DE102016111075B4 - Method for determining the height of a fluid level and microscope suitable therefor - Google Patents

Abstract

Method for determining the height of a fluid level of a chamber (7) arranged in a microscope (10) and filled with a fluid (8), wherein an upper reference object (1) floating on the surface of the fluid (8) and one on the Bottom of the fluid-filled chamber (7) located or sunk on this floor lower reference object (2) are introduced into the chamber (7) or are designed as components of the chamber (7),
wherein the chamber (7) is introduced into the object space of the microscope (10) equipped with a lens (6),
wherein successively the focus positions of the upper reference object (1) and the lower reference object (2) are determined with the microscope and the height of the fluid level is calculated from the difference of the determined focal positions.

Description

The present invention relates to a method and a device for determining the height of a fluid level of a arranged in a microscope and filled with a fluid chamber, wherein the chamber is adapted for introduction into a microscope equipped with a lens. In particular, according to the invention corresponding fluid volumes are to be calculated in the chamber filled with the fluid. In this case, the fluid itself represents a sample to be examined with the microscope, or the fluid contains a sample to be examined with the microscope.

From the prior art special measuring chambers or calibration vessels for receiving suspensions, solutions, liquids, etc., hereinafter referred to as "fluid", known in the ml range, on the basis of which an exact determination of the recorded volume is possible Chambers suitably calibrated and / or marked. In principle, such chambers can also be produced in the μl range. To determine the volume of such small volumes, a transfer to a corresponding measuring chamber is necessary, which involves the risk of fluid loss, even by the original vessel through adhesion effects remaining liquid.

Furthermore, three-dimensional measurement, for example height and volume measurement, are known from 3D video microcosm. Such systems require a high calibration, computation and image processing overhead.

So revealed the DE 102014216227 A1 a microscope, in which the lid thickness or bottom thickness of a test sample to be examined from the difference of the boundary layers to be assigned focus positions is determined.

The EP 000001761747 B1 describes a confocal measuring method for determining a liquid volume in a titer chamber, wherein the positions of a reference surface and the phase boundary of the liquid are determined.

In the DE 102007043741 A1 a device for measuring liquid interfaces is indicated by means of a zoom lens in order to determine the liquid volume.

The object of the present invention is therefore to specify a method and a microscope with a device for determining the height of a fluid level, in particular for determining a fluid volume in a chamber filled with a fluid, which overcome the abovementioned disadvantages and in particular independently Calibration vessels can work.

This object is achieved by a method and a microscope according to the independent patent claims. Advantageous embodiments will become apparent from the respective dependent claims and the following description

The inventive method is used to determine the height of a fluid level of a arranged in a microscope and filled with a fluid chamber. It comprises the following steps: Two reference objects are provided, wherein an upper reference object floats on the surface of the fluid, ie floats in the fluid-filled chamber. The upper reference object is thus referred to as "float" for short. A lower reference object is located at the bottom of the fluid filled chamber or has dropped to the bottom of the fluid filled chamber. The second object is therefore also referred to as "anchor" for short.

The upper and / or the lower reference object can already be designed as components of the chamber or be introduced into the chamber. Of course, an introduction into the already filled with the fluid chamber is possible. It should be understood that "float" means a reference object that floats on the surface of the fluid in the chamber, while "anchor" means a reference object located in the fluid-filled chamber on the bottom of that chamber sinks or is stuck there. By "anchor" is not meant that this object is designed for anchoring in the sense of a ship anchor. As explained below, the lower reference object can not be a physically extended object, but a calibration mark located on the chamber floor.

The chamber is introduced into the object space of the microscope equipped with a lens and placed, for example, on a microscope stage of the microscope. Since the present invention is particularly aimed at measuring fluid volumes in the ml and μl ranges, the fluid-filled chambers contemplated herein are typically sufficiently small to be viewed microscopically. In any case, the fluid under consideration is often a sample which is examined microscopically. The fluid may also contain the sample to be tested, for example, living cells in nutrient solution. In the course of this study, a volume determination is often of interest. Further details can be found below.

