DE3518527C2 - - Google Patents

Description

The invention relates to a fluorometer for measuring light induced changes in the fluorescence yield of a sample to be examined according to the Preamble of claim 1.

Such a fluorometer is known from US-PS 38 11 777. At The known device uses mechanical means (rotating hole disc) used to generate measuring light pulses, to protect the Photodetector in front of light of higher intensity (hereinafter referred to as strong light) and for the synchronization of the measuring light pulse source, Strong light source and photodetector. There are fundamental Limitations on shape, duration, frequency and reproducibility Availability of the measuring light pulses, as well as the use of any light swell and the electronic processing of the measurement signals. The got te facility is unable to make very quick changes to the To detect fluorescence yield, as z. B. following one Flash of light in green plants related to photosynthesis occur. Furthermore, the related perforated disk measuring principle imposed on the strong light pulse character. This makes applications excluded, in which the action of continuous, unsung weak strong light is a requirement, such as B. Investigations of the Chlorophyll fluorescence when exposed to natural sunlight on a green leaf. In the known device, it is due the inertia of the mechanical components not possible, the measuring pulse change frequency quickly. This property is of great prak tical importance to light sensitivity on the one hand objects in quasi-darkness (low Pulse frequency, low integrated measuring light intensity) and others rapid changes in the fluorescence yield induced by strong light (with high pulse frequency and correspondingly increased integrated measuring light intensity).

A similar fluorometer is also known from US-PS 38 30 222. For this known device has the same restrictions as above for the US-PS 38 11 777 executed.  

In the device described in US-PS 40 84 905 that serves same, continuous light that the chlorophyll fluorescence plant Licher examination objects changed, also as a measuring light for excitation of fluorescence. The detector system cannot distinguish between the two the measuring light excited fluorescence on the one hand and scattered strong light or the fluorescence excited by external light on the other hand if this as longer-wave radiation passes the filter in front of the photo penetrate detector. This means that this device is not suitable external, unfiltered light for illuminating the vegetable subs object to use. Another disadvantage of this known fluoro meters consists in the fact that, due to the given measuring head geometry Use an optical filter between the internal LED light source and the object under examination is prevented, so that in practice he Significant part of the measurement signal on the longer-wave portion of the measurement light is to be attributed, which from the object under investigation to the transparent LED resin body and the optical filter in the Photodetector is reflected.

In GB-PS 21 31 165 a device for measuring extremely small, repetitive pulse light signals described. This facility, which is based on a special light amplification process, is but not capable of weak light pulses with simultaneous overload measurement of non-pulsed light signals of high intensity.

In DE-OS 33 03 510 is a computer-controlled chlorophyllfluoro meters described. This facility uses conventional means for exposure and signal acquisition via optical fiber optics. Are the measuring light and the strong light, which show the changes in chloro phyllfluorescence, identical. The measuring system is also through External light interference disturbed. An exposure of the herbal pro be with unfiltered light (e.g. sunlight) is in the known on direction not possible.  

The pulse fluorometer described in DE-PS 29 14 147 works with strong pulse light from a flash discharge lamp, which in the case of photosensitive examination objects to change them during of the measuring process. These changes occur to an even greater extent when the pulse flash frequency is high. This is the familiar one direction unable to detect temporal changes in fluorescence To track the prey of light-sensitive objects under investigation. Next this device is not suitable for overlaying the pulse signals separate the external signals when the intensity of the extraneous light tion lies in the size range of the pulse flash intensity and if fast Changes in the intensity of external exposure occur.

When observing certain objects to be examined, however, it is necessary to use measuring light of very low intensity with simultaneous superimposition of very strong external radiation that changes rapidly in intensity. An example from photobiology is the observation of light-induced chlorophyll fluorescence changes (Kautsky effect), where ratios of measuring light / strong light from ¹ / ₁₀ ⁴ to ¹ / ₁₀ ^{7 are} required.

The present invention is therefore based on the object of a fluoro to create meters, which uses a sufficiently weak measuring light, the even very light-sensitive objects to be examined do not change and that is still able to selectively choose between the weak, from the measuring light excited fluorescence and much stronger superimposed signals differentiate.

This problem is solved with a fluorometer according to the Oberbe handle of claim 1 by its characteristic features.

