Apparatus for total internal reflection microscopy

An apparatus for total internal reflection microscopy of a sample, comprising a microscope objective lens; an excitation beam path for passing light through the objective lens to said sample; and a coupling element arranged in a back focal plane of the objective lens or in a plane which is conjugate to said back focal plane; said coupling element comprising a first area for relaying light to the objective lens for total internal reflection illumination of said sample and a second area; wherein said second area is capable of separating light emitted by said sample and passing through said excitation beam path in reverse direction from said excitation beam path; wherein said second area is spatially separate from said first area and does not overlap with said first area; and wherein a distance between said optical axis of the objective lens and that boundary of said first area which is nearer to said optical axis of the objective lens is selected such that the light beams passing from said first area into the objective lens are imaged by the objective lens at angles onto said sample for which total reflection of these light beams occurs.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an apparatus for total internal reflection microscopy having an objective lens and a coupling element for illuminating the specimen through the objective lens in a fashion that allows evanescent field-illumination, epi-illumination or both.

2. Description of Related Art

The principle of total internal reflection (TIR), which prevents a light beam exceeding a given angle of incidence from leaving a medium having a higher refractive index into a medium having a lower refractive index is increasingly utilized for epi-fluorescence microscopy (“total internal reflection fluorescence (TIRF)”). Therein the fact is utilized that the electromagnetic field of the totally reflected light beam extends into the medium having the lower refractive index and is able to excite fluorescent molecules there. The penetration depth of this so-called evanescent field depends on the wavelength “λ” and the angle of reflection “α” and usually is about several hundred nanometers. Thus it is possible to distinguish fluorophores located close to the reflecting interface from those located further away from it. An angle sufficiently large for total reflection within the substrate can either be achieved by coupling the excitation light beam laterally into the support substrate, or by utilizing special immersion objective lenses having an extraordinarily high numerical aperture. Such objectives allow to focus light onto the specimen at an angle exceeding the threshold angle of total internal reflection.

Lasers are usually used as light sources for TIR epi-fluorescence. A diffraction limited laser focus is projected into the rear focal plane of an appropriate objective lens having a sufficiently high numerical aperture. Upon passing through the objective lens the laser light is collimated, whereby the exact focus position in the back focal plane (pupil) of the objective lens determines the angle of incidence of the beam of light on the sample according to equation 1:
sinα=r/(n0f)

The condition for total internal reflection, on the other hand, is given by equation 2:
n0sinα=n1
whereinr: distance of the laser focus from the optical axis;n1: refractive index of the specimen medium;n0: refractive index of the substrate or the immersion medium;f: focal distance of the objective lens

For positioning the laser focus in the desired focal position within the pupil of the objective lens the laser light usually is coupled into the beam by means of a beam splitter element. However, if the laser beam does not hit the beam splitter at the right angle or if the beamsplitter is not tilted correctly, laser light may enter areas of the pupil of the objective lens, which do not result in total reflection.

The use of a laser as a preferred light source for TIR epi-fluorescence is a consequence of the low illumination depth of the TIR arrangement. Usually only a few fluorophores are excited in the narrow evanescent field, hence the resulting signals are usually very weak. If it is not possible to increase the sensitivity of the detector, the excitation energy has to be increased for achieving a good signal to noise ratio. On the other hand, when utilizing sufficiently strong lasers, even minute changes of the adjustment of the laser beam may result in a laser beam, which doesn't undergo total internal reflection. Instead it may pass through the specimen and reach the experimenter's eye where it may cause significant and harmful damage. U.S. patent application Ser. No. 2002/0097489 A1 discloses a microscope system for TIR illumination wherein exclusively white light is used, which passes through an annular aperture prior to being coupled into the illumination beam by reflection via a separate beam splitter. This beam splitter serves to combine normal epi-illumination light with TIR illumination light. A drawback of this microscope is its relatively complicated design.

It is an object of the invention to provide for an apparatus for TIR microscopy having a coupling element for TIR illumination and simultaneously for light from another light source for epi-illumination, wherein a reliable protection from faulty operation during adjustment is achieved, wherein the apparatus is also particularly suitable for the use of lasers having a high output power, and wherein the apparatus has a particularly simple design.