Finally, the focus positions of the upper reference object, the Float, and the lower reference object, the anchor, determined with the microscope. In this case, a selected interface, for example, the underside of the anchor and the top of the float are anfokussiert. Flat designs of the float and the anchor are therefore advantageous. First, the focus position of the upper reference object and then that of the lower reference object can be determined or vice versa. In order to determine the focus position, microscopes on which fixed focal length objectives are mounted displace either the microscope stage and / or the objective or the entire objective nosepiece with the objectives in the focusing direction (z direction) until the relevant reference object lies in the objective focus. Alternatively, in the case of a variable focal length objective, the latter is changed until the relevant reference object is in focus. The focal positions of the float, ie the upper reference object, and the armature, ie the lower reference object, are detected and the difference in the detected focal positions becomes the height of the Fluid level calculated. For this purpose, the focus adjustment is performed so that the assigned position is known or can be determined for each adjustable focus position and the respective focus position is determined. For this purpose, for example, encoders for detecting the rotational movement of the focus drive or optical or mechanical position measuring devices come into question. With negligible expansion of the upper and lower reference objects in the focus direction, the height of the fluid level results in a simple manner from the amount of the difference of the determined focal positions. With not insignificant expansion of the upper and lower reference object in the focus direction corresponding corrections are necessary, which can make the expert in a simple manner with known dimensions of these dimensions.

Suspensions or aqueous or lipid solutions are often used as fluids. Accordingly, it is advantageous if the upper and the lower reference object are hydrophobic (water repellent) and / or lipophobic (fat repellent). This prevents a change of upper and lower reference object in the fluid medium and the fluid itself.

The present invention may be used to advantage for calculating the volume of fluid in the chamber filled with the fluid, and then calculating the fluid volume from the calculated height of the fluid level with the geometry of the chamber known. If, for example, the geometry of the chamber is a cylinder, the fluid volume results from the known base area of the cylinder (π • r ² , where r = radius of the circle of the base of the cylinder) multiplied by the calculated height of the fluid -level. In the case of a cuboidal chamber, the base area results from the width and depth of the cuboid and the fluid volume correspondingly from the product of the height of the fluid level with this base area. For other geometric shapes (cylinders, pyramids, etc.), analog calculations are readily possible for the skilled person.

It is advantageous if the reference objects used as the upper reference object and as the lower reference object are those which differ in shape and / or color. As a result, the objects can be matched to the lens so that a very accurate, in particular μm-accurate determination of the distance between the float and anchor is possible. This can e.g. be achieved in that the anchor is formed as a rectangle when it lies on the bottom of the chamber and has a minimum height in the micron range, while the float is circular and also in the floating state has a height in the micron range.

Alternatively, an objective with the smallest possible depth of focus is used, which lies in particular below the dimension of the upper reference object in the focus direction. "Dimension" is the average dimension in the z direction of the upper reference object.

It is also advantageous if the upper and / or the lower reference object are or are provided with fluorescent dyes. In this way, the distance between the upper and lower reference object can be determined microscopically by means of a fluorescence microscope. It is particularly useful here if different fluorescent dyes are selected.

By means of the abovementioned embodiments of anchor and float, it is possible, in particular, to automate the method of calculating the distance between the two reference objects by approaching the relevant reference objects in succession. In the case of different shapes of the reference objects, in particular the use of a pattern recognition, with different colors the use of a color recognition and with different fluorescent dyes, in particular the use of a corresponding wavelength detection is advantageous.

Since, as already stated, the lower reference object can be embodied as a reference object located at the bottom of the fluid-filled chamber, it may be advantageous to use a calibration mark located on the chamber floor as the lower reference object. The lower reference object in this case represents less a physically extended object than the aforementioned calibration mark. It only has to be done a float can be used to implement the invention. For standardized chambers, the xy coordinates of the calibration mark (lower reference object) can thus be used in the chamber. For standardization, a calibration mark must be provided on the bottom or on the wall of the chamber for cylindrical volume supports (eg petri dishes) and a corresponding calibration mark on the fixture (the microscope stage) in order to always achieve the same orientation and positioning of the chamber, so that the xy Coordinates of the lower reference object can always be clearly determined. This allows the automatic approach of the lower reference object with the focus of the lens. For chambers with a rectangular base also calibration marks on the chamber bottom and the holding device can be attached. Alternatively, two xy coordinates are deposited which cover the two possibilities of introduction to the fixture for the position of the fixed lower reference object.

In order to avoid markings and alignment coordinates, it is particularly advantageous to fix the lower reference object in the middle or to fix it directly on the surface of the chamber floor, so that the x-y position is always known.

