DE102006042088B4 - Apparatus and method for forming an immersion film - Google Patents

Abstract

Device (12) for forming an immersion film from an immersion liquid between a lens (20) of a microscope objective (10) and a sample carrier,
with an objective-fixed element (26, 30) defining an immersion film region (28) in which the immersion film is to be formed,
a sensor device (32, 34, 36, 38, 40, 42) which is suitable for detecting the nature of the immersion film in the immersion film region (28),
a feed device (44, 46) for feeding immersion liquid into the immersion film area (28),
and means for controlling the supply of immersion liquid in response to the detection by the sensor means (32, 34, 36, 38, 40, 42), the sensor means (32, 34, 36, 38, 40, 42) controlling the Nature of the immersion film based on its electrical conductivity or its inductive and / or capacitive effect detected in an alternating electromagnetic field, or the sensor device detects the nature of the immersion film using a temperature-dependent resistor, which is arranged in the immersion film area.

Description

The present invention relates to an apparatus and a method for forming an immersion film from an immersion liquid between a lens of a microscope objective and a sample carrier.

Immersion liquids are liquids whose refractive index is close to the refractive index of the glass used for sample carriers and lenses. When an air gap between an entrance or exit lens of a microscope objective (hereinafter called "objective lens" for short) and a sample carrier is filled with such an immersion liquid, the numerical aperture of the objective increases. This leads to an increased light intensity and also allows an increased resolution of the lens.

Usually, the immersion liquid is applied by hand with the aid of a pipette to the objective lens or the sample carrier. However, this is difficult with high-resolution microscopes, because in these the focus of the lens is very close to the objective lens, for example, at a distance of 0.2 mm thereof. In high-resolution microscopes, therefore, the distance between the objective lens and the sample carrier will be only fractions of a millimeter, and it is not readily possible to introduce immersion liquid into such a narrow gap with a pipette from the outside. Instead, the objective must be moved away from the sample carrier before the immersion liquid is applied, and then moved back to the sample carrier. This movement can be realized, for example, by pivoting or raising and lowering the lens. As a result, the application of the immersion liquid is particularly complicated.

A further difficulty arises in automated microscopes, in which the lens is automatically scanned over the sample carrier in order to be able to automatically examine a large number of samples in a short time. If the sample carrier is moved relative to the objective, it may be that the immersion film breaks and the microscope is disturbed in operation. In addition, automated microscopes are used to capture images of samples, such as living cells, over a period of time. In this case, the immersion medium will partially evaporate, and it must therefore be renewed from time to time, which is also expensive if done by hand.

Especially in automated high performance microscopes, where the use of an immersion medium is cumbersome, but it would be particularly advantageous to use an immersion liquid due to the thereby achievable improved image quality, in particular the increased light intensity and the increased resolution.

An apparatus for forming an immersion film of the type mentioned is in the WO 02/093232 A2 disclosed. The known device comprises a supply tube, which is attached laterally to the lens and through which an immersion liquid is supplied to the vicinity of the center of the objective lens. From the outlet opening of the feed tube, the immersion liquid is then drawn by capillary forces into the gap between the objective lens and the sample carrier.

To ensure that there is always enough immersion liquid, this is continuously supplied in abundance in the known device, and excess immersion liquid is collected in a gutter, which is provided on a lens head of the lens, and sucked with a suction device.

The DE 103 33 326 discloses an apparatus for forming an immersion film of an immersion liquid between a lens of a microscope objective and a sample carrier. The device contains an objective-resistant element in the form of a protective device, which comprises a capillary channel for receiving excess immersion liquid. In the prior art device, the immersion liquid is continuously supplied and discharged, resulting in a uniform flow of the immersion medium in the immersion film area. The supply of immersion liquid is not controlled or regulated, but it is proposed to adjust the removal amount by controlling the suction negative pressure so that the volume of the immersion film remains substantially constant. It is not disclosed how the volume of the immersion film can be determined.

The US Pat. No. 6,980,293 B1 discloses an apparatus for supplying immersion liquid in which the temperature of the immersion liquid supplied can be regulated. With the temperature-controlled immersion liquid samples can be tempered, the properties of which are highly dependent on the temperature. Examples are biological samples whose reactions are to be studied, which in turn depend strongly on the temperature of the sample.