SUMMARY OF THE INVENTION

This object is attained by an apparatus for TIR microscopy in accordance with the invention. The invention is beneficial in that laser light beams, which have been coupled into the apparatus under conditions which do not result in TIR—and which hence may escape from the set up with virtually no attenuation—are masked out already by the coupling element. A further benefit is the simple design, wherein the coupling element is used not only for coupling the TIR excitation light into the microscope, but simultaneously also serves to couple normal epi-illumination light into the microscope and/or for decoupling light emitted by the sample from the microscope. Thereby simultaneously another essential requirement of practical TIRF systems is achieved: it is possible to realize the option to supplement information obtained by TIRF methods by classical epi-illumination methods by simultaneously illuminating the sample with normal epi-illumination light or by illuminating the sample with normal epi-illumination light shortly after illumination of the sample with light for TIRF excitation.

FIGS. 1A and 1Bshow schematically a microscope objective lens10, which is directed towards a sample12. The sample is supported by a transparent substrate14(usually made of glass). An immersion medium16is present between the substrate14and the microscope objective lens10. A coupling element24is located in the back focal plane of the microscope objective lens.

The arrangement shown inFIGS. 1A and 1Bis a system for total internal reflection (TIR) microscopy wherein TIR illumination is achieved by excitation beams19, which are imaged onto the sample12by the microscope objective lens10. The interface between the substrate14, having a refractive index equal to that of the immersion medium16(usually ca. 1.5) and the sample12may serve as the interface where TIR occurs. Biological samples usually are embedded in water and have a refractive index of 1.33 to 1.36. The resulting threshold angle of total reflection is 62.5° to 65°.

The incident light beams19for the TIR illumination are brought into focus18at a first TIR illumination light transmitting area20of the coupling element24and are subsequently collimated by the microscope's objective lens10such that they point towards the substrate14. The first area20is slit-shaped. The angle of incidence α between the (quasi-) parallel beams leaving the objective lens10and the optical axis15is determined by the radial distance of the focus18in the back focal plane of the objective lens from the optical axis15. The larger the distance from the axis15, the larger is the angle of incidence α1 see also equation 1.

The first area20of the coupling element24is transparent for the laser light, with the boundaries of the first area20being selected such that only laser light is transmitted which can reach—due to its radial distance from the optical axis15—the substrate at angles which warrant TIR to occur. This is beneficial in that thereby it is ensured that no laser light may pass through the sample which could possibly deteriorate the signal to noise ratio of the measurement or which could endanger the operator.

The coupling element24is tilted relative to an axis13perpendicular to the optical axis15. The axis13passes through the first area20and a further area22, whose position is symmetrical to area20with respect to the optical axis15. The coupling element24comprises a second area25in its central inner part. In case this second area25of the coupling element is reflective for the selected epi-illumination light, a widefield epi-illumination beam may be combined with the TIRF illumination beam. Both then, after passing the objective lens, illuminate the sample. Given that this beam occupies a circular area in the objective's back focal plane, which corresponds to angles of incidence below the critical angle, it serves for normal epi-fluorescence illumination e. In an inverted version of the above set up the laser light for TIR may be reflected by a slit-shaped reflecting area on the coupling element and the classical epi-illumination light is transmitted.

The second area25may be utilized in an analogous manner for reflecting light emitted by the sample12due to the TIR- or widefield epi-illumination from the optical axis15. To this end the second area25would be reflective for the emitted light.

Totally reflected laser light21is refocused by the objective lens10and passes through area22.

According to the alternative embodiment shown inFIG. 2a coupling element124is located in a conjugate plane of the back focal plane of the microscope objective lens10. This arrangement has several advantages: it facilitates the separation of the emitted light from the excitation beam and it makes the coupling element124more easily accessible than it is the back focal plane of the objective.

The back focal plane of the objective and all conjugate planes thereof allow the combination of beams, which are meant to reach the sample under different angles, by the fact that the beams occupy different regions of this plane. Thus different illumination light beam paths may be combined in these planes without the use of beam splitter elements which are usually employed for this purpose. In the example shown inFIG. 2the incident laser light119for TIR illumination from the light source L is focused onto a first area120of the coupling element124, with the first area120being transparent for the TIR illumination light. The focal spot118achieved thereby is imaged into a focus126in the back focal plane of the microscope objective lens10by utilizing two lenses123and127. The further optical beam path is identical to that shown inFIG. 1. By providing a reflective second area125a beam for wide field epi illumination may be combined with the focused beam used for TIR-illumination.

