DE102017203742A1 - Method and device for measuring the spatial extent of the illumination spot image function - Google Patents

Abstract

The invention relates to a method and a device for measuring the spatial extent of the illumination spot image function in multi-photon microscopy. The task is to measure the spatial extent of the illumination spot image function with little effort and high accuracy. The measuring device, should work robustly without great adjustment effort and be usable by a small handy design especially for the universal use of routine measurements as well as in the service area. According to the invention, the distribution of a laser beam (2) to be evaluated by a polarization-independent or a polarization-dependent beam splitter (3) to a linear detector (4) and a non-linear detector (5) and by the quotient of a measurement signal (S1) of the linear detector (4) and a measurement signal (S) of the nonlinear detector (5), which in single and multiphoton interactions arise, the spatial half width determines from which information about the spatial extent of the illumination point image function in the multi-photon microscopy are derivable.

Description

The invention relates to a method and a device for measuring the spatial extent of the illumination spot image function in multi-photon microscopy. The spatial illumination spot image (PSF) measurement methods currently used in the high-end area of multi-photon microscopy are mostly based on scanning the spatial illumination spot image function using ultra-small beads or ultra-thin fluorescent layers (Brakenhoff) and require a large amount of technical expertise Effort to implement such methods. It must be prepared suitable samples, which are usually not too long lasting and affected by bleaching. Z-stacks must be measured and the data must be evaluated with FIT algorithms.

It is known that the measurement of the pulse duration of short light pulses up to a duration of 10ps by means of fast photodetectors directly and below this limit to about 0.5ps by means of streak cameras and in the picosecond and subpicosecond range indirectly via correlation methods. So is out of the DE 199 44 913 A1 a method and apparatus for pulse duration measurement of very short light pulses, in particular for pulse duration measurement of femtosecond and picosecond laser pulses with a beam splitting by the sub-beams of the same optical properties are separated and detected with respect to their signals in the method for pulse width measurement of very short light pulses, each different from depend on the pulse duration of the evaluated light pulses and that the detector signals of the partial beams are evaluated in relation to each other.

The apparatus for pulse duration measurement of very short light pulses comprises a beam splitter for splitting the light beam to be evaluated into two sub-beams to be detected with the same optical properties and two light receivers each having output signals different from the pulse duration for the sub-beams separated by the beam splitter. The light receivers are connected to an evaluation stage for quotient formation of the output signals of the light receivers.

Based on this prior art, the present invention seeks to provide a method for measuring the spatial extent of the illumination point image function in multi-photon microscopy, which allows the measurement of the spatial extent of the illumination point image function with little effort and high accuracy. Furthermore, a corresponding measuring device is to be proposed, which works robustly and without great adjustment effort and can be used in the service sector, in particular for the universal use of routine measurements, by means of a small, handy design.

To achieve this object, the invention proposes a method in which the spatial half-width is determined by the quotient of a measurement signal of a linear detector and a measurement signal of a nonlinear detector, which arise in one-photon interactions or multiphoton interactions. Information about the spatial extent of the illumination spot image function in multi-photon microscopy can be derived from these quotients. The measuring device (or in short: device) for measuring the spatial extent of the illumination point image function comprises a beam splitter for splitting the light beam to be evaluated into two partial beams to be detected. A laser beam emerging from a calibration lens is distributed by a polarization-independent or by a polarization-dependent beam splitter to a linear detector and to a nonlinear detector. The signals respectively determined by the linear detector and the nonlinear detector and amplified in amplifiers are provided or can be made available to an evaluation unit for forming a quotient. The quotient formation is provided for determining the spatial extent of the illumination spot image function. The use of photodiodes as linear and non-linear detectors (e.g., SiC) results in a broad spectral range of use given by the electronic band structure of the semiconductors. After measuring the two signals, the spatial, in particular 2D, expansion of the illumination point image function (2D-FWHM) can then be determined by means of a computer by quotient formation. For this purpose, after the measurement of the two signals by quotient formation, the spatial extent of the illumination point image function is determined by means of analog or digital evaluation units. A computer may be a known PC, but also a computer unit (eg CPU), which may also be integrated in or connected to an evaluation unit. The measuring device according to the invention is insensitive to climatic fluctuations, an adjustment is not necessary and thus accounts adjustment times. Furthermore, the measuring device has a small handy design.