When using extremely short light pulses, the periodic fluorescence stimulate, the pulse intensity can be relatively high without the sub search object in the time average of strong exposure to light sets, provided the distance between two measuring pulses is relative to the pulse is great. Short pulse signals can also be advantageously used Separate selective amplification systems from superimposed external signals.

Advantageous refinements are characterized in the subclaims:  

The use of a PIN photodiode as a photodetector is advantageous for Use at high pulse frequencies and at high intensities.

Switching from low to high pulse frequencies causes an increase signal / noise ratio.

Shorting the photodiode amplifier is required to Irradiation of very strong, short flash exposure an oversaturation of the Prevent amplifier system.

The automatic zero value compensation of the measurement signal serves to suppress kung the non-variable from the variable part of the fluorescent light.

According to claim 6 to use a two-armed fiber bundle attaches Advantages that the main elements of the measuring head are flexible and spaced can be arranged by the object under examination, with high utilization of the given measuring light and the resulting fluorescence.

The central openings according to claim 7 allow a particularly compact Arrangement of the individual parts and the associated high efficiency of the Light emission and the fluorescence collection.

The use of an LED according to claim 8 with higher light intensity makes there is no need for an additional strong light source. It is advantageous that this Light source can be switched inertially and synchronously.

According to claim 9 to use a one-armed fiber bundle brings the advantage partly that this is pressed onto the object under investigation that can and that the entire fluorescence radiation without loss of scatter in the measuring head can be guided.

By arranging the strong light source according to claim 10 immediately below the examination subject and without the interposition of an optical filter there is a particularly intense exposure to light.

One field of application of the invention relates to the measurement of light-induced Ver changes in chlorophyll fluorescence (Kautsky effect) on living plants.  

These changes allow an insight into the photosynthetic behavior of the living plants and thus a diagnosis of the plant condition. Every damage to the plant is expressed in a characteristic ver change in the fluorescence induction curve.

The invention is illustrated below with reference to the drawings Described embodiments of the fluorometer. In the Shows drawings

Fig. 1 is a functional block diagram of the fluorometer,

FIG. 2a, b a schematic diagram of the fluorometer,

Fig. 3 is a time chart for explaining the switch positions in the selec tive clipping amplifier,

Fig. 4 is a block diagram of the automatic zero point compensation,

Fig. 5 shows an embodiment of the measuring head M with a two-armed fiber optics,

Fig. 6 is a schematic representation of a Meßkopfausführung with measuring light guide through central openings in the PIN photodiode, and in that arranged before the optical filter,

Fig. 7 is a schematic representation of a Meßkopfausführung with an armed fiber optics,

Fig. 8 is a schematic representation of a measuring head design with two LEDs arranged one behind the other with measuring light and strong light functions, and

Fig. 9 is a measurement curve of the time-dependent change in chlorophyll fluorescence yield at the dark / light transition.

The fluorometer shown in Fig. 1 in a functional block diagram consists of a measuring head M , a control and regulating device STR and optionally switchable devices for automatic zero value compensation 23 and for strong light exposure 18 a , 18 b , 19th A specific measurement of the pulse fluorescence is achieved by the combination of optical filters 4, 6 and selective amplifier elements 8, 11 . The LED light source 1 is operated by the pulse driver 2 via the decimal counter 12 with ¹ / ₁₀ the frequency of the clock generator 9 and irradiates the sample 5 to be examined through the first optical filter 4 , which only allows light to pass below half a predetermined wavelength. The short-wave excitation light excites long-wave fluorescence in the sample 5 in common mode and in phase. The second optical filter 6 , which is located between the sample 5 and the photodetector 7 , allows the long-wave fluorescence to pass while it absorbs the shorter-wave excitation light. The longer-wave portion of the simultaneous exposure to a light source referred to below as a strong light source of high intensity 19 or in the case of uncontrolled incidence of extraneous light on tre tending scatter, reflection and fluorescence radiation reaches the photodetector 7 via the optical filter 6 and generates a measurement signal there , which is superimposed on the pulse fluorescence signal. The total measurement signal is fed to the preamplifier 8 , which is designed as a fast pulse amplifier and thereby largely separates the clocked pulse fluorescence signal and the non-clocked strong or external light signal. When using strong light sources with steep switching edges, the separation of the pulse signal and the rapidly changing interference signal is not yet sufficient. A complete separation takes place only in the subsequent selective section amplifier 11 . For the function of the amplifier system, it is crucial that both the pulse driver 2 and the selective cut-out amplifier 11 , as well as the on / off switch 18 a , 18 b of the strong light source, and the switch S 3 in the preamplifier 8 ( FIG. 2a) are functionally coupled to the clock generator 9 via the decimal counter 12 and the logic switching elements in the means for synchronization 10 in a time-defined manner.

