Abstract:
The invention makes it possible to adjust the light intensity of a laser scanning microscope laser beam in an economical manner and with high accuracy. A separate acousto-optic component can be omitted in that a light modulation section such as an electroabsorption modulator (EAM) or a semiconductor amplifier (SOA) is arranged directly at the laser diode, advisably at one of its front sides. It is nevertheless possible to control the light intensity economically and with high accuracy because the important parameters of the laser beam remain unchanged when the optical output power is changed by the light modulation section. The light modulation section is preferably formed integral with the laser diode in at least one material layer.

Description:
The present application claims priority from PCT Patent Application No. PCT/EP2009/004265 filed on Jun. 12, 2009, which claims priority from German Patent Application No. DE 10 2008 028 707.5 filed on Jun. 17, 2008, the disclosure of which is incorporated herein by reference in its entirety. 
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The invention is directed to a laser scanning microscope (LSM) with a laser diode. 
     2. Description of Related Art 
     Laser scanning microscopes (LSM) are used for confocal recording of images of a sample by a laser beam which usually scans in a zigzag shape. Prior to a scanning process, individual regions to be recorded (regions of interest, or ROI) can be determined. To avoid unnecessary stress on the sample, the laser light should be switched on as accurately as possible upon entering the region to the scanned. This is also true for the peripheral regions of the sample, which are generally not to be imaged, in the area where the laser beam changes direction because the speed at which the laser beam moves over the sample is slowest in this area and the radiation loading is therefore at its highest. In addition to processes for switching on and switching off from and to the zero level, changes in the beam intensity between two intensity levels other than zero are also required depending on the application. 
     In the prior art, the light intensity of a laser beam of a diode laser in an LSM can be controlled with high accuracy by means of an acousto-optic component, for example, an AOM (acousto-optic modulator) or an AOTF (acousto-optic tunable filter). This is disclosed, for example, in DE 197 02 753 A1. However, a component of this kind requires a relatively large installation space and is costly. 
     In order to dispense with an acousto-optic component while nevertheless enabling adjustment of the light intensity on the sample, the optical output power of the laser diode can be controlled directly by changing the electric current. In this way, both the attenuation function and the modulation function can be achieved in a manner analogous to the use of an acousto-optic component. 
     However, laser diodes which are modulated directly by diode current have drawbacks with respect to imaging. The most severe drawback is that important laser parameters become unstable when it becomes necessary in a certain application to reduce the diode current in the range of the laser threshold. Particularly noteworthy parameters are the polarization, which can fall from values greater than 100:1 to well below 10:1, and the spectral width of the laser beam—the diode transitions seamlessly from laser mode to LED mode so that the spectral width can increase to several nanometers. Stability and power calibration are also critical because the diode becomes unstable in the current intensity range around the laser threshold, which leads to excessive noise. Further, the threshold current exhibits a variation over the lifetime of the laser diode so that it is necessary to calibrate the power in the threshold range at regular intervals. Moreover, a change in current through the laser diode within the framework of direct modulation, also above the laser threshold, results in a slight shift of the center wavelength of the emitted spectral line. This can lead to a change in the beam direction due to the dispersion of the prisms used for beam shaping or in the microscope M or scanning unit S, which impairs the accuracy of exposure. 
     Another application of a LSM is fluorescence lifetime imaging (FLIM). In this case, short laser pulses of durations typically from 20 ps to 100 ps are used as illumination. In the prior art, these short laser pulses are generated by means of a laser diode controlled by electric pulses, so-called gain switching. In so doing, the electric pulses are adapted to the respective individual laser diode so as to generate optimal optical pulses (i.e., with a full width (FWHM) from 20 ps to 100 ps, steep edges, insignificant or nonexistent shoulders, and no long afterglows). Changes in intensity are caused by neutral density glass because changes in intensity due to a change in the electric control parameters (e.g., the electric pulse height and pulse width or CW bias current) necessarily entail changes in the optical pulse shape and can therefore be carried out only within a narrow, carefully selected range. 
     SUMMARY OF THE INVENTION 
     It is the object of the invention to improve a laser scanning microscope of the type mentioned above in such a way that the light intensity of the laser diode can be controlled economically and with high accuracy. 
