Counting compartment and method for sample analysis

Abstract

The invention relates to a counting chamber for a microscope, adjustable in height. The method measures a cell count at two different chamber heights and deduces the concentration of particles without an exact calibration of the absolute height, as the height difference gives a measurement of the missing volume and therefore an estimate of the particle concentration.

Claims

1. A flow-through counting compartment for analyzing samples comprising cells or particles, said counting compartment comprising: (1) a lid or top part comprising a top measurement window; (2) a bottom part comprising a bottom measurement window; and (3) at least two different depths provided by the bottom and top measurement windows, wherein the bottom and/or top measurement window comprises one or more steps.

2. The flow-through counting compartment of claim 1, wherein the at least two different depths provided by the bottom and top measurement window are achieved by (i) the bottom and/or top measurement window comprise step(s); or (ii) one of the bottom or top measurement window is placed with inclination; or (iii) one of the bottom or top measurement window is placed parallel to the other measurement window and is provided with an inclined plane on the side facing the sample liquid; or (iv) moving the lid or top part and/or the bottom part of the counting compartment.

3. The flow-through counting compartment of claim 1, further comprising: means for moving the lid or top part; an inlet and outlet; a heating unit for controlling the temperature of the sample to be analyzed, and/or a positioning unit for moving the counting compartment.

4. The flow-through counting compartment of claim 3, wherein the means for moving the lid or top part comprise magnetic means that are in the lid or top part and/or the bottom part.

5. The flow-through counting compartment of claim 1, wherein the bottom or top measurement window are made of a transparent material.

6. The flow-through counting compartment of claim 1, wherein the at least two different depths provided by the bottom and top measurement window are in the range of about 5 to about 100 m, and/or wherein the difference between the two different depths provided by the bottom and top measurement window is at least the size or height of a monolayer of the cells or particles of the sample to be analyzed.

7. A method for analyzing a sample comprising cells or particles, using the flow-through counting compartment of claim 1 comprising the step of determining the difference between the value measured at the at least two different depths of the counting compartment.

8. The method of claim 7, comprising the steps of (1) providing a sample comprising cells and/or particles to be analyzed; (2) providing the counting compartment; (3) loading the counting compartment with the sample, wherein the lid or top part is in open position; (4) closing the counting compartment by closing the lid or top part; (5) determining the sample value to be measured at the first depth of the at least two different depths and at the second depth of the at least two different depths; (6) determining the difference between the two values measured in step (5) and obtaining the sample value; (7) opening the counting compartment by opening or lifting the lid or top part; and (8) rinsing the counting compartment and thereby removing the sample.

9. The method of claim 8, wherein the sample is homogenized.

10. A counting device for analyzing samples comprising cells or particles comprising: a counting compartment, a control unit, filling means, removing means, a light microscope and/or a fluorescence microscope, and, one or more sensors, wherein said counting compartment comprises: (1) a lid or top part comprising a top measurement window; (2) a bottom part comprising a bottom measurement window; and (3) at least two different depths provided by the bottom and top measurement windows, wherein the bottom and/or top measurement window comprises one or more steps.

11. The counting device of claim 10, further comprising processing, analyzing and/or evaluating system and software.

12. The counting device of claim 10, which is automated.

13. The counting device of claim 10, comprising a phase contrast microscope comprising a motorized positioning unit, and/or a heating unit; and a white-light interferometer sensor, current sensor, inductive sensor, capacitive sensor or optical sensor.

14. A method for analyzing samples comprising cells or particles, comprising the step of controlling the depth of a counting compartment or sensor measurement, comprising the steps of: (1) providing a sample comprising cells and/or particles to be analyzed; (2) providing a counting compartment comprising a marking in the lid and a marking in the bottom or in its measurement windows (which allows z axis-calibration), and/or providing a counting compartment or device comprising a sensor (such as a white-light interferometer sensor, current sensor, capacitive sensor or laser sensor), (3) loading the counting compartment with the sample, (4) measuring the distance between the marking in the lid and the marking in the bottom or in its measurement windows (i.e. z axis-calibration) or by measuring the depth with the sensor; and then determining the depth of the loaded counting department; optionally comprising the use of a correction value or factor; (5) determining the sample value(s) to be measured, (6) opening the counting compartment by opening or lifting the lid or top part; (7) rinsing the counting compartment and thereby removing the sample.