According to FIGS. 2a and 2b, the clock generator 9 controls the pulse driver 2 and the selective cutout amplifier 11 via a decimal counter 12 . Furthermore, the same decimal counter 12 delivers synchronizing pulses for controlling the means for synchronization 10 at one of its outputs. The fluorescence pulses are received by the photodetector 7 , processed in the preamplifier 8 and the impulse signal is fed to the selective section amplifier 11 . In the preamplifier 8 the RC high-pass filter R 1 , C 1 separates the fast pulse signal from slower or continuous interference signals. The switch S 3 is used for short-circuiting the amplifier input to prevent over-saturation of the amplifier system when using flashes of light. In the selective cut-out amplifier 11 , which is connected downstream of the pre-amplifier 8 , control two switching pulses obtained from the clock generator 9 via the decimal counter 12 at its output "0" after suitable delay in the delay elements V 1 and V 2, two switches S 1 and S 2 , for the purpose of temporally selective takeover of the fluorescence pulse signal coming from the preamplifier 8 .

The relative temporal position of the light pulse 3 of the LED 1 , the signal 13 coming from the amplifier 8 and the switching pulses coming from the delay elements V 1 and V 2 are shown in FIG. 3.

Downstream of the switches S 1 and S 2 are two storage capacitors C 2 and C 3 , which take over the cut-out signal values 15 and 16 ( FIG. 3) for each measurement pulse and store them at the two inputs of a downstream instrument amplifier 14 until the next measurement pulse. At the output of an instrument amplifier 14 , the measurement signal is present as a difference signal between the extreme values 15 and 16 of the charge and discharge pulse, which is a measure of the respective fluorescence yield of the object under investigation.

The clock generator 9 is switched by means of the switch 21 by a pulse from the means for synchronization 10 from a low to a high frequency and vice versa. Switching the clock generator 9 from low to high frequency serves to increase the signal / noise ratio. The associated increase in measuring light intensity is negligible in its effect with simultaneous irradiation of strong light.

So that the steep signal edges occurring when switching on and off strong light sources with steep switching edges cannot coincide with the signals 13 in time, 12 decimal counters generated sync pulses are used to switch the strong light source on and off, a logic circuit L 1 ensuring that the synchronizing pulses only trigger a switch-on trigger pulse when the start button 17 is actuated and a second logic circuit L 2 in conjunction with a digitally adjustable delay element 20 selects a synchronous pulse to trigger the switch-off trigger pulse.

At the output of the instrument amplifier 14 in the selective cut-out amplifier 11 ( FIG. 2a) there is an optionally switchable device 23 for automatic zero value compensation of the measurement signal. The auxiliary device according to Fig. 4 consists of a zero-value compensation button 24 on one Be actuation a binary counter 25 is driven. This is a resistance chain 26 connected downstream, which generates variable voltages to compensate for the present measurement signal at its output. These voltages are compared in a comparator 27 with the given measurement signal voltages and the comparison result causes the binary counter to an up or down count and the associated correction of the compensation voltage at the output of the resistance chain 26th This counting process continues until the zero value compensation is reached. The compensation value is retained as soon as the button 24 opens again. The compensation voltage and the measurement signal are subtracted from each other at the differential amplifier 28 and the difference signal is evaluated.

In Fig. 5 an embodiment of the measuring head M with two-armed fiber optics (optical fiber bundle 31, 32 ) is shown. In the shielding housing 29 , the light from a light-emitting diode (LED) 1 falls through the first optical filter 4 onto the one optical fiber bundle 30 and irradiates the sample 5 . Furthermore, the shielding housing 29 contains a photodetector 7 with the two-th optical filter 6 , which receives the fluorescent pulse signal through the second optical fiber bundle 31 . The fibers of the two optical fiber bundles 30, 31 are combined and mixed at a common outlet end 32 .

