DE3905348A1 - Arrangement for measuring the irradiation power occurring in a laser scanning microscope or the light power reflected and diffusely scattered on the irradiated object or the difference between the two powers - Google Patents

Abstract

In the arrangement, the photoelements 1 and 2 are arranged in such a manner that the light in the optical beam path between light source and object either passes through both photoelements or is reflected at them or passes through one photoelement and is reflected at the other, and that either both photoelements are connected together electrically in anti-parallel to form a two-port network or both photoelements are electrically connected on one side with respectively one input of the weighted-summing current/voltage converter 3, while their counter-electrodes are respectively at a constant reference potential. By means of the weighted summation or difference formation, with a corresponding selection of amount and sign of the weighting factors, an output signal proportional to the irradiated, reflected or absorbed light power results. The measuring arrangement is particularly suitable for integration in a microscope objective or for the construction of a measuring probe independent of the microscope. <IMAGE>

Description

The invention relates to an arrangement for measuring the a laser radiation microscope or reflected on the irradiated object and diffused light output or the difference between the two Services. Laser scanning microscopy comprises one group non-destructive imaging processes based on local Differences in optical and optically stimulated interactions processes and enables the examination of lateral Structures of technical and biological micro objects.

In the known laser scanning microscopes, the most Object reflected laser power (e.g. Wilke, V .: Scanning 7, 1985, 89-96) and / or the primary irradiated Light output analogous to be in DE-PS 21 40 335 written constant light photometer with separate photoelek trical transducers, such as photo elements, photodiodes or Measured photomultipliers, the separation of the Lei shares with the help of optical beam splitters, that each transducer is only from the reflected or only  is hit by the primary laser radiation. A Be the difference between the primary irradiated and the right inflected light output, which is not trans Parent objects of the absorbed radiation power ent speaks and specifically in the photoacoustic and photother paint laser scanning microscopy for separation on the one hand thermal and thermoelastic and on the other hand pure Optical contrasts can be achieved either by Subtraction of the electrical output quantities of the corresponding the photo receiver or provided a con constant laser power by forming the reflection sig nal complementary signal. Because here in particular the investigation of highly reflective objects in comparison small differences to the output quantities are formed a very precise sensitivity adjustment of the photoreceivers required. The disadvantage here is that the transmission degree of imaging optics between beam splitter and object the sensitivities of both photo receivers or at con constant laser power the sensitivity of the Re flex light detector and the primary arriving at the object Radiant power affected to different degrees, so that transmittance changes when changing lenses or due to contamination of optical surfaces, the measuring accuracy for severely impair the absorbed radiation power or require a new adjustment.

Another disadvantage of the known non-confocal arrangement voltage lies in the high background signal in an image in reflected light that comes from reflections on everyone between Beam splitter and object-located glass-air interfaces results. Because beam splitter to maintain acceptable picture however, errors only converge in telecentric or weakly th or divergent beam path can be used, is at least the amount of reflected light caused by the lens in principle not to be eliminated in the known arrangements.  

The aim of the invention is to increase the measurement accuracy and ease of handling a laser Scanning microscope, especially when determining the absorber radiated power of highly reflective objects like a contrast enhancement in the non-confocal Scanning light laser scanning microscopy.

The invention has for its object in a laser Scanning microscope the influence of opti surfaces between light source and lens or at Changes in transmission occurring lens changes on the sensitivity adjustment especially when measuring the degree of absorption to avoid and the stray light below reason to lower in reflection mode.

This object is achieved by an arrangement for measuring the radiation radiation occurring on a laser scanning microscope or the light output reflected and diffusely scattered on the irradiated object, or the difference between the two services, consisting of two scattering layers which are sufficiently transparent or sufficiently reflective at the laser wavelength used -Photoelements 1 and 2 as well as optionally a weighted summing current / voltage converter 3 solved, according to the invention the Photoele elements 1 and 2 are arranged so that the light in the optical beam path between the light source and the object either passes through the photo elements or both is reflected from them or a photo element is shone through and is reflected at the other that the light-sensitive surfaces of both photo elements completely enclose the incident beams and that either both photo elements are electrically connected antiparallel to a two-pole or he two photo elements are electrically connected on one side to each input of the weighted summing current / voltage converter 3 , while their counter electrodes are each at a constant reference potential.

