet: electron diffraction patterns) of ice crystals in a dilute aqueous solution (PBS) at various cooling rates are shown. All cooling rates (relative measurements) were recorded using a 50 μm thermocouple. The actual cooling rates are much higher. (A) Sample frozen at a measured cooling rate of about 1000 K / s (Figure 8), obtained with a prior art device adapted to an inverted microscope. 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 is composed only of hexagonal ice, a size that would displace molecular components inside the cells and destroy them. (B) and (C) show samples frozen at measured cooling rates of about 2500 K / s and 3500 K / s, respectively. Individual ice crystals were observed. At a cooling rate of about 3500 K / s, the ice crystals have a diameter smaller than 50 nm. (D) shows a cell sample containing 30% dextran (a cryoprotectant) frozen at a measured cooling rate of approximately 3500 K / s. Cuboidal ice crystals with diameters less than 10 nm were formed. The arrow points in the direction of the cell membrane. [Figure 10] A cross-sectional view of one embodiment of the device (100) according to the invention is shown. The following means are shown: lifting cylinder (200), cooling tank (300), pressure tank (400), piston tip (510), heat exchanger (600), measuring device (1000), e.g. an inverted microscope, and gas outlet (23). The closed expansion chamber (30) forms the heat exchanger (600) together with the carrier substrate (10). The gas outlet (23) further comprises a quick coupling for an exhaust hose. [Figure 11]Figure 11 shows an external view of the embodiment of the device (100) according to the invention shown in Figure 10. Means (800) for adjusting and locking the height are shown. [Figure 12] 1 shows the bottom of the embodiment of the device (100) according to the invention shown in Figure 10. In particular, the piston tip (510), the heat exchanger (600) and the diamond window (700) are shown. [Figure 13] The lower part of the device (100) according to the invention is shown, specifically the gas outlet (23), together with a quick coupling for an exhaust hose as shown in FIG. [Figure 14] FIG. 11 shows a detailed view of the top of the pressure tank (400) as part of the apparatus (100) of the present invention shown in FIG. 10. Shown are threaded holes (450) for assembly components such as pressure gauges, rupture disks, gas supply lines, etc. [Figure 15] A top view of the threaded holes (450) of one embodiment of the device (100) according to the invention shown in Figure 10 is shown. The means (800) for adjusting and locking the height, the threaded holes (450) and the holes (210) for the lifting cylinders are shown. [Figure 16] A more detailed view of the lift cylinder (200) of one embodiment of the device (100) according to the invention shown in Figure 10 is shown. In particular, the piston guide (520) is shown. [Figure 17] Widefield fluorescence microscopy of EGFP-transfected HeLa cells before (first three images) and during cryofixation (rightmost image). Scale bar: 10 μm. [Figure 18] Widefield fluorescence image of the fluorescent dye Rhodamine 6G in cell culture medium after ultrarapid freeze-arrest under a microscope (far left). The sample was mostly homogeneous, apart from highly fluorescent structures that were already present before freeze-arrest. Arrows: expansion cracks. Rightmost three images: The same sample during rewarming. The cracks disappeared completely and subsequently, upon removal of the fluorescent dye, ice crystals became visible (arrows). The ice crystals then first grew and then melted at higher temperatures, leading to an expansion of the liquid phase between the ice crystals (illustrated by the arrows in the rightmost image). Scale bar: 10 μm [Figure 19]Examples of MCF7 cells expressing TC-PTP-mCitrine imaged by confocal scanning microscopy at the indicated times before freeze arrest (images 1 to 3 from the left). Right half: the same cells imaged by confocal microscopy (sum of 10 frames) and widefield microscopy (top: single frame, bottom: sum of 100 frames). Bottom right image: super resolution radial fluctuation (SRRF) reconstruction of 100 frames. Scale bar: 10 μm. [Figure 20A] MCF7 cells expressing R-PTP-γ-mCitrine and Alexa647-labeled Snap-EGFR are shown, previously imaged by confocal laser scanning microscopy at the indicated times (scan time of 3.75 s) after application of 100 ng / mL EGF (single frames, images 1 to 3) and during freeze arrest (total of 10 frames, 37.5 s, image 4). Right, top: confocal scans of a larger area including several cells. Second row: SRRF reconstructions of 100-frame widefield recordings of the same cells (left) and an example of a cell fixed without prior EGF stimulation. Third row: Enlargement of the boxed area 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 arrest (bottom). Left image: individual frames, right image: sum of 10 frames. Graph: corresponding cumulative background corrected line profile color-coded by frame number. [Figure 21A] CLSM and STED nanoscopy of Alexa647-Snap-EGFR in live (top) and freeze-stopped MCF7 cells (bottom). CLSM of cells (first column, pixel size: 100 nm). Detail of boxed region (pixel size: 40 nm) with indicated laser illumination intensity. CLSM scan (second column) and STED scan (third column). Live cells: single scan. Freeze-stopped cells: total of 10 scans. [Figure 21B]Alexa647 photobleaching during STED scans at room temperature (rt) or cryo-stopped (cryo) at the indicated depletion laser powers. λ: decay rate determined by exponential fit (mean + / - standard deviation, N=5). [Figure 21C] STED spatial frequency spectra normalized by corresponding CLSM of different regions containing vesicles (cryo: n = 11; chemical fixation: n = 8; rt raw: n = 6). Horizontal lines: frequency ranges where STED information content is significantly higher than CLSM (Student's t-test); vertical lines: digital resolution. [Figure 21D] Top row: 3D-CLSM of freeze-arrested MCF7 cells. Bottom row: Detailed high-resolution scans (pixel size: 40 nm) of the indicated areas in the top row: CLSM (left column), STED (middle column), and merged (right column). [Figure 22] Representative intensity-weighted mean fluorescence lifetime (τ) images obtained by confocal FLIM of Cos7 cells stably expressing LIFEA2 without (left column) and with (right) clustered ephrinA1 stimulation at room temperature (rt, top) or under cryo-arrest (cryo, bottom). Bars: τ (ns). [Diagram 23] Confocal FLIM of LIFEA2-expressing Cos7 cells after clustered ephrinA1 stimulation for the indicated times before and during ultrarapid freeze arrest. Top panel: fluorescence photon counts, bottom panel: τ (ns), graph: corresponding τ histograms at room temperature and freeze arrest. [Figure 24A] Normalized cumulative fluorescence decay histograms of LIFEA2 at room temperature and freeze arrest with (rt:B, N = 6; n = 9; cryo:D, N = 7; n = 16) and without (rt:A, N = 6, n = 10; cryo:C, N = 2, n = 11) clustered ephrinA1 stimulation. [Figure 24B] Imaginary (Im) and real (Re) parts of the phasor (R) derived from the first harmonic of the fluorescence decay data in Figure 24 A. Dashed line: linear regression of the phasor. [Diagram 25]Fluorescence photon counts (upper panel) and fluorescence intensity-weighted fraction of active conformations of LIFEA2 (lower panel) at different z-heights (rows) obtained by confocal FLIM upon freeze-arrest after 14 min stimulation with clustered ephrins. Bars: fraction of LIFEA2 in active conformations (α). [Figure 26] A representative series of 10 consecutive STED scans of a 5 × 5 μm area of ​​Alexa647-Snap-EGFR in EGF-stimulated (100 ng / ml) MCF7 cells under freeze arrest is shown. Images are displayed as the sum of two scans and gamma corrected (γ: 0.24) to accentuate dim fluorescence. Plasma membranes with protrusions are visible as continuous fluorescent areas separating cytoplasmic fluorescence with endocytic structures from extracellular autofluorescence (extracellular). The series was acquired at maximum laser intensity (0.42 W / μm2). Ice crystals larger than 80 nm (digital resolution), which would exclude fluorophores, scatter light, and appear as dark spots, were not detected in the merged images, nor did they form during the scan due to possible heating by the STED beam. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0332] Example 1: Design and operation of an exemplary embodiment of the device of the present invention The main components of the cooling device (100) are the pressure tank (400) that holds N2 and helium, and the carrier substrate (10) that acts as a heat exchanger unit. The pressure tank (400) was made of stainless steel, and the joints were sealed with copper gaskets. The pressure tank (400) was cooled externally by liquid N2, which was filled in a cooling tank (300) made of steel plate and insulated with Styrodur®. The pressure tank (400) contained a custom-made piston, controlled pneumatically, that acted as a valve (22). The piston was sealed at the shaft with a spring-loaded PTFE sealant. The tip of the piston was made of high density polyethylene (HDPE) that, when closed, forms a seal against the stainless steel of the tank and at the same time withstands pressure. When the piston rises, the pressurized fluid is released to the upper side (11) of the carrier substrate (10). The supply lines (20), which act as connecting parts, were kept as short as feasible and insulated from the inside by HDPE to minimize heating of the coolant from the metal parts. In the heat exchanger unit, the mixture is directed through a nozzle at the outlet opening (21) directly onto the upper side (11) of the carrier substrate (10) containing the diamond window, after which the mixture spreads out and is directed to an exhaust outlet.

