JP7223597B2 - Methods for identifying cell division - Google Patents

Description

The present disclosure relates to a method for identifying cell division and a method and system for measuring proliferation activity of a cell population using the method.

In recent years, attempts have been made to transplant various cells in order to repair damaged tissues and the like. For example, attempts have been made to utilize fetal myocardial cells, skeletal myoblasts, mesenchymal stem cells, cardiac stem cells, ES cells, etc. for the repair of myocardial tissue damaged by ischemic heart diseases such as angina pectoris and myocardial infarction. (Non-Patent Document 1).

As described above, the establishment of a new treatment method based on regenerative medicine has increased opportunities to utilize various cells.
Conventionally, the growth activity of adherent cells is measured by counting the number of cultured cells over time and creating a growth curve, or by measuring the number of cells in a cell population and the percentage of each cell cycle using flow cytometry, etc. and estimated and evaluated based on these results (Non-Patent Document 2). Techniques have also been developed for understanding the proliferative activity and state of a target cell population based on the brightness, morphology, particle size, migration speed, cell count, and localization pattern of cells in a culture vessel, etc., using image analysis. (Non-Patent Document 3, Patent Documents 1 to 6).

Japanese Patent Application Laid-Open No. 2002-218995 Japanese Patent Application Laid-Open No. 2017-140006 Japanese Patent Publication No. 2017-023055 Japanese Patent Publication No. 2016-185106 JP 2014-085950 A JP 2014-085949 A

Shimizu et al., Circ Res. 2002 Feb 22;90(3):e40-e48 Remvikos Y1 et al., Br J Cancer. 1991 Sep; 64(3): 501-507. Seungil Huh et al. IEEE Transactions on Medical Imaging. 2011 March: VOL.30:NO.3:586-596

However, since the cell number and cell cycle measurement of actual cultured cells can distinguish between live and dead cells, it is possible to accurately measure proliferative activity. However, since it is subjected to a microscope or a flow cytometer after staining, not only is complicated processing required, but there is also the problem of damaging or destroying cells. On the other hand, when dividing cells are identified by image analysis based on the brightness, morphology, etc. of cell images, dead cells, dust, or impurities become noise, resulting in a lack of accuracy.

The present inventors focused on the fact that adherent cells enhance the intensity of reflected light when dividing and attenuate the intensity after division, and by using this property as an indicator of cell division of adherent cells, It has been found that cells can be distinguished from dead cells, debris or impurities. In the course of further intensive research, it was found that by using such an index, cell division of adherent cells can be accurately identified even under high-density conditions. It enabled the measurement and led to the completion of the present invention.

That is, the present invention relates to:
[1] A method for identifying cell division of adherent cells by measuring the enhancement of reflected light intensity of adherent cells and the attenuation to the intensity before enhancement.
[2] The method according to [1], wherein the attenuation of the reflected light intensity from the enhanced intensity to the pre-enhanced intensity is 20 minutes or more and 2 hours or less.
[3] The method according to [1] or [2], which is caused by morphological changes in adherent cells.
[4] The method according to any one of [1] to [3], wherein the measurement is performed using time-lapse images.
[5] A method for measuring the proliferation activity of an adherent cell population, comprising:
The method according to claim 1, comprising counting spots whose reflected light intensity has increased in unit time and has attenuated to the pre-enhancement intensity.
[6] Further, the step of counting the spots where the reflected light intensity is enhanced during the unit time and the enhanced reflected light intensity is maintained, and
Including the step of subtracting the number of spots where the counted reflected light intensity increased and the increased reflected light intensity was maintained from the number of spots where the counted reflected light intensity increased and attenuated to the intensity before the increase, [5 ] method.
[7] The method according to [5] or [6], wherein the attenuation of the reflected light intensity from the enhanced intensity to the pre-enhanced intensity is 20 minutes or more and 2 hours or less.
[8] The method according to any one of [5] to [7], for evaluating proliferative activity in a 90% or more confluent adherent cell population.
[9] The method according to any one of [5] to [8], which is caused by morphological changes in adherent cells.
[10] The method according to any one of [5] to [9], wherein the measurement is performed using time-lapse images.
[11] A system for measuring the proliferative activity of an adherent cell population, comprising:
A storage unit for storing a culture vessel for an adherent cell population, a measurement unit for measuring changes in the intensity of reflected light from the adherent cells, a storage unit for storing the measurement results, and analysis of the results obtained by the measurement unit. and an analysis unit that calculates the growth activity of the adherent cell population in the culture vessel,
The above system, wherein the analysis comprises counting spots that have increased in reflected light intensity and have decayed to the pre-enhancement intensity during a unit time.
[12] The analysis further comprises counting spots in which the reflected light intensity has increased and the increased reflected light intensity has been maintained during the unit time;
subtracting the number of spots where the counted reflected light intensity increased and the increased reflected light intensity was maintained from the number of spots where the counted reflected light intensity increased and attenuated to the intensity before the increase, [11 ] system.
[13] The system according to [11] or [12], wherein the analysis unit further includes means for detecting cell candidate regions of individual adherent cells.
[14] The system according to any one of [11] to [13], wherein the housing includes a light source.
[15] The system according to any one of [11] to [14], wherein the measuring section includes an imaging section.
[16] The system according to any one of [11] to [15], wherein the imaging unit is capable of acquiring time-lapse images.