     According to the invention, a light modulation section is arranged directly at the laser diode, advisably at one of its front sides. In particular, the light modulation section can be associated with the laser diode as an integral component part. 
     By means of the solution according to the invention, a separate acousto-optic component can be omitted. Nevertheless, it is possible to control the light intensity economically and with great accuracy because the important parameters of the laser beam (beam profile, wavelength, pulse shape, polarization, noise) remain unchanged when the optical output power is changed by means of the light modulation section. Further, an appreciable reduction in the required installation space is achieved with a light source of this kind. Different measuring processes and evaluating processes can be advantageously combined with a laser scanning microscope that is outfitted according to the invention. 
     A first preferred embodiment form of a light modulation section of the type mentioned above is an electroabsorption modulator (EAM) serving as an optical switch/dimmer. An EAM is an optical modulator which is based on semiconductor material and which is usually based on the Franz-Keldysh effect or the quantum-confined Stark effect. The use of EAMs is state of the art in optical data communications. By applying and/or adjusting an electrical field, the absorption can be controlled in a semiconductor with a suitable band gap (without excitation of electrons), which results in a corresponding change in transmission. For technical applications, it is particularly relevant that this effect is stable under different operating conditions. First EAMs are commercially available (for example, Model OM5642W-30B by OKI). EAMs have the advantage over acousto-optic modulators that they can be operated with DC voltage, whereas AOMs must be controlled by electric signals with high frequencies, typically 80 MHz to 120 MHz, and high power, typically 50 mW to 200 mW. 
     In a second preferred embodiment form, the light modulation section is advantageously a semiconductor optical amplifier (SOA). The semiconductor optical amplifier can advisably be constructed as a tapered amplifier. Both variants allow an economical intensity modulation and have a low variation in the rise time (delay) of the light intensity. The semiconductor-based light modulation section can advantageously be constructed monolithically with the laser diode for an integral construction. 
     The arrangement of the light modulation section directly at the laser diode can be realized in two alternative ways. The light modulation section is preferably formed integral with the laser diode in at least one material layer. A light source of maximum compactness is provided in that the laser diode and the EAM are both made of a semiconductor wafer. Diodes of this kind are also already commercially available (OKI OL5157M) as so-called distributed feedback lasers (DFB) for optical telecommunications. Further, in an advantageous manner, there is no need for a free-space section between the laser diode and the modulation section. 
     As an alternative to an integral construction of the laser diode and light modulation section, the light modulation section can also be advantageously arranged as a separate component part at a front side of the laser diode. This alternative also allows a compact construction and makes it possible to dispense with a free-space section. 
     In a preferred embodiment, the light modulation section or the integrated unit comprising the laser diode and light modulation section is provided with a direct (fixed) fiber coupling (pigtail). The direct fiber coupling has the advantage that a laser module coupled to an LSM in this way is compact, stable, and requires no adjustment on the part of the user. Moreover, it allows for simple integration into an existing device concept. In an alternative embodiment form, a fiber manipulator is mounted in a compact manner directly at a front side of the light modulation section. 
     The LSM is advantageously outfitted with a control unit which operates the laser diode permanently and significantly above a laser threshold. Fluctuations in beam parameters can be prevented in this way. 
     A control unit is advantageously provided which adjusts the optical output power of the laser diode by means of the integrated light modulation section. This control unit may be the same as or different than the control unit for modulating the current of the laser diode. 
     An LSM according to the invention can advantageously be used for fluorescence lifetime imaging (FLIM) in which the laser diode is used in pulsed mode. The high shape fidelity of the pulses modulated by means of the light modulation section with respect to the electric control signal makes it possible to carry out fluorescence excitation with greater accuracy. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows a laser scanning microscope with a laser diode according to the prior art; 
         FIG. 2  shows a laser diode unit with a light modulation section; and 
         FIG. 3  shows a laser scanning microscope according to the invention with a laser diode. 