[0333] The use of a mixture of helium and N2 gave much better cooling performance than either gas alone. N2 alone liquefies rapidly at −196 °C and 50 bar, which reduces the pressure in the vessel and slows its release. As a result, the cooling rate was much slower. Helium cannot be liquefied under these conditions, but its heat capacity as a gas alone is too low, which results in incomplete and slow freeze fixation. N2 was charged into the apparatus for 1 min by first filling it with nitrogen from an internal source (about 7 to 8 bar), introduced through a copper spiral immersed in liquid nitrogen. The pressure was then increased by helium from a pressure 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, TX, 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 the nitrogen supply to prevent high pressure from entering the copper spiral. The entire apparatus was driven by an electronically controlled lifting cylinder (1000 N, Drive-System Europe GmbH). The microscope is mounted on a frame on top of an epifluorescence microscope in such a way that it can be elevated to mount the specimen using a microscope slide (Stockholm, Wörthersee, Germany).

[0335] Before the shot, the cells were always heated to 37°C by two 50 W heating bands (Friedr.Freek GmbH, Menden, Germany) attached to a heat exchanger unit and controlled by a PID controller (KT4, Panasonic Corporation, Kadoma, Japan).

[0336] To keep the temperature at -196 °C after rapid cooling, N2 from an internal supply (about 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 in the vessel falls below the pressure in the copper coil. This allows liquid nitrogen to flow through the vessel over the diamond, keeping the vessel at a temperature close to -196 °C.

[0337] Around the objective lens, a transparent flexible foil was attached to the stage by custom 3D printed connectors. The chamber inside this foil was filled with a constant low flow of room temperature dry helium throughout the experiment. The temperature of the objective lens was controlled by an objective lens heater (Bioptechs Inc., Butler, PA, USA). The objective lens heater was always set to 25 °C. Imaging was performed for 30 to 60 min. During the experiments, the temperature of the objective lens was typically between 7°C and 10°C. [Example 2: Temperature measurement using the device according to the present invention] A 50 μm non-insulated wire thermocouple was attached to the underside (12) of the carrier substrate (10) of the device (100) according to the 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 parts reached a temperature close to 77 K. The pressure tank (400) was then filled with 50 bar of liquid nitrogen and helium. A drop of HEPES-buffered DMEM on a microscope cover slide was deposited on the underside (12) of the carrier substrate (10) by water deposition so that the drop surrounded the thermocouple. The wire thermocouple was not flat, so a gap was left between the carrier substrate and the wire, resulting in a cylindrical sample with a diameter of 4 mm and a thickness of about 100 μm. The temperature measurement was started and cooling was started 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 with the device according to the invention at pressures of 15 bar, 25 bar and 50 bar A 50 μm non-insulated wire thermocouple was attached to the underside (12) of the carrier substrate (10) of the device (100) according to the 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 parts reached a temperature close to 77 K. The pressure tank (400) was then filled with liquid nitrogen and helium at 15 bar, 25 bar or 50 bar, or with 50 bar helium without liquid nitrogen. A drop of HEPES-buffered DMEM on a microscope cover slide was deposited on the underside (12) of the carrier substrate (10) by water deposition, so that the drop surrounded the thermocouple. The wire thermocouple was not flat, so a gap was left between the carrier substrate and the wire, resulting in a cylindrical sample with a diameter of 4 mm and a thickness of about 100 μm. The temperature measurement was started and, during the measurement, the outlet valve (22) was opened to start cooling. The cooling rate decreased significantly at lower pressures, and the lowest cooling rate was observed in the absence of liquid nitrogen (Figure 7C). Example 4: Temperature measurement using a prior art device adapted to an inverted microscope The apparatus disclosed in EP2877828B1 was arranged and adapted to an inverted microscope by extending the supply lines to the upper side of the carrier substrate. A thermocouple was attached to the underside of the carrier substrate. Temperature measurements were performed as described in Example 2. The results of this measurement are shown in Figures 7B and 8. Example 5: Microscopy of Adherently Grown Mammalian Cells An inverted microscope equipped with a temperature control device according to the invention was arranged. First, the cooling tank (300) and the permanent cooling device were cooled with liquid nitrogen until the vigorous boiling of the liquid nitrogen ceased and the parts reached a temperature close to 77 K. The pressure tank (400) was then filled with 50 bar of nitrogen and helium. The cover slide with the attached and grown cells was attached to the underside (12) of the carrier substrate (10) by water attachment. For this, most of the cell culture medium was removed and the cells remained in a thin layer of about 15 μm. The stage was lowered onto the objective of the microscope and the live cells could be observed. At the time point of interest, the valve was opened to rapidly freeze-arrest the cells. After this, detailed microscopic measurements of the frozen-arrested cells could be carried out. Example 6: Spatially resolved spectroscopic investigation of living cells. ·method [Resolution and localization accuracy calculations] The resolution and localization accuracy were calculated taking into account the effects of the imaging system, molecular motion, quantum yield of the fluorophores, and photostability. Briefly, motion was modeled as diffusion of molecules through a liquid with a low Reynolds number, so that the diffusion coefficient D of an object was obtained as a function of temperature (T) and radius (r) through the Stokes-Einstein equation:

[0338]

number

[0339] where 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-arrest (CA), the viscosity increases by more than 15 orders of magnitude, so the object was considered to be at rest. The photophysical properties of the fluorescent proteins (quantum yield and photostability) were taken from the literature, and their changes upon freeze-arrest were taken from the data presented here (QY CA / QY RT =1.2, (emitted photons) CA / (emitted photons) RT >10).