The photo elements are advantageously ver together tied or at least one of them is in La with one this scanning microscope firmly existing optical element connected to a unit.

The photo elements are advantageously sufficiently trans Parent thin-film systems on the lens surfaces of a micro lens attached.

In the arrangement according to the invention, at least one of the Photo elements as an optical mirror surface in a mirror or mirror lens objective can be arranged.

Advantageously, both are and at least two resistances influencing the weighting factors, the functional components of the weighted summing current / Voltage converters are too, with the same microscope objective connected to a unit and carries the microscope objective if necessary, electrical contact on its outer surface surfaces with the photo elements and if necessary with the weighted summing current / voltage converter or components integrated in the lens are.

The arrangement of both photo elements in the illuminating beam path ensures that they also meet the light reflected on the object again, so that their photo currents I ₁ and I ₂ correspond to the relationships I ₁ = E ₁ P + E ₂ PR and I ₂ = E ₃ P + E ₄ PR with the current sensitivities E ₁ , E ₂ , E ₃ and E _{4 of} the photo elements and the reflection coefficient R of the object surface based on the irradiation power P of the laser that can be measured on the object or the light power PR reflected on the object in focus each from one of the primary incident and one of the reflected light power on the object proportional portion, whereby due to the weakening of light by the photo elements, the inequality E ₂ E ₃ < E ₁ E ₄ applies. The photo elements can be operated electrically both in the short circuit and pre-tensioned in the reverse direction.

Since in expressions I ₁ (P, PR) and I ₂ (P, PR) the relative proportions of the primary radiated and reflected power are different due to the inequality, it is always possible to use a linear combination α I ₁ + β I ₂ to form with the rational constants α and β , which is proportional only to the radiation power P or only the reflected power PR or the difference between the two powers P (1 - R) .

The weighted summing current / voltage converter may serve this purpose, the output voltage U = α I ₁ + β I ₂ resulting in U = ( α E ₁ + β E ₃ ) P + ( α E ₂ + β E ₄ ) PR . A voltage proportional to the irradiation power is obtained when β = - α E ₂ / E ₄ , a reflected power per proportional voltage for β = - α E ₁ / E ₃ and a voltage proportional to the difference between the two powers under the condition α E ₁ + β E ₃ = - ( α E ₂ + β E ₄ ) or β = - α (E ₁ + E ₂ ) / (E ₃ + E ₄ ). When using a current / voltage converter which forms several linear combinations of the input signals which are weighted differently in accordance with the above relationships, the power components mentioned can also be measured in parallel. With direct anti-parallel connection of the photo elements, the individual photocurrents subtract from one another, so that the total current results in I = I ₁ - I ₂ = (E ₁ - E ₃ ) P + (E ₂ - E ₄ ) PR . For E ₂ = E ₄ this current is proportional to the irradiation power P , for E ₁ = E ₃ the reflected power and for E ₁ + E ₂ = E ₃ + E ₄ the difference between the two services. Here, the sensitivities of the Photoele elements must be matched once so that the desired proportionality results. This coordination is tied to a fixed transmittance of the photo elements and the optical path between them and to a certain light transmission of the distance between the photo elements and the object. Therefore, this variant is particularly suitable for integrating the photo elements into the lens used or for building a measuring probe independent of the microscope in the form of a transparent plane-parallel plate as a support for the photo elements, which is inserted at any point between the light aperture of the microscope and the object. The handling of the measuring arrangement is particularly simple if, in addition to the photo elements, the weighted sum current / voltage converter or the weighting-determining parts thereof are integrated into the lens used and are adjusted to a standard sensitivity that is the same for all lenses at the output. When using such prepared lenses in a lens changer, this is advantageously mechanically coupled to an elec trical switch which contacts the lens currently in the beam path or its electrical assemblies.