Furthermore, it is advantageous if the upper and the lower reference object are connected to each other or connected via a flexible connection. This can be done for example by means of a microfiber. This ensures that the float and anchor are always within the field of view of the reference lens, so when refocusing from anchor to float, or vice versa, that object and its focus can be found quickly. In particular, a fixed anchor on or in the bottom of the chamber may be connected to the float.

The invention further relates to a microscope with a corresponding device for determining the height of a fluid level of a arranged in the microscope and filled with a fluid chamber comprising a befindliches in the chamber upper reference object, floats in the fluid-filled chamber on the surface of the fluid, and a lower reference object located in the chamber, which is designed as an anchor and is located in the fluid-filled chamber at the bottom of the chamber. Here, the microscope is equipped with a lens and has a microscope stage, which receives the fluid-filled chamber in the object space of the microscope. The microscope comprises a focusing device, which focuses the objective successively on the upper reference object (float) and the lower reference object (armature) and determines the first focus position of the upper reference object and the second focus position of the lower reference object. Furthermore, the microscope comprises a computing unit which calculates the height of the fluid level from the difference between the two corresponding focal positions of the upper reference object and the lower reference object. Further explanations of the microscope according to the invention can be found in the above explanations of the method according to the invention and analogously transferred to the microscope.

The arithmetic unit can be a microprocessor-based arithmetic unit or a part thereof or integrated into it. Since in modern microscopes, the focus position is usually detected electronically anyway, it is usually only necessary to implement the invention to calculate by means of a corresponding software module from the difference of the determined focus positions of armature and float the height of the fluid level, as above in Connected with the inventive method is executed.

Advantageously, the microscope for calculating the fluid volume is formed in the chamber filled with the fluid, for which purpose the arithmetic unit calculates the fluid volume from the calculated height of the fluid level with known geometry of the chamber. Reference should be made to the statements in connection with the method according to the invention in order to avoid repetition.

Advantageously, the upper and lower reference object differs in shape and / or color. Again, reference is made to the above statements in connection with the method according to the invention.

Advantageously, the objective of the microscope has a depth of focus which lies below the dimension of the upper reference object (float) in the focusing direction. Again, reference is made to the statements in connection with the method according to the invention in order to avoid repetition.

Advantageously, the upper and / or the lower reference object each have fluorescent dyes, wherein the microscope is designed as a fluorescence microscope. Further explanations of this embodiment can be found in the above statements on the corresponding embodiment of the method according to the invention.

Advantageously, the chamber has a calibration mark on the bottom of the chamber as the upper reference object. The lower reference object in this case is not designed as a physically extended object. Also to this is on the above Referred to in the context of the method according to the invention.

Finally, it is advantageous if the upper reference object and the lower reference object are connected to one another via a flexible connection. For this purpose, the flexible connection can either lead from the calibration mark on the bottom of the chamber to the upper reference object, ie the float, or else connect the anchor to the float as a physically extended object. Also with regard to further explanations of this embodiment, reference is made to the above statements in connection with the method according to the invention.

As applications of the invention are the calculations of evaporations, for example, when a fluid-filled Petri dish is placed as a chamber in an incubator. By determining the volume with the aid of the invention before and after the incubation, the evaporated volume can be determined. In addition, a volume consumption can be determined by absorption of fluid by microorganisms. Furthermore, it is possible to directly determine a vaporized volume after laser applications. Of course, the invention presented here is also suitable for measuring smaller volumes of fluid independently of any microscopic examination or treatment of the sample, that is to say of the fluid. The invention is particularly suitable for the determination of volumes in the ml or μl range, however, larger volumes are to be determined as far as the size of the chamber allows a determination according to the invention of the height of the fluid level by means of a microscope.

Further advantages and embodiments of the invention will become apparent from the description and the accompanying drawings.

It is understood that the features mentioned above and those yet to be explained below can be used not only in the particular combination given, but also in other combinations or in isolation, without departing from the scope of the present invention.

The invention is illustrated schematically with reference to an embodiment in the drawing and will be described in detail below with reference to the drawing.

list of figures

• 1 schematically shows the upper and the lower reference object, which are connected via a connection,
• 2 shows schematically an embodiment of a microscope according to the invention,
• 3 very schematically shows the essential components of the device for determining the height of a fluid level in a cuboid chamber in an upright microscope,
• 4 shows very schematically the essential components of a device for determining the height of a fluid level in a cylindrical chamber in an inverted microscope and
• 5 shows very schematically the essential components of the device for determining the height of a fluid level in a cuboid chamber in an upright microscope according to another embodiment.