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

[0341]

number

[0342] becomes:

[0343]

number

[0344] Here, v MB corresponds to the mean square displacement (4DT) of the diffusion process. The calculation, procedure described is appropriate when a single point-like light source is used as a label for the imaged structure. In contrast, if the structure of interest is perfectly labeled, additional steps are required. Considering uniform labeling, there is one light source at each point of the structure, so the observed object becomes the 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 room temperature at a rate of (1 / 500 ns). This value ensures that the fluorophore is not re-excited upon activation and was found to be in agreement with other values ​​reported in the literature. Furthermore, it was considered that due to the reduced collection and transmission efficiency, only 10% of the emitted photons reach the detector.

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

[0346]

number

[0347] N photons are sampled from a distribution consisting of two Gaussian distributions defined by The images were separated by a distance d. The results were binned (size a) to simulate pixelation. A Gaussian mixture model was used to recover these Gaussian distributions, and the contrast was defined as

[0348]

number

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

[0350]

number

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

[0352]

number

[0353] If the distance between the two structures is greater than , the structure is considered to be decomposed.

[0354]

number

[0355] and the resolution of N was defined as the minimum d at which 95% of the realizations are resolved. The localization accuracy was calculated as a function of N, a, and the detector noise. Importantly, the PSF was replaced by the EPSF to account for motion blur. [Measuring cooling rate] The cooling performance was measured by a 50 μm Constantan copper thermocouple (Omega Engineering Inc., Norwalk, CT, USA; response time: 3 ms) attached to a 1 μL drop of aqueous buffer solution. The total thickness of the sample was about 100 μm, as inferred from the drop covering the entire area of ​​the attached cover slide due to the thickness of the thermocouple. The thermocouple was connected to a computer via a USB data acquisition module (Omega Engineering Inc., Norwalk, CT, USA) and recorded the L Readouts were taken at a frequency of 1000 Hz (digital resolution: 2 ms) using abview software (National Instruments, Austin, TX, USA). [Calculation of thermal diffusivity limited cooling rate] To relate the measured cooling rates to those limited by the thermal diffusivity of the aqueous sample, heat flux limited cooling from one surface (i.e., cooling of the aqueous solution from the diamond) is based on the 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 Wm -2, the 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 taken with a pipette covering the entire cover slide. The freezing rate for samples containing a 50 μm thermocouple was calculated assuming that the thermocouple was measuring 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 to be 10 to 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] where c is the heat capacity (water: 4 Jcm -3 K -1 ), m is the mass of the sample (0.25 mg).

[0361] When combined, we get the following result:

[0362]

number

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

[0364]

number

[0365] Despite ignoring heat transport along the thermocouple and the different thermal conductivities and capacities of the thermocouple and the aqueous solution, the resulting diffusivity-limited and measured temperature change rates were very similar. [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 not express 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, CA). MCF7 cells were then transfected with this construct and PiggyBac transposase (System Bioscience) at 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 an additional week in regular cell culture medium, cells were selected for high R-PTP-γ-mCitrine expression by FACS.

[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, CA). MCF7 cells were then transfected with this construct and PiggyBac transposase (System Bioscience) in a 1:1 ratio. Transfection was performed 24 h after seeding using FuGENE® 6 (Roche Diagnostics) as 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, CA). MCF7 cells were then transfected with the constructs and PiggyBac transposase (System Bioscience) in a 1:1:2 ratio. Transfection was performed 24 h 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 h. After an additional week in normal cell culture medium, cells were sorted by FACS for high R-PTP-γ-mCitrine expression. We observed that MCF7 cells that do not express SNAP-EGFR cannot survive for more than a few days. Labeling of Snap-EGFR was performed by incubating cells with 0.5 μM Snap-Surface Alexa 647 (New England Biolabs GmbH, Frankfurt, Germany) for at least 60 min. Constant recycling of EGFR resulted in labeling of EGFR not only in intracellular compartments but also in the plasma membrane. Cells were labeled with Snap-Surface Alexa 647 and confirmed by microscopy that >99% of these cells also expressed SNAP-EGFR. [Cell culture, transfection, and preparation] HeLa (ATCC no. CCL-185), Cos7 (ATCC no. CRL-1651), and MCF7 (ATCC no. HTB-22) cells were obtained from ATCC. Cells were authenticated by short tandem repeat (STR) analysis and were free of mouse, rat, and hamster (Leibniz Institute DSMZ) DNA sequences. Cells were routinely tested for mycoplasma infection using the MycoAlert mycoplasma detection kit (Lonza, Basel, Switzerland). They were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 200 mM L-glutamine, and 1% non-essential amino acids and cultured at 37 °C in 95% air and 5% CO2.

[0368] For ultrarapid freeze-stop experiments, cells were plated at 1 × 10 per well on 4 mm cover slides in 3.5 cm plastic Petri dishes. 5 From 2 × 10 5 Cells were seeded or 4 mm cover slides at a concentration of 4 x 10 4 The cells were mounted in a chamber made of biocompatible silicon (4-well microinsert; ibidi GmbH, Grafelfing, Germany) filled with 20 μL of cell culture medium at 10 cells / mL. The latter approach had the advantage that no medium was left on the bottom surface of the cover slide. Cells were seeded at least 1 day before the experiment. The cover slide was mounted on a custom 3D-printed holder to facilitate mounting by removing excess liquid before the experiment and depositing the remaining aqueous solution directly onto the diamond heat exchanger. Experiments at 37 °C were performed in glass-bottom cell culture dishes (MatTek Corporation, Ashland, MA, USA) with 1 × 10 cells per dish. 5 From 4 × 10 5 This was carried out in cells.

