Serum albumin and immunoglobulin G (IgG) were clearly detected in blood vessel s but not in parenchyma of living mouse cerebellum under normal conditions [5]. The serum protein s were detected also in the brain parenchyma around the blood vessels when the specimens were quickly frozen after resection. The difference of serum protein immunolocalization was attributed to their rapid leakage across the blood–brain barrier caused by anoxia /ischemia . In addition, the immunoreactivity of serum proteins was drastically reduced with transcardial perfusion fixation followed by alcohol dehydration , indicating that the serum proteins were washed out during perfusion [5]. Later studies also demonstrated that the in situ localization of serum proteins and soluble molecules, including glycogen , was significantly affected by conventional fixation methods but well maintained with IVCT [19–22]. The previous studies suggest that preservation of soluble molecules in living animals is one of the important benefits of IVCT under the light microscopic observation [23].

The soluble component s are easily translocated or lost during tissue preparation procedures of chemical fixation methods. Thus, freeze substitution was considered more effective to preserve localization of such soluble molecules [24, 25]. It is possible that the freeze substitution completed by the organic solvent s at low temperature s decreases diffusion and loss of the soluble molecules in cells and tissues [26]. The soluble components are cross-linked by fixatives in the organic solvents to avoid translocation. This cross-linkage may result in enhanced immunoreactivity, compared with chemical fixation methods [27]. Loosening of intermolecular and intramolecular cross-linkage may also facilitate access of antibodies to the epitopes and enhances immunoreactivity following quick-freezing method s [28, 29]. IVCT would be the most suitable approach in case ischemic/anoxic stress can produce serious effects in morphology as well as molecular localization in cells and tissues of living animals.

Since the time duration required for complete fixation which would include time for penetration and chemical reaction for cross-linkage [30] is unclear in chemical fixation methods, rapid changes of morphology and molecular distribution are generally difficult to capture. On the other hand, immediate immobilization of the target molecules at the moment of freezing is possible by cryofixation . A study which effectively showed this property of cryofixation was related to the rapid phosphorylation of rhodopsin [31]. The study demonstrated that 30 s exposure of mouse retina to light after dark adaptation, but not 10 s exposure, dramatically increased the immunoreactivity of rhodopsin phosphorylated at ³³⁴Ser. Since some molecular components can be immunohistochemically detected with quick freezing and freeze substitution better compared with other conventional methods, IVCT would be a useful approach in analyses of dynamic and rapid molecular change s in sections of living animal organ s .

In conventional perfusion fixation methods, perfusion pressure needs to be regulated for morphofunctional studies since the pressures of perfusion can affect the morphology of living animal organ s [30, 32]. In resected tissues, the blood circulation is essentially stopped and most blood vessel s collapse before completion of fixation [23]. With IVCT, functioning organs of living animals can be directly cryofixed without perfusion or tissue resection to capture dynamic changes under different hemodynamic condition s [12, 33]. This merit of IVCT was clearly shown in kidneys and livers at the light microscopic level [19–21]. Molecular markers facilitated histological analyses of blood or lymphatic vessels under physiological and pathological conditions [34–36]. Extrinsic molecules such as fluorescent dye s are useful to visualize the functional morphology and permeability of blood vessels, and IVCT followed by freeze substitution would well maintain those soluble molecular markers [37–40]. IVCT would also be useful to visualize alterations of intramembranous components in flowing erythrocyte s [41]. In conclusion, one of technical advantages of IVCT which is distinct from conventional tissue preparation methods is the rapid preservation of morphology and molecular conformation.

IVCT immobilizes every component in vitreous ice crystal s and is useful to capture quickly changing tissue and cellular morphology under normal blood circulation . IVCT is not a method to examine the time-lapse changes in single target organs of living animals. However, different functional or structural molecules on serial or same sections can be simultaneously analyzed. In addition, these molecules can be observed in the context of the histological structures. Therefore, combination of IVCT and other methods, such as intravital imaging , freeze-fracture replication, or X-ray microanalyses for electron microscopy , will be effective to obtain novel information about living animal organ s [41, 46]. One of the examples is the combination of IVCT and Raman microscopy , which showed the different molecular organization of the mouse retina [47]. Various morphofunctional information can be obtained from the iced blocks depending on the procedures employed after IVCT.

It is challenging to use IVCT in larger animals including humans, since the liquid IP cryogen poured over the target organs also touches adjacent tissues and causes severe freezing damage. To solve these problems, a new cryotechnique , named “cryobiopsy ,” was recently developed by utilizing a home-made device, cryoforceps [17]. Cryobiopsy did not kill the host mice but revealed open sinusoids and flowing erythrocyte s , as observed with IVCT [17, 48]. There were tissue areas with compression deformation around the tissue edges of the cryoforceps, but the areas adjacent to such compressed tissues reveals native morphology [17, 39]. Cryobiopsy can be used to obtain specimens from larger animals including humans with minimal ischemia /anoxia and would contribute to new pathological analyses in clinical medicine .

IVCT has been widely used in studies dealing with various organs of living animals, both at the electron and light microscopic levels. IVCT should be applied to not only standard microscopic analyses but also new analytical fields which involve animal experimental models as well as materials in clinical medicine . New devices, such as gastric endoscopes with cryoforceps and cooling system, are needed to efficiently apply the cryobiopsy system to the human biopsy.