In 1 is schematically an upper reference object, hereinafter referred to as a float 1 referred to, and a lower reference object, hereinafter as an anchor 2 indicated. Both are connected 3 here a microfiber, connected together. The length of the connection 3 depends on the one hand on the maximum height of the fluid level and on the other hand on the diameter of the visual field of the microscope used to determine the height of the fluid level. At a high fluid level, the length must be sufficient for the float 1 can float on the surface of the fluid. On the other hand, the length of the connection must be 3 be sized at given fluid levels so that the float 1 not out of sight In this way it is possible in a reliable way quickly to the float 1 or on the anchor 2 to focus.

2 schematically shows an upright microscope 10 with a device for determining the height of a fluid level in a cuboid chamber 7 , A possible embodiment of the chamber 7 is in 3 illustrated and explained in more detail there. The microscope 10 here an upright microscope, as used for example for the laser microdissection, has a microscope stage 11 on top of a fluid filled chamber 7 in the object space of the microscope 10 receives. By means of a focusing device 12 can the lens 6 of the microscope 10 successively to the lower reference object 2 and the upper reference object 1 be focused. This is done with a lens 6 fixed focal length the distance between lens 6 and microscope stage 11 or chamber 7 changed while at a lens 6 adjustable focal length alternatively or additionally, the focal length can be changed accordingly. Accordingly, the first focus position of the upper reference object and the second focus position of the lower reference object are determined, and then the height of the fluid level is calculated from the difference of the two corresponding focal positions. This is an arithmetic unit 13 provided, which is part of the general control unit of the microscope 10 can be.

With the in 2 illustrated microscope 10 For example, one may be in the fluid of the chamber 7 sample or the fluid itself as a sample in transmitted light illumination. For example, it may be lysed cells that have previously been excised from a tissue section by laser microdissection. For optical viewing of a sample, the microscope points 10 an eyepiece 14 on, in a known manner with the tube of the microscope 10 connected is.

3 shows very schematically the essential components of a device for determining the height H of a fluid level in an upright microscope 10 according to 2 is arranged. A cuboid chamber 7 is located in the holding device of a microscope stage 11 of the upright microscope 10 , Above the chamber 7 is the lens 6 , The chamber 7 is thus located in the object space of the microscope, with the focus of the lens 6 can be set to different focal planes of the object space. In the chamber 7 itself is a fluid, here for example an aqueous suspension. The width of the chamber 7 is with x , the depth of the chamber 7 is with y The base area results from this as x • y. To the volume of the fluid 8th in the chamber 7 it is necessary to calculate the height H to determine the fluid level.

For this purpose, for example, first on the anchor 2 at the bottom of the chamber 7 focused. For this purpose, in a known manner, the microscope stage 11 in its position in the focus direction by means of an adjusting device 12 be adjusted in z-direction, ie parallel to the optical axis. Additionally or alternatively, in the case of a microscope objective 6 variable focal length can be adjusted so that the armature 2 in focus. A combination of both approaches is possible. This results in the focal position and thus the focal length 4 of the anchor 2 , which is also called the second focal length 4 of the lower reference object 2 referred to as. In a completely analogous way, the focal length becomes 5 of the swimmer 1 also known as the first focus length 5 of the upper reference object 1 referred to as.

The amount of difference between the two focal lengths 4 and 5 gives the height H the fluid level in the chamber 7 , The volume V results from this to V = h · x · y.

For easier and clear focus on swimmers 1 or anchor 2 Both objects in this embodiment are of different shape and color, allowing them to be uniquely and quickly identified.

4 shows a further embodiment for determining the height H a fluid level in a chamber 7 in an inverted microscope, this chamber 7 according to 4 has a cylindrical shape. The inverse microscope is shown only schematically by the lens 6 below the chamber 7 is shown. Like reference numerals again designate like elements as in FIG 3 , In this respect, a further explanation of these elements is omitted here. The difference between focus length 5 between microscope objective 6 and swimmers 1 and focus length 4 between lens 6 and anchor 2 gives the height here H , Alternatively, the focus length 4 from the focal length 5 deducted and then the amount will be formed. The volume V results from the product of height H and base of the cylinder π • r ² .

In the described embodiments, it has been assumed that the dimensions of float 1 and anchor 2 in focus direction at least opposite the height H are negligible. If this is not the case, corrections would have to be made to the dimensions of the float 1 and anchor 2 consider. This is possible for the skilled person in a simple manner.