Dead cells, dust, or impurities mixed in cultured cells exhibit high reflected light intensity in the same manner as cell division. and changes in reflected light intensity resulting from impurities in the culture vessel, which enables accurate identification of cell division and measurement of proliferation activity of cell populations. Therefore, in research and medical settings, it is possible to accurately measure proliferative activity, etc. without the need for complicated processing, and it is easy to select the appropriate culture time according to the cell population, and it can be applied to appropriate treatment. becomes. Since cell division or mitotic phase can be accurately measured even under high-density conditions where relatively many dead cells are present, there is no need for excessive culturing, and deterioration of cell quality due to excessive culturing can be prevented. Furthermore, the method of the present invention enables non-invasive measurement of cell division or mitotic phase with high accuracy. can be provided to

FIG. 1 is a diagram of adherent culture of skeletal myoblasts observed over time with a phase-contrast microscope. It shows that the reflected light intensity increases during cell division (3) and that the intensity attenuates after division (4, 5). In the figure, the dashed line indicates before division, the wavy line indicates during division, and the straight line indicates after division. FIG. 2 is a diagram of about 30% confluent skeletal myoblasts observed over time with a phase-contrast microscope. It shows that the reflected light intensity increases when individual adherent cells undergo cell division, and the intensity attenuates after division. In the figure, the dashed line indicates before division, the wavy line indicates during division, and the straight line indicates after division. FIG. 3 is a diagram of about 90% confluent skeletal myoblasts observed over time with a phase-contrast microscope. FIG. 4 shows that reflected light intensity increases during cell division even under highly confluent conditions, and cell division can be identified. The wavy lines in the figure indicate dividing cells.

The present invention will be described in detail below.
Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications and other publications and information referenced herein are hereby incorporated by reference in their entirety. In addition, if there is any discrepancy between the publications referred to in this specification and the description in this specification, the description in this specification shall take precedence.

The present invention relates to a method for identifying cell division of adherent cells by measuring reflected light intensity of adherent cells.
The inventors found that the reflected light intensity of adherent cells changed during mitosis. Observation of adherent cells under constant incident light irradiation revealed that the intensity of reflected light from adherent cells increased toward the mitotic phase, reached a peak at the time of mitosis, and then decreased after mitosis. confirmed.

Adherent cells that extend in the planar direction on the culture substrate gradually change into a rounded three-dimensional shape toward cell division (division phase). Such morphological changes cause the adherent cells to have a vertical length that results in reflection of light along the contours of the top surface of the adherent cells, further enhancing such reflected light as it becomes more rounded. . As described above, the adherent cells assume a rounded three-dimensional shape during cell division, and such a change in shape can be interpreted as an increase in the intensity of reflected light. In addition, daughter adherent cells generated by cell division return to the form before cell division by extending in the plane direction, and the reflected light enhanced by this is attenuated to the intensity before cell division.

