DE102012016347A1 - Microscope for single plane illumination microscopy (SPIM) has sample chamber having screw-in insert fittable annular opening in which sealing ring for fitting detector and/or illumination lens and/or lens mount or holder is placed - Google Patents

Abstract

The microscope has illumination device having illumination light source (3) and illumination beam path for illumination of sample (1) with light sheet via illumination lens. A detector detects light emitted from sample. An imaging optical system maps sample on imaging lens (7) in imaging beam path to detector. A liquid filled sample chamber (10) has light transmissive windows having screw-in insert (5) fittable in thread (8) formed in annular opening in which a sealing ring for fitting a detector and/or illumination lens and/or lens mount or holder is placed.

Description

The invention relates to a microscope which comprises an imaging objective for imaging a specimen on a detector and means for illuminating the specimen with a light sheet in the focal plane of the imaging objective or in a defined plane in the vicinity of this focal plane. The means for illumination comprise a coherent light-emitting illumination source.

A microscope, wherein the illumination beam path and detection beam path are arranged substantially perpendicular to each other, and wherein the sample with a light sheet in the focal plane of the imaging lens, d. H. perpendicular to its optical axis, is designed for the examination of samples according to the method of the selective plane illumination microscopy (SPIM). In contrast to confocal laser scanning microscopy (LSM), in which a three-dimensional sample is scanned point by point in individual, different depth planes and the resulting image information is subsequently assembled into a three-dimensional image of the sample, the SPIM technology is based on of wide field microscopy and allows visualization of the sample based on optical sections through individual planes of the sample.

The sample is placed in the overlapping area of illumination and detection. Fluorescence signals, which are excited by the illumination light sheet, are imaged onto a camera over the entire field of view of the detection objective. Due to the rectangular illumination with a thin light sheet only a small part of the axial extent of the detection optics is illuminated and thus produces an optical section. In order to observe a different area in the sample, the sample, regardless of the optics, is driven through the light sheet with a sample positioning unit.

Among the benefits of SPIM technology is the increased speed at which image information is collected, the lower the risk of bleaching of biological samples and the increased penetration of the focus into the sample.

In principle, in SPIM technology, fluorophores which are contained in or introduced into the sample are excited with laser light, which is shaped into a so-called light sheet. With the light sheet, a selected plane in the depth of the sample is illuminated in each case and an image of this sample plane in the form of an optical section is obtained with an imaging optical unit. Substantially equivalent to such a stimulus with a static light sheet is the rapid reciprocation of a thin, rotationally symmetric laser beam in the focal plane of the imaging lens. Effectively, d. H. in the time average over the period of observation, thus results in the shape of a leaflet.

For example, the SPIM technology is described in Stelzer et al., Optics Letters 31, 1477 (2006) , in Stelzer et al., Science 305, 1007 (2004) , in the DE 102 57 423 A1 and in the WO2004 / 0530558 A1 ,

In 1 First, the basic structure of a SPIM microscope is shown. The light of a lighting source 1 is about a lighting optics 2 shaped into a light sheet and onto a sample three directed. Sample and light sheet are in the focal plane of an imaging lens 4 , The optical axis of the imaging lens 4 is perpendicular to the direction from which the sample three is illuminated. The illumination optics 2 typically includes a plurality of optical elements that illuminate the coherent light of the illumination source 1 collimate and form a light sheet. In the prior art, the illumination optics comprises 2 As a rule, a cylindrical lens whose flat side facing the sample and whose curved side points in the direction of the illumination source. Below are several examples of lighting optics 2 are explained, with which the generation of a light sheet with increased depth of field and reduced shadow is possible over known from the prior art arrangements.

problem

A big advantage of optical microscopy is the observation of live samples over a longer period of time. Due to the unique geometry of the optics, the requirement to move the sample completely freely and to observe the sample under physiological conditions, a special sample chamber design has prevailed ( ). The sample chamber is filled with a liquid (usually buffer such as PBS), the sample is immersed in this liquid for observation as well as the detection objective. To ensure that no liquid escapes from the sample chamber, the interface between the detection objective and the sample chamber must be tightly sealed. Different water immersion lenses have very different geometrical dimensions ( ), as an alternative to the immersive lens should also be possible to observe with an air lens through a cover glass in the sample chamber.

This means that the sample chamber must be specially designed for the detection objective and a simple change between different detection objectives is not possible. But that is exactly what is necessary. For different samples, and also for different sample size, a change of the lens is unavoidable because the magnification and / or numerical aperture must be adapted to the sample. In order to construct a universally applicable optical disc microscope, which can be used with very different samples, there must be an easy way to change the lenses. Otherwise, a separate sample chamber must be constructed for each lens. This is awkward for the user (he must always have the right chamber for each lens), very expensive (sometimes a recalculation of the optics design is necessary) and expensive.

shows possible used lenses in a SPIM setup. 2 (a) For example, an air lens 5 × NA 0.16, which requires a cover glass in the sample space. 2 B) -D) are exemplary water submersible lenses with a NA 1.0 and different magnification magnification: (b) 20 ×, (c) 40 × and (d) 63 ×.

solution

The problem presented is solved by the features of the independent claims.