The advantages of the arrangement according to the invention exist against over the prior art in that by minimizing the optical path between object and photoelectric beam lungsmeßwandlern against independence of the measurement signal about changes in transmittance of the entire upstream Light path is achieved, leading to greater stability and too detection limit shifted to lower values the measurement of the absorbed light output as well as a substantial reduction in the background signal and thus to an expanded dynamic range when measuring the re inflected light output and that continues with Inte gration of the photo elements in microscope lenses and solid Adjustment to a specified standard measuring sensitivity or when inserting the photo elements between the lens and There is no need to compare objects when changing lenses and there with the operating effort is reduced.

In the drawings, the invention order shown in different variants.

Fig. 1 shows the photo elements 1 and 2 , the weighted sum-current / voltage converter 3 , the glass plate 4 , the lens 5 and the object 6th In

Fig. 2, the photoelements 1 and 2 and the object 6 and the lens 7 are shown.

Fig. 3 shows the photo-elements 1 and 2, the object 6, the metal mirror 8 and 9, the differential-current / voltage converter 10 and the voltage source 11.

example 1

Fig. 1 shows a possible implementation of the arrangement according to the Invention. Here, the Si photo elements 1 and 2 are deposited as large-area homogeneous thin-film systems on both sides of a plane-parallel glass plate 4 , the layer thicknesses being so small that the optical transparency is above 50%. The glass plate is arranged between the microscope objective 5 and object 6 as an optical window in a special sample holder for carrying out photothermal examinations so that it is irradiated by the convergent light beam of the laser scanning microscope without limiting effects in its light-sensitive area. It has the thickness and refractive power of a normal cover glass, so that no additional aberrations occur when using a correspondingly corrected microscope objective.

Since both photo elements are hit by both the light running towards the object and the light reflected there, their photocurrents I ₁ and I ₂ settle in accordance with the relationships I ₁ = E ₁ P + E ₂ PR and I ₂ = E ₃ P + E ₄ PR with the sensitivities E ₁ and E ₃ related to the measurable radiation power P on the object and the sensitivities E ₂ and E ₄ related to the reflected or diffusely scattered light output PR emanating from the object each from one of the primary irradiated and one of the objects reflected radiation power proportional proportion, whereby due to the weakening of light when passing through the photo elements E ₂ / E ₁ < E ₄ / E ₃ applies. The photo currents of both photo elements are, as can be seen in Fig. 1, with opposite polarity in the inputs of the weighted summing current / voltage converter 3 with the adjustable weighting factors K ₁ , K ₂ , l ₁ , l ₂ , m ₁ , m ₂ fed in, so that the voltages U ₁ = K ₁ I ₁ - K ₂ I ₂ = (K ₁ E ₁ - K ₂ E ₃ ) P + (K ₁ E ₂ - K ₂ E ₄ ) PR , U ₂ = l ₁ I ₁ - l ₂ I ₂ = (l ₁ E ₁ - l ₂ E ₃ ) P + (l ₁ E ₂ - l ₂ E ₄ ) PR and U ₃ = m ₁ I ₁ - m ₂ I ₂ = (m ₁ E ₁ - m ₂ E ₃ ) P + (m ₁ E ₂ - m ₂ E ₄ ) PR set.

To measure the primary radiated power, K ₁ and K _{2 are selected in} accordance with the condition K ₂ = K ₁ E ₂ / E ₄ and a measurement sensitivity U ₁ / P = K ₁ (E ₁ - E ₂ E ₃ / E ₄ ) is obtained. To measure the reflected power, one chooses l ₁ and l ₂ according to the condition l ₂ = l ₁ E ₁ / E ₃ and obtains a measuring sensitivity U ₂ / PR = l ₁ (E ₂ - E ₁ E ₄ / E ₃ ). To measure the difference between the primary radiated and reflected power, select m ₁ and m ₂ according to the condition m ₂ = m ₁ (E ₁ + E ₂ ) / E ₃ + E ₄) and obtain a measuring sensitivity U ₃ / P (1 - R) = m ₁ (E ₁ E ₄ - E ₂ E ₃ ) / E ₃ + E ₄ ).