The invention has for its object to provide a device and a method of the type mentioned, which allow an immersion film between the lens of a microscope objective and a sample carrier automatically and reliably form, even if the microscope objective and the sample carrier quickly relative are moved to each other and / or if long-term studies on a sample are made.

This object is achieved in a device of the type mentioned above in that it comprises an objectively fixed element which defines an immersion film area in which the immersion film is to be formed, a sensor device which is suitable for detecting the nature of the immersion film in the immersion film area, a feed device for introducing immersion liquid into the immersion film area and means for controlling or regulating the supply of immersion liquid as a function of the detection by the sensor device. Further, it is achieved by a method according to claim 16.

Unlike the above-mentioned prior art, in which the immersion liquid is supplied through the feed tube at a point near the objective lens and is substantially trusted to form a sufficient immersion film between the microscope objective and the sample carrier by the capillary forces the device of the invention is an objectively fixed element which defines an immersion film region in which to form the immersion film. The nature of the immersion film in this immersion film area is detected by the sensor device. In this case, the "nature" of the immersion film does not mean a large number of detailed properties of the immersion film. Rather, it is important that it can be determined whether there is a sufficient immersion film in the immersion film area for the purpose of immersion microscopy. For example, if it is determined by means of the sensor device that the immersion film in the immersion film region becomes thin or begins to crack, the supply of additional immersion liquid can be caused by means of the control or regulating means.

The immersion film between the sample carrier and the objective lens is not limited to the immersion film region, but may also extend beyond it by capillary forces. However, the immersion film area is the area in which the constitution of the immersion film is checked by the sensor device. In addition, since the immersion film portion is provided in an objectively fixed member, it has the further beneficial effect of assisting the entrainment of the immersion film when the sample is moved relative to the objective, as will be explained below with reference to an embodiment.

In an advantageous embodiment, the lens-fixed element is designed as an adapter, which is suitable to be attached to a microscope objective. Such an adapter can easily be retrofitted to existing microscopes, which are currently working without immersion liquid or the immersion liquid is applied by hand.

In an advantageous embodiment, the sensor device detects the nature of the immersion film based on its electrical conductivity. For example, the electrical conductivity in a direction transverse to the thickness direction of the immersion film may be measured over a portion of the immersion film area. If the thickness of the immersion film decreases in places, this can be detected by a decrease in conductivity. In an advantageous development, the means for controlling or causing the supply of immersion liquid, when the conductivity of the immersion film in a direction transverse to its thickness direction below a lower threshold, and terminate the supply of immersion liquid, when said conductivity exceeds an upper threshold.

However, the embodiment just described requires a conductive immersion medium. The inventors have found that the conductivity of so-called deionized water is fair enough to achieve the detection described above. However, if still purer water or other non-conducting immersion liquid is to be used, the sensor device must respond to other properties of the immersion film. For example, in an alternative embodiment, the sensor device can detect the nature of the immersion film based on its inductive and / or capacitive effect in an electromagnetic alternating field.

In an advantageous development, the sensor device further comprises a device for measuring the temperature of the immersion liquid. Due to the temperature, the measured values of the sensor device can be corrected or scaled. For example, the conductivity of liquids depends on their temperature. In order to be able to conclude more reliably from a measured conductivity on the nature of the immersion film, it is advantageous to also detect the temperature and to compensate for the temperature-induced influences on the measurement result or to calculate out.

In a particularly advantageous development, the lens-fixed element comprises a plate and the immersion film region is formed by a recess in the plate. Since the plate is lens-fixed, the immersion film is held in the recess of the plate and thus in the region of the objective lens when the sample carrier relative to the lens is moved, as is the case for example in the case of sample scanning or so-called "screening". This is a significant improvement over a design in which the immersion film is held only by capillary force between the objective lens and the sample carrier.

Preferably, the plate is made of an electrically non-conductive material, in particular plastic, a ceramic or Teflon. Particularly advantageous is a plate which consists of a board material, in particular of glass fiber fabric and epoxy resin. Such board plates are mass-produced and therefore inexpensive available. In addition, electrodes and / or leads of the sensor device can be easily formed on board plates in a conventional manner.