Preferred developments are listed in the dependent claims.

• – einer Beleuchtungseinrichtung, umfassend eine Beleuchtungslichtquelle (3) und einen Beleuchtungsstrahlengang zur Beleuchtung der Probe (1) mit einem Lichtblatt, vorzugsweise über ein Beleuchtungsobjektiv
• – einer Detektierungseinrichtung zur Detektierung von Licht, das von der Probe (1) abgestrahlt wird,
• – einer Abbildungsoptik, die die Probe (1) über ein Abbildungsobjektiv (7) in einem Abbildungsstrahlengang mindestens teilweise auf die Detektierungseinrichtung abbildet,
• – einer vorzugsweise mit Flüssigkeit gefüllten Probenkammer die in Beleuchtungsrichtung und in Detektionsrichtung mindestens ein lichtdurchlässiges Fenster aufweist,
• – wobei das Lichtblatt im Fokus des Abbildungsobjektivs (7) oder einer definierten Ebene in der Nähe des geometrischen Fokus des Abbildungsobjektivs im Wesentlichen eben ist und wobei das Abbildungsobjektiv (7) eine optische Achse aufweist, die die Ebene des Lichtblattes in einem von Null verschiedenen Winkel, bevorzugt senkrecht schneidet,

weiterhin aber auch eine entsprechend gemäß der Erfindung ausgeführte Probenkammer für die SPIM Mikroskopie, die mindestens ein lichtdurchlässiges Fenster in Detektionsrichtung und/oder Beleuchtungsrichtung aufweist
sowie einen Objektivwechseladapter oder Adapter für Linsenfassungen- oder Halterungen für die SPIM Mikroskopie gemäß der Erfindung und ein erfindungsgemässes Verfahren zum Objektivwechsel oder Wechsel von Linsenfassungen- oder Halterungen in der SPIM Mikroskopie,
wobei mindestens in einem lichtdurchlässigen Fenster eine Aufnahme, vorzugsweise mit einem Gewinde versehen, vorgesehen ist in die mindestens ein vorzugsweise einschraubbarer Einsatz (Adapter) einpassbar ist der eine vorzugsweise ringförmige Öffnung aufweist in der ein Dichtungsring zur Einpassung eines Dektektionsobjektives und/oder Beleuchtungsobjektives und/oder einer Linsenfassung- oder halterung eingebracht ist. Anstatt jeweils die ganze Probenkammer an das Detektionsobjektiv anzupassen wird erfindungsgemäss die Schnittstelle ausgetauscht.The invention relates to a microscope for SPIM microscopy, consisting of

• A lighting device comprising an illumination light source ( three ) and an illumination beam path for illuminating the sample ( 1 ) with a light sheet, preferably via an illumination objective
• A detection device for detecting light emitted by the sample ( 1 ) is radiated
• - an imaging optics, the sample ( 1 ) via an imaging lens ( 7 ) is imaged at least partially on the detection device in an imaging beam path,
• A sample chamber, preferably filled with liquid, which has at least one light-permeable window in the illumination direction and in the detection direction,
• - wherein the light sheet in the focus of the imaging lens ( 7 ) or a defined plane in the vicinity of the geometric focus of the imaging lens is substantially planar and wherein the imaging lens ( 7 ) has an optical axis which intersects the plane of the light sheet at a non-zero angle, preferably perpendicular,

furthermore, however, also a sample chamber designed according to the invention for SPIM microscopy, which has at least one light-permeable window in the detection direction and / or illumination direction
as well as an objective change adapter or adapter for lens mountings or holders for SPIM microscopy according to the invention and a method according to the invention for the objective change or change of lens mountings or holders in SPIM microscopy,
wherein at least in a translucent window, a receptacle, preferably provided with a threaded, in the at least one preferably screw-insert (adapter) can be fitted which has a preferably annular opening in a sealing ring for fitting a Dektektionsobjektives and / or illumination objective and / or a lens mount or bracket is introduced. Instead of respectively adapting the entire sample chamber to the detection objective, the interface is exchanged according to the invention.

three shows an inventive lens adapter as an interface, in (a) as a side view of the lens adapter, in (b) as a front view of the lens adapter. The middle part 13 remains free or translucent. 3 (c) is an application of the lens adapter using the example of the ZEISS 63 × NA 1.0 detection lens It is an adapter 5 shown, which is selected to match the lens and is screwed into the sample chamber. The respective adapter contains a plastic or rubber ring 6 and thereby close tightly with the detection lens, 7 , here's an example of this 2d) in from. With the adapter you get the necessary flexibility. So it is possible to use water immersion lenses of different sizes with the sample chamber, it can also be used with coverslips (or even very different glass thicknesses).