[0369] Transient transfection of HeLa cells with EGFP (GFP-N1 vector, Clontech Laboratories Inc., Mountain View, CA, USA) was performed 6 to 24 h after cell seeding using 1 μg of DNA per well and FuGENE® 6 (Roche Diagnostics) as 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 for 20 min at 37 °C, which 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 on a commercial confocal laser-scanning STED microscope (Expert Line, Abberior Instruments, Göttingen, Germany) equipped with 488 nm, 561 nm, and 640 nm excitation lasers and STED lasers with wavelengths of 595 nm (nominal 1 W) and 775 nm (1.25 W) and integration of FLIM hardware and software. The system was further equipped with an MT20 illumination system (Olympus Deutschland GmbH, Hamburg, Germany) and an Orca-R2 camera (Hamamatsu Photonics, Hamamatsu, Japan) for wide-field imaging.

[0371] Wide-field fluorescence imaging of HeLa cells expressing EGFP and rhodamine 6G solution, as well as donor-only controls for FLIM, was performed using a 40× 0.6NA objective (LucPlanFLN; Olympus Deutschland GmbH, Hamburg, Germany). All other imaging was acquired with a 40× 0.95NA objective (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, measured illumination power at the sample plane 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, measured power at the sample plane: 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 / s. This relatively low frame rate allowed us to successfully reconstruct super-resolution images from the reduced fluorescence fluctuation dynamics under cryogenic conditions.

[0373] STED imaging was performed using a 640 nm laser excitation and a 775 nm laser for stimulated emission depletion. Samples were scanned with a pixel length of 40 nm and a dwell time of 15 us. Individual frames were aligned by a rigid registration algorithm using the open source program Fiji (Schindelin et al. 2012, Nature Methods 9, 676-682) to correct for sample drift. To achieve a highly sensitive doughnut-shaped depletion beam under cryogenic conditions, spherical aberration had to be corrected by a correction collar on the objective lens. [Image analysis] General image processing, including quantification of fluorescence intensity, extraction of line profiles, and calculation of Manders coefficients, was performed using the open source program Fiji (Schindelin et al. 2012 Nature Methods, 9:676-82.). Quantification of bleaching was performed by fitting the fluorescence intensity with a monoexponential fit using Microsoft Excel (Microsoft Corporation, Redmond, WA, USA). When the fluorescence intensity could not be fitted with a monoexponential fit because the fluorescence converged to zero, the first monoexponential part of the data was fitted. Super-Resolution Radial Fluctuation (SRRF) analysis was performed using the NanoJ plugin to 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 FLIM datasets] Each photon counting data set was registered to correct for possible stage drift by applying a rigid-body translation-only transformation. After computing the photon counting image stacks, a discrete Fourier transform-based algorithm (Reddy et al. 1996, IEEE Transactions on Image Processing 5, 1266-1271) implemented in Python (imreg_dft 2.0) was used to find the shifts in the X and Y directions of each frame relative to the first frame. These values ​​were rounded to the nearest integer. A new, registered photon counting data set was assembled by shifting each photon in each frame by a pre-determined number of pixels. The first frame after registration was Photons outside the image defined by the frame were removed.

[0374] The registered photon counting datasets were corrected for background and instrument response function (IRF) directly from the data as previously described (Grecco et al. 2009, Opt. Express 17, 6493-6508). Briefly, background was estimated in two ways. First, all frames in the image stack were summed to generate a photon counting image. After removing noise using a 9-pixel median filter implemented in Python (SciPy 1.5.2), the location of the minimum (P) was found. Then, the total background was calculated as the average value around P pixels from the original image using a 21- to 51-pixel window (depending on the image). Background was also estimated from the photon counting histogram. Again, after median noise removal (21-bin window), the background per bin was estimated as the average value of the original histogram around the minimum of the denoised one (51-bin window). This value was then multiplied by the number of bins and extrapolated to the whole image. The consistency of both background values ​​was cross-checked. The 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). Briefly, this is a weighted, translation-invariant Haar wavelet transform denoising algorithm for phasor images that significantly reduces bias and mean squared error.

