EP4288762A1 - Dispositif et méthode de mesure photométrique en masse - Google Patents

Dispositif et méthode de mesure photométrique en masse

Info

Publication number
EP4288762A1
EP4288762A1 EP22713512.6A EP22713512A EP4288762A1 EP 4288762 A1 EP4288762 A1 EP 4288762A1 EP 22713512 A EP22713512 A EP 22713512A EP 4288762 A1 EP4288762 A1 EP 4288762A1
Authority
EP
European Patent Office
Prior art keywords
laser light
examined
light
frequency
laser
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22713512.6A
Other languages
German (de)
English (en)
Inventor
Axel Schönbeck
Roman Schnabel
Roland Thünauer
Jens Bosse
Jan Südbeck
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Leibniz Institut Fuer Virologie
Universitaet Hamburg
Original Assignee
Leibniz Institut Fuer Virologie
Universitaet Hamburg
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from DE102021102634.2A external-priority patent/DE102021102634A1/de
Priority claimed from DE102021127117.7A external-priority patent/DE102021127117A1/de
Application filed by Leibniz Institut Fuer Virologie, Universitaet Hamburg filed Critical Leibniz Institut Fuer Virologie
Publication of EP4288762A1 publication Critical patent/EP4288762A1/fr
Pending legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/47—Scattering, i.e. diffuse reflection
    • G01N21/4788—Diffraction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/41—Refractivity; Phase-affecting properties, e.g. optical path length
    • G01N21/45—Refractivity; Phase-affecting properties, e.g. optical path length using interferometric methods; using Schlieren methods
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/47—Scattering, i.e. diffuse reflection
    • G01N21/49—Scattering, i.e. diffuse reflection within a body or fluid
    • G01N21/51—Scattering, i.e. diffuse reflection within a body or fluid inside a container, e.g. in an ampoule
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/47—Scattering, i.e. diffuse reflection
    • G01N2021/4704—Angular selective
    • G01N2021/4707—Forward scatter; Low angle scatter