In an advantageous development, the supply device comprises a channel which is formed in the plate and communicates with the recess. The channel may be formed by a trench in the plate, which is covered with another plate. The integration of the channel in the plate results in a very flat design, which is particularly advantageous in high-performance microscopes, in which a very small distance between the lens and the sample carrier must be adhered to.

In an advantageous development, the supply device is reversible in its conveying direction, so that immersion liquid can be removed from the immersion film region. This may be advantageous, for example, to suck the immersion liquid, if it is determined by means of the sensor device that too much immersion liquid was supplied. Additionally or alternatively, the conveying direction can be reversed in order to suck off a large part of the immersion liquid before the objective is removed from the sample carrier in order to prevent contamination of the microscope with immersion liquid.

Further advantages and features of the present invention will become apparent from the following description in which the invention with reference to an embodiment with reference to the accompanying drawings is explained in more detail. Show:

1 a plan view of a microscope objective on which an apparatus for forming an immersion film is attached according to a development of the invention,

2 a partially sectioned side view of the microscope objective and the device of 1 and

3 an enlarged section 2 ,

1 shows a plan view of a microscope objective 10 on which a device 12 is attached to form an immersion film. 2 shows a side view of the lens 10 and the device 12 in which the right half of the device 12 along the line AA of 1 is shown cut, and 3 shows the through the circle in 2 highlighted section in an enlarged view.

How best in 2 can be seen, includes the lens 10 a lens body 14 and a lens head 16 on which the device 12 is attached. The lens head 16 has an inner conical surface 18 , in the center of which is an objective lens 20 is provided, which is an entrance or exit lens of the lens 10 can act. Radial outside connects to the inner conical surface 18 an annular horizontal surface 22 of the lens head 16 at. The horizontal surface 22 is from an outer conical surface 24 surround.

The device 12 includes a plate 26 in which a recess 28 is trained. When the device 12 , as shown in the figures, on the lens 10 attached, the inner conical surface protrudes 18 through the recess 28 in the plate. The recess 28 the plate 26 forms an immersion film area, which is described below.

The plate 26 is with a ring 30 connected to the lens head 16 surrounds when the device 12 on the lens 10 is attached.

In the embodiment shown, the plate consists 26 from a commercially available board material consisting of glass fiber fabric and epoxy resin, and also referred to as FR4 or FR5. On the surface of the plate 26 are electrodes 32 respectively. 34 formed parallel to the edge of the recess 28 are arranged. The electrodes 32 and 34 are via associated lines 36 respectively. 38 with connections 40 respectively. 42 connected.

The electrodes 32 . 34 and the wires 36 . 38 are made by conventional etching techniques on the board material from which the plate 26 is formed, trained and are thus extremely easy and inexpensive to manufacture. The electrodes 32 . 34 and the wires 36 . 38 are preferably made of a copper layer which is covered by another layer of hard gold.

Especially in 1 and 3 is visible in the plate 26 a channel 44 formed at one end with the recess 28 and at that another end with a hose 46 communicated. The channel 44 is formed by a trench, which is in the plate 26 is milled. On both sides of the trench is the thickness of the plate 26 slightly reduced. In this area of reduced thickness is another, thinner plate 48 set flush with the surface of the plate 26 closes and covers the ditch to the channel 44 to complete at the top. The plate 48 is in 2 for the sake of clarity once again 48 ' in a side view and under 48 '' shown in a plan view.

The device 12 is formed in the embodiment shown as an adapter that can be placed on a variety of commercially available lenses. In the embodiment shown, the underside of the plate 26 on the outer edge of the ring 30 glued to the device or the adapter 12 by means of the ring 30 on the lens head 16 the lens 10 to fix.

The following is the function of the device 12 described. Through a peristaltic pump (not shown) an immersion liquid, for example deionized water, through the hose 46 and the channel 44 in the plate 26 in the recess 28 in the plate 26 fed. This will make the recess 28 filled with the immersion liquid. In the operation of the microscope is opposite the lens 20 , ie, in the representations of 2 and 3 above the lens head 16 a sample carrier (not shown) containing the samples to be examined by the microscope. The focus of the lens 10 is about 0.2 mm above the lens 20 , and in this very small distance in the operation of the sample carrier (not shown in the figures) is arranged. The in the recess 28 introduced immersion liquid fills the gap between the lens due to the adhesion forces 20 and the sample carrier (not shown). That is, an immersion film is formed between the lens 20 (and a surrounding part of the inner conical surface 18 ) and the sample carrier (not shown).