[0376] The average fluorescence lifetime of the FRET donor mCitrine fused to TC-PTP or R-PTP-γ expressed in MCF7 cells measured increased from 3.04 + / - 0.01 ns at room temperature to 3.71 + / - 0.02 ns (mean + / - standard deviation) under freeze arrest (p < 0.0001 using Student's t-test), but remained close to monoexponential. In measurements with LIFEA2, the clearly resolved non-exponential fluorescence decay profile under freeze arrest showed a mixture of active and inactive conformations with clear mean FRET efficiencies and a fraction of LIFEA2 containing non-absorbing mCherry acceptors. The resulting denoised cumulative phasor plots were therefore analysed by global analysis using a three-component model derived as an extension of a previously described two-component model (Verveer et al. 2000, Science 290, 1567-1570). These three spatially invariant states are (1) a sensor with a fixed fraction of donor alone (sensor with a non-absorbing acceptor; fraction under freeze-stop: 0.2) and fluorescence lifetime (3.71 + / - 0.02 ns), (2) a sensor in a closed (active) conformation with a fluorescence lifetime (0.9 + / - 0.2 ns) and corresponding FRET efficiency (0.75 + / - 0.25), and (3) a sensor in an extended (inactive) conformation with a fluorescence lifetime (3.5 + / - 0.2 ns) and corresponding FRET efficiency (0.05 + / - 0.01), each of which is represented by a phasor. Thus, each observed phasor is the sum of the three phasors weighted by the corresponding normalized number of photons from each state. The complete phasor data of the images was therefore fitted with a linear combination of the three phasors and the corresponding photon fractions 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 of the photon fraction in each state to the fraction of molecules was done by dividing each by the corresponding fluorescence lifetime (a measure of quantum yield) and normalizing the sum to 1. The fraction of active conformations was normalized by dividing by (1 - fraction of donor only), to account for the fraction of sensors containing non-absorbing acceptors. Fluorescence microscopy of cryofixed HeLa cells expressing EGFP EGFP-transfected HeLa cells were prepared for cryomicroscopy as outlined above. Wide-field fluorescence imaging of these EGFP-expressing HeLa cells revealed that It was shown that after cryopreservation with the device of the present invention, EGFP fluorescence and cell morphology were preserved, with no detectable ice crystals that could result in areas of no fluorescence (Figure 17). Fluorescence microscopy of rhodamine 6G solution A solution of rhodamine 6G in cell culture medium with a thickness of 15 μm was prepared and cryopreserved and thawed using the device described herein. Wide-field fluorescence imaging of the fluorescent solution showed that no detectable ice crystals were formed during and after cryofixation. When the solution was thawed, ice crystals formed (Figure 18). Confocal imaging of MCF7 cells expressing TC-PTP-mCitrine To investigate whether complex subcellular organization is preserved after ultrarapid freeze-arrest, the extensive and dynamically maintained endoplasmic reticulum (ER) (Guo et al. 2018, Cell, 175:1430-42.e17) was imaged before and after freeze-arrest by 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-arrest demonstrated that the ER was highly preserved after ultrarapid freeze-fixation.

[0377] The high photostability of the fluorophores at cryogenic temperatures allowed us to acquire 100 widefield fluorescence images of the same arrested cell, enabling super resolution radial fluctuation (SRRF) reconstructions with improved resolution and contrast (Gustafsson et al. 2016, Nature Communications, 7:12471) (Figure 19), demonstrating the preservation of delicate ER organelles after cryofixation. Confocal imaging of MCF7 cells co-expressing Alexa647-SNAP-EGFR and R-PTP-γ-mCitrine To evaluate how ultrarapid freeze-arrest can improve the study of molecular organization in dynamic cellular systems, the spatial distribution of a protooncoprotein and a functionally related candidate tumor suppressor protein were imaged by multimodal fluorescence imaging. For this, MCF7 cells co-expressing fluorescent fusion constructs of the receptor tyrosine kinase epidermal growth factor receptor (EGFR, Alexa647 labeled as Snap-EGFR: Alexa647-SNAP-EGFR) and receptor-like protein tyrosine phosphatase gamma (R-PTP-γ) fused to mCitrine (R-PTP-γ-mCitrine) (Stanoev et al. 2018, Cell Systems, 7:295-309.e11) were mounted on a cryo-microscope according to the present invention. Stimulation with epidermal growth factor (EGF, 100 ng / ml) allowed the vesicular transport dynamics of both proteins to be tracked at room temperature, and subsequent freeze-arrest allowed us to precisely capture the spatial pattern of the interrelationship of both proteins (Figure 20A).

[0378] SRRF reconstruction of 100 widefield fluorescence images of the same arrested cells was also possible (Figure 20A). These reconstructions revealed a non-uniform distribution of EGFR along the lateral side of the plasma membrane (Masip et al. 2016, Nature Methods, 13) were revealed as patches of approximately 400 nm. Dual-color SRRF revealed a high degree of colocalization of R-PTP-γ and Alexa647-SNAP-EGFR in these patches in resting cells. Both proteins also showed colocalization in endosomal structures, but this was lost after 15 min of EGF stimulation, with both proteins already separated at the plasma membrane (Figure 19). This colocalization analysis, obtained at cryogenic temperatures, revealed a nanoscale organization of the EGFR / R-PTP-γ complex that co-migrates within the endosomal system. These results are consistent with the recycling of the EGFR / R-PTP-γ complex, which maintains a 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 separation indicates that the EGFR-EGF dimeric complex can no longer interact with R-PTP-γ, which is then rerouted to late endosomes to generate 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 constitutes a special case among super-resolution techniques, since the fluorescence intensity is directly related to the density of the fluorescent marker, resulting in a quantitative molecular pattern. In this laser-scanning nanoscopy technique, resolution beyond the diffraction limit is achieved by stimulated emission of excited fluorophores using a second high-intensity donut-shaped laser beam. As a result, high requirements on photostability, fluorescence quantum yield and density of fluorescent labels hinder STED imaging of sparse fluorescently tagged molecules in live cells.