A further embodiment of the measuring head M is shown in FIG. 6. 1 shows an LED, 4 a first optical filter, 35 a PIN photodiode with a central opening 33 , and 36 a second optical filter with a central opening 34 in a shielding housing 29 , through the opening 37 of which the fluorescence of the sample 5 to be examined is excited . The arrangement of the PIN photodiode 35 and the optical filter 36 , each with central openings, enables irradiation of the sample 5 from a short distance, with the associated optimal use of the given LED light intensity and, at the same time, effective collection of the fluorescent light.

Fig. 7 shows an embodiment of the measuring head M analogous to FIG. 6 but each with a coupled to the opening 37 , one-armed optical fiber bundle 38 , which is pressed onto the sample 5 without a gap. Due to this arrangement, fluorescence measurements on difficult-to-access research objects are made possible, while at the same time making optimal use of the given LED light intensity and effectively collecting the fluorescent light.

In FIG. 8, the measuring head is M analogous to that of FIG. 6, however, is located between the opening 34 and the opening 37, a further LED 39 with a transparent resin body. The other LED 39 can be irradiated by the LED 1 in this order. In addition, the further LED 39 delivers unfiltered strong light for exposing the sample 5 up close. At the same time, a sufficient case of fluorescent pulse light on the PIN photodiode 35 is ensured in this arrangement.

Fig. 9 shows a chlorophyll fluorescence induction curve of a spinach leaf measured with the fluorometer during the transition from dark to light. In the present measurement example, the intensities of measurement light / strong light / saturation pulses behave like ¹ / ₁₀ ⁴ / ₁₀ ⁶ . The strong light causes changes in the fluorescence yield in connection with the course of photosynthesis in the leaf; the saturation pulses are used to determine the maximum fluorescence yield.

Claims (10)

1. Fluorometer for measuring light-induced changes in the fluorescence yield of a sample to be examined with a measuring head in a shielding housing, a control and regulating device, an excitation light source, means for generating light pulses from the excitation light source, a photodetector for receiving the fluorescence pulses, a first optical filter between Excitation light source and the sample to be examined, a second optical filter between the sample and the photodetector, a high-intensity light source for light-induced change of the sample, and with means for synchronizing the excitation light source, the high-intensity light source, the photodetector and a signal amplifier, characterized in that

• - The excitation light source is a light emitting diode ( 1 ) (LED);
• - The means for generating the light pulses ( 3 ) of the LED ( 1 ) consist of a pulse driver ( 2 ) which periodically supplies rectangular current pulses in the µsec range;
• - The first optical filter ( 4 ) is transparent below one of the wavelengths predetermined by the LED emission spectrum;
• - The second optical filter ( 6 ) is transparent above this predetermined wavelength;
• - A preamplifier ( 8 ) is connected to the photodetector ( 7 ), the preamplifier ( 8 ) for separating the periodically rectangular current pulses in the µsec range from overlaid, slower interference signals, an RC high-pass filter (R 1 , C 1 ) having;
• - The control and regulating device (STR) a clock generator ( 9 ) for generating control pulses for the pulse driver ( 2 ) and for a selective cut-out amplifier ( 11 ), and the means for synchronizing ( 10 ) the light pulses ( 3 ) of the LED ( 1 ), the light source of high intensity ( 19 ) and blanking pulses for short-circuiting the preamplifier ( 8 );
• - The selective section amplifier ( 11 ) has two switches (S 1 , S 2 ), which are controlled by the clock generator ( 9 ) via switching pulses which are generated by means of a decimal counter ( 12 ) and by means of delay elements (V 1 , V 2 ), So that a temporally selective takeover of the incoming signal from the preamplifier ( 8 ) ( 13 ) on two storage capacitors (C 2 , C 3 ) at the two inputs of an instrumentation amplifier ( 14 ) is ensured, the switches (S 1 , S 2 ) are closed only for short periods of time in the maximum of one charge pulse ( 15 ) and in the minimum of one subsequent discharge pulse ( 16 );
• - The means for synchronization ( 10 ) when actuating a start button ( 17 ) trigger pulses for actuating an on / off switch ( 18 a, b) of the high-intensity light source ( 19 ), the generated by the clock generator ( 9 ) Switching pulses by means of the decimal counter ( 12 ) are shifted in time so that they fall in the middle between two light pulses ( 3 ) of the LED ( 1 ), and a logic circuit (L 1 ) ensures that the switching pulses only Trigger trigger pulse when the start button ( 17 ) is actuated, and wherein a second logic circuit (L 2 ) in conjunction with a digitally adjustable delay element ( 20 ) selects a switching pulse to trigger the switch-off trigger pulse.