On the other hand, when the adherent cells die and are detached from the culture substrate, they float in the culture medium in a round shape, and thus the dead cells also enhance reflected light in the same way as cell division. Also, not only dead cells but also dust or impurities floating in the culture solution newly enter the observation field of view, which also enhances the reflected light in the same manner as cell division. However, dividing cells, unlike dead adherent cells or debris or impurities, decay as daughter cells develop after the reflected light enhancement as described above. Therefore, it is possible to identify the target that has been attenuated after the reflected light is enhanced as the reflected light derived from cell division. It is possible to distinguish from reflected light changes due to impurities in the .

In the present invention, "adherent cells" or "adherent cell population" refer to cells or cell populations that can be adherently cultured to a culture substrate by seeding in a culture vessel and incubating. Although not limited to these, somatic cells (e.g., cardiomyocytes, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, renal cells, adrenal cells, periodontal ligament cells, gingival cells, periosteal cells, skin cells, synovial cells, chondrocytes, blood cells, etc.), somatic stem cells (e.g., cardiac stem cells, mesenchymal stem cells, etc.), pluripotent stem cells, (embryonic stem cells, iPS (induced pluripotent stem) cells, etc.) and blast cells (fibroblasts, skeletal myoblasts, osteoblasts, etc.).

The culture vessel is not particularly limited as long as it allows adherent cells to proliferate, and those commonly used in the art can be used. Base materials for such base materials include, but are not limited to, polyethylene, polypropylene, Teflon (registered trademark), polyethylene terephthalate, polymethyl methacrylate, nylon 6,6, polyvinyl alcohol, cellulose, silicon, polystyrene. , glass, polyacrylamide, polydimethylacrylamide, metals (eg, iron, stainless steel, aluminum, copper, brass) and the like. The culture substrate may include the surface of a cell culture scaffold as well as one surface of a culture vessel (for example, the bottom surface of the vessel).

The surface of the culture substrate may be subjected to processing that is advantageous in the production of sheet-like cell cultures, including processing for enhancing cell adhesiveness, processing for facilitating detachment, and the like. Examples of such processing include, but are not limited to, corona discharge treatment, ultraviolet irradiation treatment, coating with hydrophilic compounds such as collagen gel and hydrophilic polymers, collagen, fibronectin, laminin, vitronectin, proteoglycans, and glycans. Extracellular matrices such as saminoglycan, coating with cell adhesion factors such as cadherin family, selectin family, integrin family, etc., and coating with materials whose physical properties change in response to stimuli such as temperature and light can be mentioned.

In the present invention, "enhancement of reflected light intensity" refers to an increase in reflected light intensity relative to the initial value in the measurement of reflected light intensity. An increase with respect to the initial value means that the reflected light intensity increases beyond the set value. For example, it may be 10 or more, 20 or more, 30 or more, or 40 or more with respect to the initial gradation value. Further, the increase with respect to the initial value is, for example, an increase in the reflected light intensity by a specific magnification or more with respect to the initial value, and is not limited thereto, but for example, at least 1.2 times with respect to the initial value. or more, at least 1.5 times or more, or at least 1.8 times or more.

In the present invention, "attenuation to the intensity before enhancement" means that the reflected light intensity attenuates from the peak toward the intensity before enhancement. The degree of attenuation does not have to be exactly the same as the intensity of the reflected light before enhancement. can be
In the present invention, "the enhanced reflected light intensity is maintained" means that the enhanced reflected light intensity hardly changes from the peak, or the degree of attenuation from the peak is low. A low degree of attenuation may be, for example, that the intensity of reflected light is not attenuated below a predetermined value defined as "increase of reflected light intensity" or that it is not attenuated below a specific magnification.

In the present invention, the method for measuring the intensity of reflected light during culture is not limited as long as it is possible to measure the change in the reflected light of the adherent cells. Any device that can be observed or measured can be used. In the present invention, "adherent cells" include not only adherent cells in the literal sense, but also adherent cell images that can be regarded as equivalent, and therefore any form of data that can acquire the reflected light intensity of adherent cells. and may vary depending on the device. Therefore, the reflected light intensity of the adherent cells may be, but not limited to, image data or numerical data. For example, when the measuring device is a time-lapse imaging device, the reflected light is acquired as time-lapse image data. Numerical data such as the number of spots with increased intensity and attenuated will be obtained.