To determine E ₁ and E ₃ , the arrangement is irradiated in its normal direction of use and the transmitted power P and the photocurrents I ₁ and I _{2 are} measured. To prevent the arrangement from being hit by reflected light, it is kept I ₁₁ = E ₁ P and I ₂₁ = E ₃ P or E ₁ = I ₁₁ / P and E ₃ = I ₂₁ / P. If you now irradiate the arrangement in the opposite direction to the normal beam direction with an irradiation power P and again prevent the arrangement from being hit by reflected light, this corresponds to the returning beam in the usual direction of use and the photocurrents I ₁₂ = E ₂ P and I ₂₂ = are obtained E ₄ P or E ₂ = I ₁₂ / P and E ₄ = I ₂₂ / P. With the example values E ₁ = 1.25 mA / W, E ₂ = 0.8 mA / W, E ₃ = 1.25 mA / W, E ₄ = 1 mA / W, K ₁ = 4 V / µA, l ₁ = 5 V / µA and m ₁ = 9 V / µA and choice of K ₂ , l ₂ and m ₂ according to the above conditions gives K ₂ = 3.2 V / µA, l ₂ = 5 V / µA and m ₂ = 8.2 V / µA and thus the measuring sensitivities U ₁ / P = 1 V / mW, U ₂ / PR = -1 V / mW and U ₃ / P (1 - R) = 1 V / mW. This means that all the named radiation components can be measured simultaneously, regardless of the optical system of the laser scanning microscope used for imaging, and you also get the reflection coefficient R and the absorption coefficient A = 1 - R for any irradiated laser power as quotients of the corresponding one Output voltages of the weighted summing current / voltage converter. The measuring arrangement is independent of focusing fluctuations due to the integral light detection and has a minimal temperature drift due to the close thermal coupling of the photo elements. Since there are no further glass-air interfaces between the measuring arrangement and the object, the formation of a background signal due to undesired reflections is excluded.

Example 2

Fig. 2 shows a variant of the arrangement for measuring the absorbed by the examination object radiation power in a laser-scanning microscope, in which the Si-photoelements 1 and 2 over each other as large-area homogeneous, sufficiently transparent thin-film systems, on the front lens 7 of a microscope objective are deposited. They are electrically connected antiparallel, so that the externally measurable photocurrent results as the difference between the two individual currents. Since they are hit by both the incoming and the reflected light when the object is irradiated as in Example 1, the individual photocurrents with the descriptions of Example 1 result in I ₁ = E ₁ P + E ₂ PR and I ₂ = E ₃ P + E ₄ PR or the resulting total current to I = I ₁ - I ₂ = (E ₁ - E ₃ ) P + (E ₂ - E ₄ ) PR . A signal proportional to the radiation power absorbed in the object P (1 - R) results for E ₁ - E ₃ = E ₄ - E ₂ and one obtains I = (E ₁ - E ₃ ) P (1 - R) . The above condition can be met by applying an additional light-reducing layer with the transmittance T on the side of the arrangement facing the object. Here the sensitivities are modified to E ′ ₁ = TE ₁ , E ′ ₃ = TE ₃ , E ′ ₂ = E ₂ / T and E ′ ₄ = E ₄ / T, and the result is (E ′ ₄ - E ′ ₂ ) / (E ′ ₁ - E ′ ₃ ) = (E ₄ - E ₂ ) / T ² (E ₁ - E ₃ -). Because of 0 < T ² <1, the condition E ′ ₄ - E ′ ₂ = E ′ ₁ - E ′ ₃ for the modified sensitivity constants can always be met if the original layer preparation meets the condition 0 < (E ₄ - E ₂ ) / (E ₁ - E ₃ ) <1 is sufficient. The closer the light is to 1, the closer the quotient (E ₄ - E ₂ ) / (E ₁ - E ₃ ) is.