2. Fluorometer according to claim 1, characterized in that the photodetector ( 7 ) is a PIN photodiode, with sufficiently fast response time for detecting the fluorescence pulses in the µsec range and with high linearity of the output current over a wide light intensity range. 3. Fluorometer according to claim 1 or 2, characterized in that the light source of high intensity ( 19 ) is a flash discharge lamp, and that the switching pulse triggered when the start button ( 17 ) is triggered by a pulse generator ( 22 ) for a defined duration Actuation of a switch (S 3 ) for short-circuiting the preamplifier ( 8 ) is generated. 4. Fluorometer according to one of claims 1 to 3, characterized in that the actuation of the on / off switch ( 18 a, b) of the light source of high intensity ( 19 ) of the clock generator ( 9 ) by means of one of the means for synchronization ( 10 ) controlled switch ( 21 ) is switched from a low to a high frequency and vice versa. 5. Fluorometer according to one of claims 1 to 4, characterized in that the control and regulating device (STR) is supplemented with an optionally switchable additional device ( 23 ) for automatic zero value compensation of the measurement signal, consisting of a clock generator ( 9 ) when actuated a zero value compensation button ( 24 ) controlled Bi närzähler ( 25 ) with a downstream resistor chain ( 26 ), which generates output voltages for compensation of the present measurement signal, which is first compared in a comparator ( 27 ) with the measurement signal to who the binary counter ( 25 ) to cause an upward or downward count, and that then in a differential amplifier ( 28 ) the diffe rence voltage is formed between the measurement signal and the compensation voltage, the differential voltage fluctuating statistically around the zero value after a sufficient counting period, and after switching off the clock pulses each because the existing compensation value is retained. 6. Fluorometer according to one of claims 1 to 5, characterized in that in the measuring head (M) the LED ( 1 ) and the photodetector ( 7 ) with the filters ( 4, 6 ) side by side in the shielding housing ( 29 ) is arranged are, and that the LED ( 1 ) and the photodetector ( 7 ) via separate fiber optic bundles ( 30, 31 ) at a common exit end ( 32 ) are merged and mixed, and that the exit end ( 32 ) of the combined optical fiber bundle at a short distance is held by the sample to be examined ( 5 ). 7. Fluorometer according to one of claims 2 to 5, characterized in that the LED ( 1 ), the first optical filter ( 4 ), the PIN photodiode ( 35 ) and the second optical filter ( 36 ) are arranged one above the other and that LED ( 1 ) through the first optical filter ( 4 ) and through central openings ( 33, 34 ) of the overlying PIN photodiode ( 35 ) and the second optical filter ( 36 ) via an opening ( 37 ) of the shielding housing ( 29 ) located sample ( 5 ) irradiated, and that the fluorescence excited there by the second optical filter ( 36 ) hits the PIN photodiode ( 35 ). 8. Fluorometer according to one of claims 1 to 7, characterized in that the LED ( 1 ) in addition to the periodic measuring light pulses also provides continuous light of higher intensity, the on / off switching device of the continuous light component of higher intensity by the means for Synchronization ( 10 ) is controlled. 9. Fluorometer according to claim 8, characterized in that the LED ( 1 ) and the PIN photodiode ( 35 ) via one end of an optical fiber bundle ( 38 ) with the sample ( 5 ) are connected, and that the other end of the optical fiber bundle is spaced apart can be pressed onto the sample so that both excitation light and continuous light of higher intensity from the LED ( 1 ) via the optical fiber bundle ( 38 ) irradiate a sample ( 5 ) located away from the measuring head and the fluorescence excited there via the same fibers of the optical fiber bundle is fed back into the measuring head. 10. Fluorometer according to claim 7, characterized in that there is a further LED ( 39 ) with a transparent synthetic resin body in the beam path between the first optical filter ( 4 ) and the sample ( 5 ), which delivers continuous light of higher intensity.