The method of the invention can be measured at any cell density as long as it is possible to measure changes in reflected light intensity of adherent cells. Although not limited to this, the cell density is 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, It may be 95% or more, 98% or more confluent. A confluency of 80% or more is preferable from the viewpoint of accurate measurement even if the morphology and outline of cells cannot be observed sufficiently, and a confluency of 90% or more is more preferable from the viewpoint that dead cells may occur.

In one aspect of the invention, the dead cells generated can be degraded over time. This can also be seen as the enhancement and attenuation of reflected light, and from the standpoint of clearly distinguishing the cell division of adherent cells from the generation and decomposition of dead cells, "the intensity of reflected light increases and reaches the intensity before enhancement. The "attenuated" time is, for example, 20 minutes to 2 hours, 30 minutes to 1 hour and 30 minutes, preferably 30 minutes to 1 hour from the viewpoint of providing more accuracy.

Another aspect of the invention includes a method of measuring the proliferative activity of a cell population using the method described above. Such methods include the above-described method, comprising the step of counting spots whose reflected light intensity increases during a unit time and then attenuates to the pre-enhancement intensity. The “proliferation activity” of a cell population means the number of adherent cells proliferating (increasing) per unit time. Specifically, although not limited to this, for example, the number of cell divisions per unit time of a cell population corresponds to the number of cells increased per unit time, so that the reflected light intensity increases during the unit time. Proliferative activity can be measured by counting cells that have potentiated and then decayed to pre-potentiation intensity. The adherent cells in the cell population do not necessarily have to be recognized as distinct cells or cell images, as long as the intensity of the reflected light is enhanced and the spots are attenuated to the intensity before the enhancement.

Cell proliferation is affected by various conditions, such as the number of seeded cells (seeded cell density), culture environment, and medium composition. Proliferative activity between samples can be compared by comparing the number of cell divisions per unit time in the samples. For example, the number of cell divisions per unit time in one field of view of a phase-contrast microscope is counted for spots where the reflected light intensity is enhanced and attenuated to the intensity before enhancement, and by applying this value to the entire culture vessel, proliferation Not only the activity but also the total number of cells in the culture vessel can be estimated. Furthermore, it is possible to predict the time required for the cell population in the culture vessel to become confluent based on the predicted total number of cells in the culture vessel and proliferative activity. A person skilled in the art can calculate information on culture required in each technical field from the obtained cell proliferation rate using a known method.

One aspect of the invention includes a method of measuring the net proliferative activity of a cell population. The net proliferative activity in the adherent cell population is the number of dividing cells minus the number of dead cells per unit time, where the number of dividing cells is measured as enhanced and attenuated spots of reflected light intensity, and the number of dead cells Since the number is measured as spots where the reflected light intensity is maintained after enhancement, the number of spots where the reflected light intensity is enhanced and attenuated to the pre-enhancement intensity, the reflected light intensity is enhanced, and the enhanced reflected light intensity is maintained Net proliferative activity can be measured by subtracting the number of spots obtained.
In this aspect, when a sheet-shaped cell culture is prepared using cells collected from a patient, the properties of the cells differ greatly depending on each patient, and the amount of dead cells generated varies depending on the number of times of culture. It is advantageous to measure the proliferative activity of

The method of the invention can be measured at any cell density as long as it is possible to measure changes in reflected light intensity of adherent cells. Although not limited to this, the cell density is 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, It may be 95% or more, 98% or more confluent. In particular, a confluency of 90% or more is preferable from the viewpoint that many dead cells begin to occur.

In one aspect of the present invention, the time "the reflected light intensity is enhanced and attenuated to the intensity before enhancement" is, for example, 20 minutes or more and 2 hours or less, 30 minutes or more and 1 hour and 30 minutes or less, giving more accuracy It is preferable that the time is 30 minutes or more and 1 hour or less.

In one aspect of the present invention, the time period for which the "enhanced reflected light intensity is maintained" is not particularly limited as long as it is a time period during which dead cells can be identified, for example, 2 hours or more, 3 hours or more, 4 hours or more, 5 Time or more, preferably 6 hours or more from the viewpoint of clearly distinguishing from spots related to cell division, and 12 hours or less, 18 hours or less, or 24 hours or less from the viewpoint of distinguishing from dust or impurities. Within 30 hours is preferable.