5 shows an analog representation 3 with a chamber 7 with rectangular base. Unlike the chamber 7 out 3 is at the chamber 7 according to 5 the lower reference object 2 designed as a calibration mark. It is therefore dispensed with a physically extended object and instead one at the bottom of the chamber 7 calibration mark 2 used as lower reference object. In particular, this calibration mark is located 2 exactly in the middle (intersection of the two diagonals) of the base of the chamber 7 , In this way, an automatic start of the calibration mark 2 possible. The upper reference object is in accordance with the embodiment 5 again as a swimmer 1 educated. The swimmer 1 Floats on the surface of the fluid 8th , Note here something of the 3 different representation due to the perspective view of the chamber 7 according to 5 , Regarding the determination of height H and the volume V of the fluid 8th in the chamber 7 be on the statements in connection with the 3 directed.

LIST OF REFERENCE NUMBERS

1

Float, upper reference object

2

Anchor, lower reference object

3

connection

4

second focus length

5

first focus length

6

microscope objective

7

chamber

8th

fluid

10

microscope

11

microscope stage

12

focusing

13

computer unit

14

eyepiece

x

width

y

depth

r

radius

H

height

Claims (14)

Method for determining the height of a fluid level of a chamber (7) arranged in a microscope (10) and filled with a fluid (8), wherein an upper reference object (1) floating on the surface of the fluid (8) and one on the Bottom of the fluid-filled chamber (7) located or sunk on this floor lower reference object (2) are introduced into the chamber (7) or are designed as components of the chamber (7), wherein the chamber (7) is introduced into the object space of the microscope (10) equipped with a lens (6), wherein successively the focus positions of the upper reference object (1) and the lower reference object (2) are determined with the microscope and the height of the fluid level is calculated from the difference of the determined focal positions. Method according to Claim 1 for calculating the fluid volume in the chamber (7) filled with the fluid (8), wherein the fluid volume is calculated from the calculated height of the fluid level with known geometry of the chamber (7). Method according to Claim 1 or 2 , wherein as the upper reference object (1) and as the lower reference object (2) such reference objects are used, which differ in shape and / or color. Method according to one of Claims 1 to 3 in which a lens with a depth of focus which is below the dimension of the upper reference object (1) in the focus direction is used as the objective (6). Method according to one of the preceding claims, wherein the upper and / or the lower reference object (1, 2) are provided with fluorescent dyes. Method according to one of the preceding claims, wherein a calibration mark on the chamber bottom is used as the lower reference object (2). Method according to one of the preceding claims, wherein the upper and the lower reference object (1, 2) are interconnected via a flexible connection (3). Microscope (10) with a device for determining the height of a fluid level of a chamber (7) arranged in the microscope (10) and filled with a fluid (8): an upper reference object (1) located in the chamber (7) and floating in the fluid-filled chamber (7) on the surface of the fluid (8), a lower reference object (2) located in the chamber (7) located in the fluid-filled chamber (7) at the bottom of the chamber (7), wherein the microscope (10) equipped with a lens (6) has a microscope stage (11) which accommodates the fluid-filled chamber (7) in the object space of the microscope (10), and comprises a focusing device (12) which projects the objective (6). successively focused on the upper reference object (1) and the lower reference object (2) and determines the first focus position of the upper reference object (1) and the second focus position of the lower reference object (2), as well a computing unit (13) which calculates the height of the fluid level from the difference of the two corresponding focal positions of the upper reference object (1) and the lower reference object (2). Microscope (10) after Claim 8 for calculating the fluid volume in the chamber (7) filled with the fluid (8), wherein the arithmetic unit (13) calculates the fluid volume from the calculated height of the fluid level with known geometry of the chamber (7). Microscope (10) after Claim 8 or 9 , wherein the upper and the lower reference object (1, 2) differ in shape and / or color. Microscope (10) according to one of Claims 8 to 10 wherein the lens (6) has a depth of focus that is below the dimension of the upper reference object (1) in the focus direction. Microscope (10) according to one of Claims 8 to 11 , wherein the upper and / or the lower reference object (1, 2) fluorescent dyes and the microscope (10) is designed as a fluorescence microscope. Microscope (10) according to one of Claims 8 to 12 , wherein the chamber (7) on the chamber bottom has a calibration mark as a lower reference object (2). Microscope (10) according to one of Claims 8 to 13 , wherein the upper and the lower reference object (1, 2) via a flexible connection (3) are interconnected.

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