     
    
    
     DETAILED DESCRIPTION OF EMBODIMENTS 
     It is to be understood that the figures and descriptions of the present invention have been simplified to illustrate elements that are relevant for a clear understanding of the present invention, while eliminating, for purposes of clarity, many other elements which are conventional in this art. Those of ordinary skill in the art will recognize that other elements are desirable for implementing the present invention. However, because such elements are well known in the art, and because they do not facilitate a better understanding of the present invention, a discussion of such elements is not provided herein. 
     The present invention will now be described in detail on the basis of exemplary embodiments. 
     Identical parts have identical reference numerals in all of the drawings. 
       FIG. 1  shows a schematic view of a laser scanning microscope  1  known from the prior art. It comprises a microscope unit M, a scanning unit S having a common optical interface by means of an intermediate image Z, and a laser module L. The scanning unit S can be connected to the photo tube of an upright microscope as well as to the side output of an inverted microscope. The microscope unit M has an objective  4  and a tube lens  9  for observing a sample  5 . 
     The scanning unit S contains collimating optics  16 , a deflecting mirror  17 , a scanning objective  22 , a scanner  23 , a main beamsplitter  24 , and imaging optics  25  for detection. A monitoring beam path is masked out by a semitransparent mirror  18  in direction of a monitoring diode  19  which is arranged in front of a neutral filter  20 . A deflecting mirror  27  behind the imaging optics  25  reflects the beam coming from the sample  5  in the direction of the pinhole diaphragm  29  which is adjustable perpendicular to the optical axis and whose diameter is variable, and downstream of which are arranged an emission filter  30  and a suitable receiver element  31 , for example, a photomultiplier (PMT). An external control unit  34  is connected to local control units  35  and  38  for the monitor diode  19  and the adjustable pinhole diaphragm  29 . 
     The separate laser module L contains a laser diode  13  as individual light source, whose laser beam is initially freely propagated after exiting from the laser diode  13  and passes through an acousto-optic component  32 , for example, an AOTF. The laser beam is then coupled into the illumination beam path of the scanning unit S by a fiber manipulator  33  and a light-conducting fiber  14 . 
     The laser diode  13  emits a wavelength of 405 nm, for example. It provides two different operating modes: a standby mode which on the one hand serves to protect the laser diode  13 , and on the other hand protects the sample from excessive radiation stress with an optical output power appreciably below the maximum value (e.g., by a factor of 10,000), and an imaging mode (e.g., with optical maximum optical power) in which the laser line serves to record images. Alternatively, the laser diode  13  can also be operated below its maximum output in certain applications. The possible applications of the imaging mode can be divided into two groups. On one hand, sample light can be recorded. In this case, it is necessary to vary the laser output in the low power range for optimally adapting the laser power to the operating conditions (absorption, emission of fluorescence dye, pixel time). On the other hand, it can be used for sample manipulation. In so doing, certain regions of the sample  5  must be irradiated at maximum laser power. In the first case, the laser power must be adjusted as continuously as possible. Further, it is desirable to adapt the illumination phases of the sample (which means a laser radiation load) to the actual image recording times in order to protect the sample. In the second case, it is necessary above all to switch the laser power on and off quickly and, as far as possible, so as to be pixel-synchronous. In both cases, the adjustment of the power of the laser beam is realized with the acousto-optic component  32  which is arranged in the free-space section between the laser and fiber. 
       FIG. 2  shows a laser diode  13  with a light modulation section  15 . The semiconductor layers of the laser diode  13  and of the light modulation section  15  are arranged on a common substrate  39  so that they form an integral unit. An air gap L is arranged between the active layer  40  of the laser diode  13  and the active layer  41  of the light modulation section  15  for electric insulation. 
       FIG. 3  shows an LSM  1  in a configuration having, by way of example, a laser diode  13  which has an integrated light modulation section  15 . Accordingly, it is also possible to adjust the light intensity/power of the laser beam with an acousto-optic component. The laser diode  13  can emit a wavelength of 375 nm, 405 nm, 440 nm, 473 nm, 488 nm or 635 nm, for example. Apart from the laser module L, the LSM  1  is constructed in the same way as the LSM  1  according to  FIG. 1 , which is already known. In alternative embodiment forms (not shown), the LSM  1  can also be outfitted, for example, with additional lasers, particularly with a plurality of laser modules L and/or a plurality of lasers in one laser module L. Further, for purposes of multichannel detection, it can have a plurality of detectors  31  which are coupled with the imaging beam path by beamsplitters, for example. As an alternative to the integral construction of the light modulation section  15 , the latter can be arranged (not shown) directly at the front side of the laser diode  13  as a separate component. 