Another aspect of the present invention includes a system for measuring proliferative activity of a cell population using the method described above. Such a system includes a storage unit for storing a culture vessel for an adherent cell population, a measurement unit for measuring changes in the intensity of reflected light from the adherent cells, a storage unit for storing the measurement results, and the measurement unit. Including an analysis unit that analyzes the obtained results and calculates the growth activity of the adherent cell population in the culture vessel, and the analysis counts the spots where the reflected light intensity has increased during a unit time and has attenuated to the intensity before the increase. The system is included, including the steps.

In one aspect of the system of the invention, it includes a system that measures the net proliferative activity of a cell population. In addition to the system of the present invention, the system includes a step of counting spots where the reflected light intensity increases and the increased reflected light intensity is maintained during the unit time, and the counted reflected light intensity is The system further comprising the step of subtracting the counted number of spots where the reflected light intensity has increased and the increased reflected light intensity has been maintained from the number of spots that have increased and attenuated to the pre-enhancement intensity.

The system of the present invention includes a container that houses the culture vessel for the adherent cell population. The enclosure can be equipped with any light source for observing changes in the optical properties of adherent cells. The light source can be arbitrarily selected from, for example, visible light, infrared light, or ultraviolet light, depending on the desired optical properties. Visible light is preferable from the viewpoint of versatility and non-invasiveness to samples. .

The system of the present invention includes a measuring portion for measuring changes in optical properties of individual adherent cells in the cell population. Such a measuring unit may be any one that includes a measuring device capable of obtaining changes in the optical properties of adherent cells. In terms of data versatility, processing versatility, and sample non-invasiveness, the measurement unit includes an imaging unit, and changes in the reflected light intensity of adherent cells are acquired as time-lapse images. preferably.

The analysis performed in the analysis unit may vary depending on the format of the obtained adherent cell data, and is not limited to this. It may be drawn, for example, the total number of cells may be estimated based on this, and the cell candidate regions are detected for individual adherent cells in one field of view, and the resulting division Viability may be estimated by overlaying with cell images.

In one aspect of the present invention, the time "the enhanced reflected light intensity is maintained" is
The time is not particularly limited as long as dead cells can be identified, and is, for example, 2 hours or longer, 3 hours or longer, 4 hours or longer, 5 hours or longer, and preferably 6 hours or longer from the viewpoint of clearly distinguishing from spots related to cell division. , within 12 hours, within 18 hours, and within 24 hours from the viewpoint of distinguishing from dust and impurities, and preferably within 30 hours from the viewpoint of clear distinction.

Cell density is 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more confluent It can be. In particular, it is advantageous at 90% or more where many dead cells begin to occur.

The present invention will be described in more detail below with reference to specific embodiments of the present invention, but the present invention is not limited to these specific examples.

Example 1. Identification of cell division in single cells using reflected light intensity Skeletal muscle suspended in DMEM/F12 medium containing 20% human serum (Thermo Fisher Scientific Inc.) in a culture dish (175 cm ² flask, Thermo Fisher Scientific Inc.) Blast cells and 3.5×10 ⁴ cells/flask were seeded and cultured at 37° C. under 5% CO ² for 5 days. After adhering to the culture dish, time-lapse images of the same cells were acquired with a phase-contrast microscope. Time-lapse images of the same cells showed that the reflected light from the adherent cells increased toward the mitotic phase, the intensity of the reflected light peaked during division, and the reflected light attenuated after cell division. It took about 40 minutes from enhancement to attenuation of the reflected light. The results are shown in FIG.

Example 2. Identification of dividing cells in a cell population using reflected light intensity. Under the same conditions as Example 1, the cells were cultured for 5 days to reach 30% confluency, and a time-lapse image of the same cell population was obtained with a phase-contrast microscope. Time-lapse images of each cell in the cell population showed that the reflected light from the adherent cells increased toward the mitotic phase, the intensity of the reflected light peaked during division, and the reflected light attenuated after cell division. It took 20 minutes to 2 hours from the enhancement to the attenuation of the reflected light in each cell, and the mode was about 40 minutes. In addition, no cell division was observed without an increase in reflected light. Therefore, by using the enhancement and attenuation of the reflected light as an index, it is possible to identify cells in the cell division or mitotic phase in the cell population, and by counting the spots showing the enhancement and attenuation of the reflected light, the cell population can be identified. It was found that the proliferative activity could be measured. The results are shown in FIG.