For the adapters presented here, in the case of the water immersion objective, the sample chamber is pressed against the detection objective. Here, a plastic ring that is squeezed onto the lens, the seal takes over. It also ensures that the lens is not damaged. For example, there are advantageously four different adapter rings, one for each lens, since the respective lens geometry is different. One of the adapter rings has instead of the aperture (opening) for the dive lens, a cover glass screwed (exchangeable), since he has to work with the 5 × air lens, for example.

In 4 is a SPIM sample chamber 10 represented the translucent windows 11 having. Along the optical axis 12 takes place the SPIM lighting, not shown here.

On one side of the sample chamber 10 is in one by means of a thread 8th screw-in insert 5 (Apapter) a lens 7 provided, by means of the sealing ring 6 firm and liquid-tight in the insert 5 is squeezed.

Since it is the lens 7 Here is an example of an air lens, indicates the use 5 a cover glass attached to it 9 on that the lens 7 before in the sample chamber 10 protects the liquid.

This method is not limited to the detection objective alone, but can also be used analogously for the illumination objectives.

Without limitation, it can also be applied to single lenses such as cylindrical lenses for generating the light sheet which are mounted in a corresponding holder or socket.

It could also be a structure with up to four lenses, immersed in the sample chamber, can be realized. It would also be conceivable that the sample chamber is solid and all lenses (up to 4) are moved into the sample chamber.

For example, if two immersive lenses are used at an angle to each other, at least one of the lenses must be movable to ensure the tightness.

Another requirement is that the screw connection between adapter and sample chamber must be tight. This can be done by an additional seal or other known measures.

Since optical disc microscopy is predominantly used in living samples, it is advantageous if the adapters and sample chamber can be sterilized. Ie. The materials must withstand common sterilization and cleaning agents (eg 70% ethanol). In addition, it would be advantageous if the materials are autoclavable.

The sealing ring should therefore be interchangeable, also because the plastic will become porous over time.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• DE 10257423 A1 [0006]
• WO 2004/0530558 A1 [0006]

Cited non-patent literature

• Stelzer et al., Optics Letters 31, 1477 (2006). [0006]
• Stelzer et al., Science 305, 1007 (2004) [0006]

Claims (8)

Microscope for SPIM microscopy, comprising - a lighting device comprising an illumination light source ( three ) and an illumination beam path for illuminating the sample ( 1 ) with a light sheet, preferably via an illumination objective - a detection device for detecting light emitted by the sample ( 1 ), - an imaging optics, which the sample ( 1 ) via an imaging lens ( 7 ) in an imaging beam path is at least partially imaged on the detection device, a sample chamber preferably filled with liquid which has at least one transparent window in the illumination direction and / or in the detection direction, wherein the light sheet is in the focus of the imaging objective ( 7 ) or a defined plane in the vicinity of the geometric focus of the imaging lens is substantially planar and wherein the imaging lens ( 7 ) has an optical axis, which intersects the plane of the light sheet in a non-zero angle, preferably perpendicular, characterized in that at least in a light-transmitting window, a receptacle, preferably provided with a thread, is provided in the at least one preferably screw-in insert ( Adapter) is fitted which has a preferably annular opening in which a sealing ring for fitting a Dektektionsobjektives and / or illumination objective and / or a lens mount or bracket is introduced. Sample chamber for SPIM microscopy, which has a translucent window in the detection direction and / or illumination direction, in which at least in a translucent window, a receptacle, preferably provided with a thread provided in the at least one preferably screw-threaded insert (adapter) is the one preferably annular opening in which a sealing ring for fitting a Dektektionsobjektives and / or illumination objective and / or a lens mount or bracket is introduced. Lens change adapter or adapter for Lens mountings or holders for SPIM microscopy, which consists of at least one receptacle in a translucent window, preferably with a thread, in which at least one preferably screwed insert (adapter) is fitted with a preferably annular opening in the a sealing ring for fitting a Dektektionsobjektives and / or illumination objective and / or a lens mount or bracket is introduced. Method for changing lenses or changing lens frames or holders in SPIM microscopy, wherein in the case of different detection and / or illumination objectives in at least one light-permeable window provided with a receptacle, preferably with a thread, into which at least one preferably screw-in insert (adapter) can be fitted which has a preferably annular opening in which a sealing ring is inserted for fitting a Dektektionsobjektives and / or illumination objective and / or a lens holder or holder, a change takes place. Device according to one of the preceding claims, wherein a plurality of inserts or adapters are provided which have a different inner diameter of the opening for adaptation to different detection and or illumination lenses and / or lens frames Device according to one of the preceding claims, wherein a liquid-tight cover glass is provided on the adapter for the use of dry lenses in the direction of the sample. Device according to one of the preceding claims, with an exchangeable sealing ring. Device according to one of the preceding claims, wherein the lenses and / or lens frames in the direction of the sample chamber for generating a contact pressure on the sample chamber in the direction of the optical axis are displaceable and lockable.

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