15. The method of claim 14, wherein the samples comprising cells or particles are semen samples blood samples, samples comprising particles, samples of microorganisms, or algae samples.

16. The method, according to claim 15, wherein the sample value to be measured is the number of cells or particles in the sample.

17. The method of claim 14, further comprising analysis of cell motility; cell morphology; cell viability; cell acrosome integrity; cell mitochondrion activity; and/or cell deoxyribonucleic acid integrity.

18. The method of claim 14, wherein controlling the depth of the counting compartment is done by z-axis calibration.

19. The method of claim 14, wherein the sensor measurement is done by using a white-light interferometer sensor, current sensor, capacitive sensor or laser sensor.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. Schematic presentation of the counting compartment with at least two different depths.

(2) (A) In one embodiment, the bottom and top measurement window each comprise a step. The counting compartment is configured such that when the lid/top part closes on the bottom part the step of each of both measurement windows is on top of each other (i.e. super-imposed), thereby forming two different depths.

(3) (B) In one embodiment, one of the bottom or top measurement window is placed with inclination.

(4) (C) In one embodiment, one of the bottom or top measurement window is placed parallel to the other measurement window and is provided with an inclined plane on the side facing the sample liquid.

(5) (D) In one embodiment, the at least two different depths are provided by movement, such as with a spindle with motor.

(6) FIG. 2. A preferred embodiment of the counting compartment.

(7) (A) The top or lid part comprising a top measurement window and micrometer screws or gauges, wherein the top or lid part is situated on the bottom part.

(8) (B) The bottom part comprising a bottom measurement window, permanent electro magnets, spacer and an outlet.

(9) (C) The top or lid part comprising a top measurement window.

(10) FIG. 3. An exemplary embodiment of the counting device.

(11) The counting device comprising a phase contrast light microscope (1), scan stage unit (2), control unit with pump (3), draw-off pump with container (4), injection pump (5), injection syringe for sample (6). The counting device is connected to a processing, analyzing and/or evaluating system and software, in particular AndroVision CASA Software (Minitb GmbH, Germany).

(12) FIG. 4. Schematic view of the arrangement of measurement fields during an analysis with an evaluating system and software.

(13) (in particular AndroVision CASA Software), wherein

(14) m number of measurement fields, and

(15) n=number of measurement fields for each depth.

(16) FIG. 5. Example of an analysis.

(17) Shown is the determination of a cell concentration in a sample.

EXAMPLES

Example 1 Measurement Conditions

(18) The counting compartment can be used for sample analyses after cleaning and rinsing.

(19) Furthermore, the compartment has to warm up on a certain temperature which can generally be achieved by using a heating unit integrated into the microscope. For semen samples the temperature should simulate physiological conditions (e.g. between 35 C. and 39 C.).

(20) Cleaning can be conducted by using alcoholic derivatives and rinsing by using a washing liquid such as BTS semen extender in case of semen samples. The amount of washing liquid should be sufficiently chosen in order to remove the amount of sample material in the counting compartment.

(21) The counting compartment has to be opened for rinsing i.e. the lid or top part is in open position.

(22) After homogenizing a sufficient amount of sample can be injected into the counting compartment. The counting compartment has to be open during sample injection. Analyses can follow.

(23) After finishing analyses the compartment is opened again and rinsed for the next sample.

(24) The procedure starts from the beginning but without the cleaning step which is only necessary before using the compartment for the first time.

Example 2 Sample Treatment

(25) In case of semen samples, the raw ejaculate is appropriately diluted with extenders, such as Androhep Plus (Minitb GmbH, Germany), Androstar Plus (Minitb GmbH, Germany), M III (Minitb GmbH, Germany) or BTS (Minitb GmbH, Germany), shortly after collecting the ejaculate. For example, an appropriate dilution can be one part raw ejaculate and nine parts of extender, depending on the animal species and expected semen concentration of the ejaculate.

(26) Homogenization of such a diluted sample, especially before using the counting compartment, can be conducted manually or mechanically.

(27) Manual homogenization includes shaking the sample and/or turning the sample in a container. Mechanical homogenization includes the usage of a vortex mechanical mixer or a magnetic stirrer.

(28) The features disclosed in the foregoing description, in the claims and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.