A one-off matched in this way into a microscope Objectively integrated measuring arrangement generated in the short circuit drove an output current which is independent of radiated Light output, reflectance of the object and changes in the optical beam path between light source and lens the difference between irradiated and reflected or back scattered light output and thus the absorbed beam power is proportional. Advantages of the arrangement are  their insensitivity achieved by the integral light measurement sensitivity to fluctuations in focus and the low Temperature drift due to the tight thermal coupling of the Photo elements.

Example 3

Fig. 3 shows a further variant of the arrangement for measuring the radiation absorbed by the object under examination in a laser scanning microscope, in which two Si photo elements 1 and 2 similarly as large-area homogeneous thin-layer systems on which act as rear electrodes, the metal mirrors 8 and 9 of a mirror lens are so abge that their partially transparent cover electrodes simultaneously form the optically effective mirror surfaces of the lens. Your cover electrodes are connected with the same polarity to the two inputs of a weighted differential current / voltage converter 10 and at the same time to two resistors which are also integrated in the mirror lens and which are functional components of the differential current / voltage converter and its for the two photocurrents Determine effective current / voltage conversion factors, while the back electrodes are connected to a voltage source 11 , the second terminal of which is at the reference potential of the current / voltage converter 10 and whose polarity is selected such that the photo elements are biased in the reverse direction. Since the photo elements are again hit by both the incoming and the reflected light and the subtraction of the photocurrents also takes place due to the selected circuit principle, a more detailed explanation of the mode of operation and the determination of the necessary weighting factors determined with the help of the resistors integrated in the lens for both photocurrents refer to Example 1. The contacting of the photo elements and resistors is done in the example by a multiple connector.

As a result, the arrangement delivers after determining the weight an output voltage that is independent of  radiated light output, reflectance of the object and Changes in the optical beam path between the light source and lens the difference between irradiated and reflect tated or backscattered light output and thus the sorbed radiation power is proportional. It is in follow the integral light measurement also insensitive against fluctuations in focus and also enables the use of opaque photo elements with conventional len semi-transparent metallic cover electrodes and thus a higher transmission of the overall lens. In addition ver avoids disrupting the arrangement with conventional observation of extraneous light, which is insufficient when transmitted mirrored photo elements.

Claims (5)

1.Arrangement for measuring the radiation power occurring on a laser scanning microscope or the light power reflected and diffusely scattered on the irradiated object or the difference between the two lines, consisting of two low-scatter, sufficiently transparent or sufficiently reflective barrier layer photo elements at the laser wavelength used ( 1 ) and ( 2 ) and optionally a weighted summing current / voltage converter ( 3 ), characterized in that the photo elements ( 1 ) and ( 2 ) are arranged so that the light in the optical beam path between the light source and the object either the photo elements shines through or is reflected on them, or a photo element shines through and is reflected on the other, that the light-sensitive surfaces of both photo elements that completely impinge on the beam bundle and that either both photo elements are electrically connected antiparallel to a bipolar or both P Hotoelements are electrically connected on one side to an input of the weighted summing current / voltage converter ( 3 ), while their counter electrodes are each at a constant reference potential. 2. Arrangement according to claim 1, characterized in that the Photo elements are connected or at least one of them with one in the laser scanning microscope which optical element firmly connected to a structural unit is. 3. Arrangement according to claim 1 and 2, characterized in that the photo elements as sufficiently transparent thin layer systems on the lens surfaces of a microscope objective are deposited.   4. Arrangement according to claim 1 and 2, characterized in that at least one of the photo elements as an optical mirror gel surface in a mirror or mirror lens lens is arranged. 5. Arrangement according to claim 1 to 4, characterized in that both photo elements and at least two the weighting factor influencing resistances that are functionally Be parts of the weighted summing current / voltage Transducers are one with the same microscope objective Unit connected and that the microscope lens ge possibly electrical contact on its outer surface bearing surfaces with the photo elements and given if with the weighted summing current / voltage Transducer or its stock integrated into the lens share are connected.