InFIG. 2two detectors6and8are schematically shown. Detector6serves to measure a signal, which is proportional to the power of the TIR illumination light119, and detector8serves to measure the power of the totally reflected light121, which, after being reflected backwards in a symmetrical fashion, passes an area122, corresponding to area120in the illumination path. Only a small fraction of both forward- and back-reflected beam is needed, it can be provided by a suitable beam splitter140. If the ratio of these two measured power values do not match, indicating that no total internal reflection occurs or occurs only partially, a protective shut-down unit reduces the laser intensity down to levels which are safe to the operator. The shut-down unit can be incorporated into a control C that is capable of maintaining the intensity of the light for total internal reflection illumination of said sample below a pre-determined threshold intensity if a ratio between the intensity of the light for total internal reflection illumination of the sample and the intensity of the light totally reflected by the sample exceeds a predetermined threshold ratio.

By choosing an appropriate material, thickness and angle of the beam splitter140, which uses a small fraction of the excitation beam to monitor its power, a wavelength-dependent beam-displacement in the plane of the coupling element124and hence of the objective's10back focal plane can be introduced, which compensates the wavelength-dependence of the penetration depth of the evanescent field in the sample12. Thus a wavelength-independent penetration depth in the sample12can be maintained.

FIG. 3shows a front view of an embodiment of a coupling element for laser light for TIR illumination. A first area transmitting the TIR illumination light is limited to two slit-like strips222and220in the outer periphery, corresponding to regions where the numerical aperture (NA) exceeds 1.35. The laser light for illumination of the sample is focused onto slit220. Slit222then serves to allow light to pass backwards after having been totally reflected at the sample. The coupling element shown inFIG. 3is adjusted in the optical axis of the microscope in a tilted orientation relative to the optical axis in the same manner as the coupling element124shown inFIG. 2. A second area reflecting the epi-illumination light comprises an inner ellipse225whose projection yields a circular area having a diameter corresponding to the numerical aperture of the objective lens utilizable by the epi-illumination. For watery media this corresponds to the range between a numerical aperture of 0 and a numerical aperture of 1.35. For achieving easy adjustment and centering it may be beneficial to provide for a transparent opening226in the inner reflecting circle. Due to its small size opening226does not significantly affect the light yield of the normal illumination light beam.

The version of the coupling element described so far is particularly suitable for the use of laser light for TIRF excitation. However, if one wishes to make use of the flexibility of a non-coherent light source, which is not limited to only a few laser lines, an intensity problem will arise. In order to overcome this problem not only a single illumination beam, but rather a bundle of beams, all having an angle larger than the angle of total reflection has to be utilized. All “utilizable” beams have to pass an annular ring in the back focal plane of the objective lens, having an inner radius rgcorresponding to the threshold angle of total reflection and having an outer radius rocorresponding to the maximum angle of the objective lens (i.e. corresponding to the numerical aperture of the objective lens).

FIG. 4shows a disc-like embodiment of a coupling element for non-coherent light. A first area, which is transparent for the light for TIR illumination, is limited to a ring322in the outer periphery corresponding to a numerical aperture of more than 1.35. The illumination light is focused onto ring322. A second area, which is reflective for the light of an epi-illumination, comprises an inner oval325corresponding to the projection of the numerical aperture of the objective lens utilizable by the epi-illumination light.

FIGS. 5A and 5Bshow an alternative embodiment of a coupling element for non-coherent light, comprising a body40having the shape of a cone having an envelope oriented at an angle β relative to the base and having a tip, which is cut at an angle Γ relative to the base light beams42for TIR illumination are prevented by the circular reflecting base surface44from reaching the sample at angles which are smaller than the threshold angle of total reflection. The elliptically shaped sectional surface46likewise is reflective for coupling incident light48for epi-illumination into the microscope objective lens10along the share50of illumination light beams.

Although the coupling element24,124preferably is tilted relative to the optical axis, this is not a mandatory feature. Rather the coupling element might be designed such that the transmission through the second area is wavelength dependent in such a manner that the light for TIR illumination is not transmitted, while the light for epi-illumination or the light emitted by the sample is transmitted.