In operation, the sample carrier (not shown) may be relative to the objective 10 be moved, for example, to automatically scan a variety of samples. The recess 28 Helps the immersion film despite this relative movement in the lens area 20 to keep. Nevertheless, in the case of relative movement, part of the immersion liquid leaves the immediate vicinity of the lens 20 lost. In addition, the immersion liquid can be dried in long-term studies, for example on living cells.

To ensure that there is always enough immersion liquid, in the embodiment shown, an electrical voltage, in particular an alternating voltage between the electrodes 32 and 34 applied, and using an unspecified electronics, the conductivity of the immersion film between the electrodes 32 . 34 , ie, the immersion film in the recess 28 , measured. Note that the immersion film is not on the recess 28 is limited, but can also be formed by capillary forces beyond this. Also does not have the entire recess 28 be completely filled with the immersion film at all times. However, the recess forms 28 an immersion film region in which the immersion film is to be formed and in which its constitution is controlled by the above-described conductivity measurement.

If the immersion film in the recess 28 becomes thin or begins to break down, this is detected by the resistance between the electrodes 32 . 34 increases or decreases the conductivity of the immersion film in the immersion film area. Once the conductivity of the immersion film falls below a lower threshold, a signal is sent to the peristaltic pump (not shown), whereupon this further immersion fluid passes through the tubing 46 and the channel 44 in the recess 28 in the plate 26 subsequently supply. This supply of immersion liquid is stopped as soon as the conductivity of the immersion film between the electrodes 32 and 34 exceeds an upper threshold. Alternatively, the peristaltic pump may also be controlled to continuously monitor for a certain amount of time after being switched on, without monitoring an upper threshold of conductivity, which is sized to replenish a sufficient amount of immersion fluid.

The electrodes 32 . 34 , the wires 36 . 38 and the electronics for determining the conductivity of the immersion film form an embodiment of the above-mentioned sensor device, which is suitable for detecting the nature of the immersion film. If the immersion liquid is non-conductive, in an alternative embodiment, it could be at the electrodes 32 and 34 An alternating field can be generated by applying an alternating electrical voltage. The electrodes act 32 . 34 as "plates" of a capacitor, which could be coupled to a resonant circuit. With a dielectric immersion medium, the capacitance of this capacitor would change depending on the thickness of the immersion film, and thus the resonant frequency of the resonant circuit. Thus, the nature of the immersion film could be detected based on a capacitive effect thereof.

In an alternative embodiment, the presence of a sufficient immersion film can be checked by a calorimetric measurement method. In this method, the different thermal conductivity of the medium surrounding the sensor is utilized. For this purpose, for example, a temperature-dependent resistor, such as a PTC or NTC resistor, is used as the sensor, which is arranged in the immersion film region. A current is applied to the sensor so that the sensor heats up. Depending on whether the sensor comes to lie in the immersion film and is thus cooled by the immersion liquid or not, the sensor will heat up less or more, whereby the resistance value of the sensor changes accordingly. By detecting the resistance value of the sensor can thus be detected whether a sufficient immersion film is present.

In an advantageous development, the pumping direction of the peristaltic pump (not shown) can be reversed, so that immersion liquid from the recess 28 through the channel 44 and the hose 46 can be sucked off. This is particularly advantageous when the lens 10 removed from the sample carrier (not shown), for example, to change the lens or to examine a new sample, because then no longer needed immersion liquid does not contaminate the microscope.