It is advantageously provided that photodiodes having a broad spectral range are provided as linear and nonlinear detectors, wherein a significantly lower light component is sufficient for the linear detector than for the nonlinear detector.

Preferably, it is further provided that the band gap of the linear detector has a Photon excitation allows and the band gap of the nonlinear detector a two-photon excitation.

The invention is explained in more detail below with reference to an embodiment schematically illustrated in a drawing.

• 1 eine Prinzipdarstellung der Messeinrichtung.

It shows:

• 1 a schematic diagram of the measuring device.

1 shows a measuring device M and an integration of the measuring method into a calibration objective 1 for a multi-photon microscope 9 , The use of photodiodes as linear or non-linear detectors 4 . 5 (eg SiC) leads to a broad spectral range of application, which is due to the electronic band structure of the semiconductors of the detectors 4 . 5 given is.

The measuring device shown in its construction M has a calibration lens 1 on. On from the calibration lens 1 emerging laser beam 2 is by a polarization independent - or in another embodiment, a polarization-dependent- beam splitter 3 for example, in the form of a glass plate divided into two sub-beams and a linear detectors 4 and to a nonlinear detector 5 directed. In this case, the proportion of light for the linear detector 4 significantly lower than that for the nonlinear detector 5 fail. The bandgap of the linear detector 4 must allow one-photon excitation. The same applies to the nonlinear detector 5 which enables a two-photon excitation. The through the two detectors 4 . 5 Signals generated by electronic amplifiers 6 . 7 amplified to electrical signals and then an analog or digital evaluation 8th in which the spatial, in particular the 2D extension of the illumination point image function is determined by quotient formation by means of computer.

• S_n - Messsignal
• C_n - Proportionalitätsfaktor, material-(detektor-)spezifisch, z.B. kann darin der Absorptionsquerschnitt enthalten sein
• P_avg - mittlere Laserleistung
• τ - Impulsdauer
• f - Laser-Repetitionsrate
• A - Beleuchtungsquerschnittsfläche: A = π w_o ² w_o ist der 1/e^2-Radius und steht in unmittelbaren Zusammenhang zur Halbwertsbreite (FWHM) $w_{FWHM}=\frac{w_0}{2\cdot \text{ln}\left(2\right)}$
  

The method according to the invention is based on the quotient formation of a linear and non-linear measurement signal, as arises in the one-photon interaction or the multi-photon interaction: $$S_n=C_n\cdot \frac{P_{avG}}{{\left(\tau \cdot f\cdot A\right)}^{n-1}}$$

• S _n - measuring signal
• C _n - proportionality factor, material (detector) specific, eg it may contain the absorption cross section
• P _avg - average laser power
• τ - pulse duration
• f - laser repetition rate
• A - illumination cross-sectional area: A = π w _o ² w _o is the 1 / e ^ 2 radius and is directly related to the full width at half maximum (FWHM) $w_{FWHM}=\frac{w_0}{2\cdot \text{ln}\left(2\right)}$
  

The above calculation of the measurement signal S _n becomes for the linear detector 4 carried out, whereby the one-photon signal S _{1 is} obtained. Likewise, the calculation for the nonlinear detector 5 executed, whereby the multi-photon signal, in this example, the two-photon signal S ₂ , is obtained. The quotient formation of the squared one-photon signal S ₁ and the two-photon signal S ₂ leads to the quotient Q _Mess : $$Q_{Mess}=\frac{S_1{}^2}{S_2}=\frac{C_1{}^2}{C_2}\cdot \tau \cdot f\cdot \pi \cdot w_O{}^2$$