Example 3. Identification of dividing cells in a confluent cell population using reflected light intensity Under the same conditions as Example 2, the cells were cultured for 10 days to become 90% confluent, and a time-lapse image of the same cell population was obtained with a phase-contrast microscope. Even in a confluent state in which the cell morphology was unclear, the intensity of the reflected light reached a peak at the time of cell division, and the reflected light attenuated after cell division, as in Example 2. It took 20 minutes to 2 hours from the enhancement to the attenuation of the reflected light in each cell, and the mode was about 40 minutes. In addition, it was considered that there were no cells undergoing division without an increase in reflected light. Therefore, in a 90% confluent state, by using the enhancement and attenuation of reflected light as an index, it is possible to identify cells in the cell division or mitotic phase in the cell population, and the spots showing enhancement and attenuation of the reflected light can be identified. It was thought that the proliferative activity of the cell population could be measured by counting. The results are shown in FIG.

Claims (14)

A method for identifying cell division of adherent cells by measuring the enhancement of the reflected light intensity of adherent cells and the decay to the pre-enhancement intensity, wherein the enhanced reflected light intensity was maintained after the enhancement of the reflected light intensity. Distinguishing cells from dividing cells as dead cells, debris or impurities . 2. The method according to claim 1, wherein the attenuation of the reflected light intensity from the enhancement to the intensity before enhancement is 20 minutes or more and 2 hours or less. Due to the morphological change of the adherent cells, the reflected light intensity is enhanced as the adherent cells become rounder, and the adherent cells extend in the planar direction, thereby attenuating the reflected light intensity to the intensity before the enhancement. 3. The method according to 1 or 2. A method according to any one of claims 1 to 3, wherein the measurement is performed using time-lapse images. A method for measuring the proliferative activity of an adherent cell population, comprising:
Including the step of counting spots whose reflected light intensity has increased during a unit time and has attenuated to the intensity before the increase,
Furthermore, the step of increasing the reflected light intensity in a unit time and counting the spots where the increased reflected light intensity is maintained;
subtracting the number of spots where the counted reflected light intensity increased and the increased reflected light intensity was maintained from the number of spots where the counted reflected light intensity increased and attenuated to the intensity before the increase, Method. 6. The method according to claim 5 , wherein the attenuation of the reflected light intensity from the enhancement to the intensity before enhancement is 20 minutes or more and 2 hours or less. 7. A method according to any one of claims 5 or 6 for assessing proliferative activity in an adherent cell population that is 90% or more confluent. Due to the morphological change of the adherent cells, the reflected light intensity is enhanced as the adherent cells become rounder, and the adherent cells extend in the planar direction, thereby attenuating the reflected light intensity to the intensity before the enhancement. 8. The method according to any one of 5-7 . A method according to any one of claims 5 to 8 , wherein the measurement is performed using time-lapse images. A system for measuring proliferative activity of an adherent cell population, comprising:
A storage unit for storing a culture vessel for an adherent cell population, a measurement unit for measuring changes in the intensity of reflected light from the adherent cells, a storage unit for storing the measurement results, and analysis of the results obtained by the measurement unit. and an analysis unit that calculates the growth activity of the adherent cell population in the culture vessel,
The analysis includes a step of counting spots whose reflected light intensity has increased during a unit time and has attenuated to the intensity before the increase, and a step of counting spots whose reflected light intensity has increased during a unit time and has maintained the increased reflected light intensity. counting steps, and
subtracting the number of spots where the counted reflected light intensity increased and the increased reflected light intensity was maintained from the number of spots where the counted reflected light intensity increased and attenuated to the pre-enhancement intensity. . 11. The system of claim 10 , wherein the analysis unit further comprises means for detecting cell candidate regions of individual adherent cells. 12. The system of any one of claims 10 or 11 , wherein the housing comprises a light source. System according to any one of claims 10 to 12 , wherein the measuring unit comprises an imaging unit. The system according to any one of claims 10 to 13 , wherein the imaging unit is capable of acquiring time-lapse images.

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