Although a preferred embodiment has been shown and described in detail in the drawings and foregoing description, this should be considered as illustrative and not restrictive of the invention. It should be noted that only the preferred embodiment is shown and described, and that all changes and modifications that are currently and in the future within the scope of the claims to be protected

LIST OF REFERENCE NUMBERS

10

microscope objective

12

Apparatus for forming an immersion film

14

lens body

16

lens head

18

inner conical surface

20

objective lens

22

annular horizontal surface

24

outer conical surface

26

plate

28

recess

30

ring

32

electrode

34

electrode

36

ladder

38

ladder

40

Connection

42

Connection

44

channel

46

tube

48, 48 ', 48' '

plate

Claims (15)

Contraption ( 12 ) for forming an immersion film of an immersion liquid between a lens ( 20 ) of a microscope objective ( 10 ) and a sample carrier, with an objectively fixed element ( 26 . 30 ) containing an immersion film area ( 28 ) in which the immersion film is to be formed, a sensor device ( 32 . 34 . 36 . 38 . 40 . 42 ), which is suitable for determining the nature of the immersion film in the immersion film region ( 28 ), a delivery device ( 44 . 46 ) for introducing immersion liquid into the immersion film area ( 28 ), and means for controlling or controlling the supply of immersion liquid as a function of the detection by the sensor device ( 32 . 34 . 36 . 38 . 40 . 42 ), wherein the sensor device ( 32 . 34 . 36 . 38 . 40 . 42 ) detects the nature of the immersion film based on its electrical conductivity or its inductive and / or capacitive effect in an alternating electromagnetic field, or the sensor device detects the nature of the immersion film by means of a temperature-dependent resistor, which is arranged in the immersion film region. Contraption ( 12 ) according to claim 1, wherein the lens-fixed element is used as an adapter ( 26 . 30 ), which is adapted to a microscope objective ( 10 ) to be attached. Contraption ( 12 ) according to claim 1, wherein the control means causes the supply of immersion liquid when the conductivity of the immersion film in a direction transverse to its thickness direction falls below a lower threshold, and terminate the supply of immersion liquid when said conductivity exceeds an upper threshold. Contraption ( 12 ) according to one of the preceding claims, in which the sensor device comprises a device for measuring the temperature of the immersion liquid. Device according to one of the preceding claims, wherein the lens-fixed element comprises a plate ( 26 ) and the immersion film area through a recess ( 28 ) in the plate ( 26 ) is formed. Contraption ( 12 ) according to claim 5, wherein the plate ( 26 ) of an electrically non-conductive Material, in particular plastic, ceramic or Teflon exists. Contraption ( 12 ) according to claim 6, wherein the plate ( 26 ) consists of a board material, in particular glass fiber fabric and epoxy resin. Contraption ( 12 ) according to one of claims 5 to 7, in which the sensor device comprises electrodes ( 32 . 34 ) and / or electrical lines ( 36 . 38 ), which as layers on the plate ( 26 ) are formed. Contraption ( 12 ) according to claim 8, wherein the layers comprise a copper layer covered with a hard gold layer. Contraption ( 12 ) according to one of claims 5 to 9, in which the supply device has a channel ( 44 ) contained in the plate ( 26 ) is formed and with the recess ( 28 ) communicates. Contraption ( 12 ) according to claim 10, wherein the channel ( 44 ) through a trench in the plate ( 26 ) formed with another plate ( 48 ) is covered. Contraption ( 12 ) according to any one of the preceding claims, wherein the supply device comprises a peristaltic pump. Contraption ( 12 ) according to one of the preceding claims, wherein the supply device is reversible in its conveying direction, so that immersion liquid can be removed from the immersion film region. Method for forming an immersion film from an immersion liquid between a lens ( 20 ) of a microscope objective ( 10 ) and a sample carrier, in which the immersion liquid into a lens-fixed element ( 26 ) formed immersion film area ( 28 ), the nature of the immersion film in the immersion film area ( 28 ) with a sensor device ( 33 . 34 . 36 . 38 . 40 . 42 ) and the supply of further immersion liquid as a function of the detection by the sensor device ( 32 . 34 . 36 . 38 . 40 . 42 ) is controlled or controlled, wherein the nature of the immersion film is detected based on its electrical conductivity or its inductive and / or capacitive effect in an alternating electromagnetic field, or the nature of the immersion film is detected by means of a temperature-dependent resistor, which is arranged in the immersion film region. Method according to Claim 14, in which a device ( 12 ) according to one of claims 1 to 14 is used.

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