     The invention consists in that a quasi-direct modulation of laser diodes with high exposure accuracy is made possible in laser scanning microscopes. For example, the integrated light modulation section  15  can be constructed as an electroabsorption modulator. In so doing, the laser diode  13  itself is not modulated but, rather, the optical output power of the laser diode  13  running at constant power is modulated in the EAM located directly after the laser diode  13  in the beam direction. 
     EAMs are available at low cost as individual components or also directly integrated with a laser diode. An EAM is a semiconductor component having a construction similar to that of a laser diode  13 . Therefore, it can be produced in approximately the same size as a laser diode  13  and can also be directly integrated with a laser diode  13 . The laser module L can accordingly be constructed in a compact manner. Because the intensity of the laser beam is modulated by means of an EAM instead of directly by the diode current, the laser diode  13  can always run in laser mode at high power (appreciably above the laser threshold). In this way, the problems mentioned above relating to current modulation (spectrum, polarization, noise) are prevented. 
     Instead of an EAM, an optical semiconductor amplifier (SOA) can be used. An SOA is constructed like a semiconductor laser diode, but its two end faces are anti-reflective. Therefore, the SOA acts only as an amplifier rather than as a laser. Its gain and, therefore, the optical output power of the laser diode  13  can be controlled by the electric current flowing through the SOA. Owing to its small size, an SOA can be compactly integrated together with the laser diode  13  in a small housing. As with the EAM, the (master) laser diode  13  is always operated far above the laser threshold. The optical power is regulated by the (slave) SOA. The advantage of this arrangement is that the output power of the laser diode  13  is not only attenuated, but can also be amplified if necessary. In this way, higher output powers can be achieved than with the laser diode  13  by itself. This arrangement is also known as a MOPA (Master Oscillator Power Amplifier). The amplifier can be designed in a variety of ways, for example, as a tapered amplifier, to reach particularly high output powers. Both the EAM and the SOA are suitable for CW mode as well as for the pulsed mode of the laser diode  13 . 
     Because of the compact construction of the laser diode  13 , whose optical output power is controllable, the light modulation section  15  can be fiber-coupled directly. In-coupling optics are dispensed with so that it is not necessary to adjust the LSM  1  prior to putting it to use. Further, since no acousto-optic component is required, no free-space section is required. In this way, the laser module L can be constructed even more compactly. 
     As an alternative to direct fiber coupling, a fiber manipulator (not shown) can be arranged directly at one front side of the light modulation section  15 . In this alternative embodiment, the fiber manipulator must be aligned before the LSM  1  is put into operation. Nevertheless, the laser module L is compact and economical. 
     While this invention has been described in conjunction with the specific embodiments outlined above, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, the preferred embodiments of the invention as set forth above are intended to be illustrative, not limiting. Various changes may be made without departing from the spirit and scope of the inventions as defined in the following claims. 
     REFERENCE NUMERALS 
     
         
           1  laser scanning microscope 
           4  objective 
           5  sample 
           9  tube lens 
           12  mirror 
           13  laser diode 
           14  light-conducting fiber 
           15  light modulation section 
           16  collimating optics 
           17  deflecting mirror 
           18  semitransparent mirror 
           19  monitor diode 
           20  neutral filter 
           22  scanning objective 
           23  scanner 
           24  main beamsplitter 
           25  imaging optics 
           27  deflecting prism 
           29  pinhole diaphragm 
           30  emission filter 
           31  receiver element 
           32  acousto-optic component 
           33  fiber manipulator 
           34  central control unit 
           35  control unit for monitor diode  19   
           38  control unit for pinhole diaphragm  29   
           39  substrate 
           40 ,  41  active layers 
         M microscope unit 
         S scanning unit 
         L air gap