While the quotient Q _{measurement is used} for measuring the pulse duration in known measuring methods, in the present invention the use of the arrangement including the signal evaluation for determining the spatial half-width is important, since the quotient Q _Mess reacts very sensitively to changes in the spatial half-width. $$w_O{}^2=F_{\lambda }\cdot \frac{S_1{}^2}{S_2}=F_{\lambda }\cdot Q_{Mess}$$

The advantage of the inventive method for measuring the spatial extent of the illumination point image function is that compared to the known method for pulse duration measurement of very short light pulses, in which the quotient formation from the two detected receiver signals for determining the pulse duration is performed both analog and digital in a computer, in the inventive method, however, by the quotient of a measurement signal S _{1 of} a linear detector 4 and a measurement signal S _{2 of} a nonlinear detector 5 , which arise in the single or multi-photon interaction, the spatial half-width is determined with higher sensitivity than in the said prior art method. The new method provides information about the spatial extent of the illumination spot image function in multi-photon microscopy.

Further advantages of the method according to the invention are that over known methods for measuring the spatial extent of the illumination point image function no elaborate detection media, such as fluorescent beads or ultrathin layer samples are necessary. Furthermore, no pixelated 2D detectors are necessary. The inclusion of z-stacks, ie the recording of 3D sample volumes is eliminated. In addition to the computationally simple evaluation, the method of quotient formation has the advantage that, in contrast to the prior art, no adjustments of the measuring device are necessary, and thus complex adjustment and setup times are eliminated. Furthermore, as in known so-called single-shot methods, in which all pixels of a three-dimensionally resolved image are recorded simultaneously, no moving components, in contrast to those methods but no spatially resolving detectors required, but only two light receivers - especially without spectral filters when diodes are used , There is no bleaching effect when using diodes, in contrast to the signal generation in a fluorescence sample.

The invention is not limited to the exemplary embodiment, but is in the method used and the measuring device M for measuring the spatial extent of the illumination spot variable. In particular, it also includes variants that can be formed by combining features or elements described in connection with the present invention. All mentioned in the foregoing description and readable from the drawing features are further components of the invention, although they are not particularly highlighted and mentioned in the claims.

LIST OF REFERENCE NUMBERS

1

Calibration objective

2

laser beam

3

beam splitter

4

linear detector

5

nonlinear detector

6

amplifier

7

amplifier

8th

evaluation

9

microscope

M

measuring device

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 19944913 A1 [0002]

Claims (7)

A method for measuring the spatial extent of the illumination _spot image function in multi-photon microscopy, characterized in that by the quotient of a measurement signal (S ₁ ) of a linear detector (4) and a measurement signal (S ₂ ) of a nonlinear detector (5), the Single- and multi-photon interactions arise, the spatial half-width is determined from the information about the spatial extent of the illumination point image function in the multi-photon microscopy are derivable. Means (M) adapted to measure the spatial extent of the illumination dot image function by means of the method Claim 1 , with a beam splitter (3) for splitting a light beam (2) to be evaluated into two partial beams to be detected, characterized in that a laser beam (2) is transmitted through a polarization-independent beam splitter (3) or through a polarization-dependent beam splitter (3) to the linear detector (3). 4) and distributed to the non-linear detector (5) such that the signals detected by the linear detector (4) and the nonlinear detector (5) and amplified in amplifiers (6 and 7) are formed by the quotient formation in an evaluation unit (8). are provided for determining the spatial extent of the illumination dot image function. Facility (M) to Claim 2 , characterized in that a calibration lens (1) is present, from which the laser beam (2) emerges or can emerge. Setup after Claim 2 or 3 , characterized in that as linear and non-linear detectors (4, 5) photodiodes are provided with a wide spectral range. Setup after Claim 4 , characterized in that for the linear detector (4) a significantly lower proportion of light is provided, as the proportion of light for the non-linear detector (5). Furnishing according to one of the Claims 2 to 5 , characterized in that the bandgap of the linear detector (4) enables one-photon excitation. Furnishing according to one of the Claims 2 to 6 , characterized in that the bandgap of the non-linear detector (5) enables two-photon excitation.

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