[0380] In fact, low depletion laser power (<42mW / μm 2 ) preserved enough fluorescent marker to acquire noisy STED images of Alexa647-Snap-EGFR in MCF7 cells at room temperature (see Figures 21A and 21B), with no statistically significant increase in information content above the diffraction limit over regular CLSM (Figures 21A and 21C). Resolution could be improved slightly by reducing motion blur through chemical fixation of the cells, allowing the accumulation of photons from five consecutive STED frames before bleaching the sample (Figure 21C). However, after ultrarapid freeze arrest of live cells, the bleaching rate decreased by about 45-fold (Figure 21b), which was consistent with the depletion laser power (420 mW / μm 2 ) and exposure time (radiation energy density: 35 mJ / μm 2 ) by 10-fold, resulting in sharp resolution of endosomal structures containing Alexa647-Snap-EGFR (Figure 21D). The amount of information increased significantly from a scale of <125 nm to a digital (pixel-limited) resolution of 80 nm (Figure 21C). At this resolution, no ice crystals were detected as areas without fluorescence in the cytoplasm of these cells (Figure 26). This was also the case when the structure and arrangement of endocytic structures were preserved after repeated STED scans at maximum depletion laser intensity. This indicates that the high-intensity STED illumination did not warm the sample enough to generate ice crystals.

[0381] In this experiment, we confirmed that cryofixation significantly prevented motion blur and bleaching, thereby enabling 3D scanning of whole cells by confocal microscopy and subsequent recording of STED images of different regions at different z levels (Figure ​(Figure2D).2D). The total acquisition time was over 15 min.

[0382] As a result, cryofixation significantly improves the signal and facilitates STED microscopy of multiple subareas within a larger volume. Confocal FRET-FLIM microscopy of Cos7 cells expressing LIFEA2 Cos7 cells expressing LIFEA2 were obtained and prepared for cryomicroscopy as outlined above. FRET-FLIM studies were performed to examine the activity state distribution of the tyrosine kinase EphA2 after cryofixation using the device of the present invention.

[0383] At room temperature, receptor activation by ligand-clustered ephrinA1 could be observed at the plasma membrane of Cos7 cells by a small (~0.2 ns) decrease in the mean fluorescence lifetime of the FRET donor mCitrine in LIFEA2 (Figure 22). However, motion blur and poor separation of the fluorescence decay profile and derived phasors severely limited the spatial and fluorescence lifetime resolution of membrane structures within the cells (Figure 22). However, at cryogenic temperatures, m The increased quantum yield of Citrine significantly improved the separation of LIFEA2 decay profiles, mean fluorescence lifetimes (τ), and associated phasors in stimulated and unstimulated cells (Figure 22). Clustered receptor activity could be resolved in plasma membrane patches and endocytic structures (Figure 22), and basal activity in the absence of exogenous stimulation was likely driven by endogenous ephrins in neighboring cells. This contrast enhancement in molecular activity imaging was particularly evident when comparing the same ephrinA1-stimulated cells before and during freeze arrest (Figure 22).

[0384] Under freeze arrest, the large separation of the cumulative phasors (Figure 24C) allows the derivation of spatially invariant fluorescence lifetimes of active (τ = 0.9 ± 0.2 ns) and inactive (τ = 3.5 ± 0.1 ns) structures by global analysis, as well as the fraction of LIFEA2 containing non-absorbed mCherry acceptors (~0.2). Using these constraints, the spatially varying molecular fraction of active LIFEA2 (α) can be derived from the phasors within each voxel of the image. Thereby, freeze arrest allowed the acquisition (times > 10 min) and reconstruction of 3D molecular activity maps of LIFEA2 in stimulated Cos7 cells (Figure 25), allowing the distinction of individual vesicles with activated receptors emerging from the plasma membrane from gradually inactivated receptors in perinuclear endosomal compartments.

[0385] Thus, under cryofixation using our device, the spatial distribution of fluorescence lifetime can be measured with greatly improved resolution, allowing unprecedented mapping of the conformational state of the receptor relative to many individual endosomes and endomembrane structures within cells (Figure 25).

Claims

1. A method for reducing the temperature of a sample (1) for inverted microscopy, comprising the steps of: (a) providing a sample (1) on the underside (12) of a carrier substrate (10) at atmospheric pressure; (b) providing liquid nitrogen having a temperature below a critical temperature and helium having a pressure of at least 2.5 MPa in a pressure tank (400); (c) supplying the liquid nitrogen under pressure from the pressure tank (400) to the top side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) so that the liquid nitrogen comes into contact with the top side (11) of the carrier substrate (10) and reduces the temperature of the sample (1); The method includes:

2. 10. The method of claim 1 further comprising: (d) tempering the sample (1) on the underside (12) of the carrier substrate (10) by passing a further liquid over the upper side (11) of the carrier substrate (10); The method includes:

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

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

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

6. An apparatus (100) for reducing the temperature of a sample (1), comprising: (a) a carrier substrate (10) having a lower side (12) for receiving a sample (1) and an upper side (11) exposed for the supply of a liquid (2); (b) a pressure tank (400) configured to hold a fluid and having an outlet valve (22) at its bottom; (c) a supply line (20) connecting the outlet valve (22) to the top side (11) of the carrier substrate (10) and configured to pass the liquid (2) at a pressure higher than atmospheric pressure; (d) an expansion chamber (30) surrounding the top side (11) of the carrier substrate (10); An apparatus (100) in which the pressure tank (400) is mounted on the top side (11) of the carrier substrate (10) in order to keep the supply lines (20) as short as possible.

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

8. 7. The apparatus (100) of claim 6, wherein the temperature of the sample (1) is reduced so as to prevent ice crystals from forming in the sample (1) or to prevent ice crystals having a diameter of more than 10 nm from forming in the sample (1).

9. 7. The apparatus (100) of claim 6, wherein the outlet valve (22) is a switchable valve, a quick-open switchable valve, an electrically or pneumatically switchable valve, or a switchable piston valve.

10. 7. The apparatus of 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) of claim 6, wherein the carrier substrate (10) is diamond.

12. 7. Apparatus (100) according to claim 6, 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.

13. The expansion chamber (30) has a dimension of 50 cm ３ From 100cm ３ The apparatus (100) of claim 6, wherein the range is

14. 7. 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 is funnel-shaped at the outlet opening (21).

15. 1. A method of fluorescence microscopy comprising: (a) providing a sample (1) comprising fluorescent molecules on the underside (12) of a carrier substrate (10) at atmospheric pressure; (b) providing liquid nitrogen having a temperature below the critical temperature and helium having a pressure of at least 2.5 MPa in a pressure tank (400); (c) supplying the liquid nitrogen under pressure from the pressure tank (400) to the top side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) so that the liquid nitrogen comes into contact with the top side (11) of the carrier substrate (10) and reduces the temperature of the sample (1); (d) irradiating the sample (1); (e) detecting fluorescence produced by the sample (1); The method includes:

16. 1. A method for performing optical microscopy by stimulated emission suppression on a freeze-stopped sample (1), comprising the steps of: (a) providing a sample (1) comprising fluorescent molecules on the underside (12) of a carrier substrate (10) at atmospheric pressure; (b) providing liquid nitrogen having a temperature below a critical temperature and helium having a pressure of at least 2.5 MPa into a pressure tank (400); (c) supplying the liquid nitrogen under pressure from the pressure tank (400) to the top side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and bringing the liquid nitrogen into contact with the top side (11) of the carrier substrate (10) and reducing the temperature of the sample (1); (d1) irradiating the sample (1) with a first pulsed laser beam focused at at least one focal region to excite the fluorescent molecules in the sample (1) to emit fluorescence; (d2) irradiating the sample (1) with a second torus-shaped laser beam having an intensity zero point within the at least one focal region to de-excite the fluorescent molecules in the sample (1); (e) detecting the fluorescence produced by the sample (1); and (f) optionally repeating steps (d1), (d2) and (e) at different positions of the focal region of the first pulsed laser beam and different positions of the intensity zero point of the second torus-shaped laser beam; The method includes:

17. 1. A method for performing time-correlated single-photon counting fluorescence lifetime imaging microscopy of a freeze-stopped sample (1), comprising the steps of: (a) providing a sample (1) comprising fluorescent molecules on the underside (12) of a carrier substrate (10) at atmospheric pressure; (b) providing liquid nitrogen having a temperature below a critical temperature and helium having a pressure of at least 2.5 MPa into a pressure tank (400); (c) supplying the liquid nitrogen under pressure from the pressure tank (400) to the top side (11) of the carrier substrate (10) by opening an outlet valve (22) located at the bottom of the pressure tank (400) and bringing the liquid nitrogen into contact with the top side (11) of the carrier substrate (10) and reducing the temperature of the sample (1); (d) periodically exciting the fluorescent molecules to emit fluorescent photons by irradiating the sample with a pulsed laser beam; (e) detecting the fluorescence photons produced by the sample (1) together with the arrival time of the photons relative to the laser pulse (1); (f) determining a fluorescence lifetime from the detected fluorescence photons and the arrival times of the photons; The method includes:

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

19. The apparatus (100) of claim 11, wherein the diamond is CVD diamond.

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