Definitions

  • the present invention relates to a device and a method for photometric mass determination, the objects to be examined being individual biomolecules and/or viruses.
  • interferometric techniques are used for the photometric determination of the mass of biomolecules and viruses.
  • Such a so-called “mass photometry” provides that the object to be measured is located near the surface of a glass sample holder. Using a high-quality but conventional laser beam, light falls through the transparent slide (sample slide) onto the object to be measured and is scattered or diffracted by it.
  • the word scattering also includes diffraction. Some of the scattered light is reflected back together with light reflected from the adjacent glass surface. Scattered light and reflected light interfere. Finally, the interference pattern is detected with a CMOS camera.
  • a basically similar setup for the interferometric scattering method is known from Kukura Philipp et al., “High-speed nanoscopic tracking of the position and orientation of a single virus”, Nature Methods, vol. 6, no. 12, December 2009, 923-27.
  • the setup shown here serves to detect the backscattered light and also to measure the virus marked with a quantum dot.
  • WO 2017/041809 A1 describes the detection of individual proteins, in which the proteins are separated with regard to specific properties such as mass, charge, shape etc. and then examined interferometrically, with the scattered light interfering with the reflected light (iSCAT).
  • GB 25 52 195 A discloses an iSCAT microscope in which the amplitude of a reference field is influenced with the aid of a filter and optimized for detection.
  • the invention is based on the object of providing a device and method for photometric mass determination which have an improved signal-to-noise ratio.
  • the object is achieved by a device having the features of claim 1 and by a method having the features of claim 14.
  • the device according to the invention is a photometric mass determination device.
  • the device according to the invention is a mass determination device which carries out a mass determination according to photometric principles.
  • the device has at least one laser source and an optical measuring device that is suitable for measuring an incident light beam.
  • the at least one laser source is aimed at the object to be examined, which can consist of a single biomolecule, a virus or the like.
  • the optical measuring device is also designed to detect scattered laser light on the object to be examined, which interferes with non-scattered laser light.
  • the laser beam has squeezed laser light. When speaking of scattered laser light, this not only refers to an interaction with structures whose dimensions exceed the wavelength of the light, but also to interactions with structures whose dimensions are smaller, sometimes significantly smaller, than the wavelength of the laser light. Thus, scattering also includes diffraction phenomena.
  • Squeezed light also known as squeezed light
  • squeezed light is a special quantum state whose fuzziness is reduced for some phases and increased for others compared to a coherent state.
  • the coherent, classic laser light has a photon number statistic, which is a Poisson distribution.
  • the distribution has a certain standard deviation around a mean value that depends on the laser power.
  • a certain power-dependent number of photons is detected per time interval. If the average laser power remains the same, the measured number of photons fluctuates from measurement interval to measurement interval around the mean value according to the Poisson-distributed counting statistics.
  • the photon count statistics correspond to the already mentioned shot noise.
  • the extremely weak signals, which are to be expected from scattering from individual molecules, correspond to only a few photons.
  • Squeezed light is a special quantum mechanical state of laser light, which shows a reduced width of the photon counting statistics.
  • This squashed distribution is also known as sub-Poissonian.
  • the standard deviation of the distribution for squeezed laser light is reduced compared to conventional lasers, whereby the average laser power and thus the signal are not affected. With squeezed laser light, there is a reduction in the shot noise and thus an improved signal-to-noise ratio.
  • back-directed laser light and scattered light falls into the optical measuring device as back-reflected laser light together with back-scattered laser light.
  • the laser light passes through the object to be examined, is partially scattered, is then thrown back together with forward-scattered light on a preferably highly reflectively coated substrate and is scattered again on the object.
  • the reflected laser light contains a large part of the incident light as well as light scattered backwards and forwards on the object. Similar to typical iSC AT setups, the reflected light is detected here, but the reflection coefficients in conventional iSC AT setups are very small, so that it is not possible to use squeezed light in a typical iSCAT setup.
  • the attachment in front of the substrate also includes the case that the object to be examined is arranged directly on the substrate.
  • the object to be examined can also be applied directly to the highly reflective substrate, for example in a liquid.
  • a cover glass provided for the object to be examined can preferably also be equipped with an anti-reflection coating, so that losses are also minimized here and the use of squeezed laser light according to the invention is possible.
  • transmitted laser light falls into the optical measuring device together with light scattered forward on the object.
  • the light shines through the slide of the object to be examined, interferes with light scattered in the forward direction and is measured together with this.
  • known measuring devices for photometric mass determination such as iSCAT, always focus on interference with the reflected light beam.
  • the method according to the invention is based on an interference of the light scattered or diffracted in the forward direction with the transmitted light beam. This procedure has the advantage, particularly when using squeezed laser light, that the quantum mechanical state with its narrow (squeezed) number of photons is retained and is not canceled out by a division into different directions. A highly reflective mirror is not required.
  • the laser light directed onto the object to be examined is guided over a spatial region of the sample at a predetermined frequency.
  • a scanning movement takes place in a scanning area. With a sample that is centered in the scanning area, the laser light well is illuminated, two scattering processes take place during one period.
  • an evaluation device is preferably based on measurement signals with twice the scanning frequency in the case of the optical measuring device. If the target is not in the middle of the scan range, the same signal is produced at two unequal frequencies, the sum of which corresponds to twice the scan frequency. If several objects are scanned, additional frequencies are generated. Signals that cannot be assigned to these frequencies do not come from objects in the scanning area, but from the laser source itself, for example, and are thus recognized as false signals. If only twice the scanning frequency is available, this is proof that a centered object is measured in an optimal manner.
  • the scan area is actively centered over the object by maximizing the signal at twice the scan frequency.
  • a control unit can be provided which is designed to adjust the scanned area of the object to be examined in such a way that a component of the signal with double the frequency compared to other components of the signal is maximized. This adjustment can take place on the one hand on the object side, or by a corresponding change in the deflection of the laser beam, so that a center point of the scanning area for the laser light is shifted.
  • the control unit ensures that the laser light is scattered twice on the object to be examined during the scanning movement. If the object to be examined is located, for example, in the edge area of the scanning laser beam, the case can arise that the scattered light is detected only once.
  • the measurement signal is then amplified at twice the scanning frequency by shifting the scanning area in relation to the object to be examined or vice versa.
  • a frequency generator which also generates twice the frequency for the predetermined frequency. Since the measurement signal of optical measuring device is a very weak signal, it is important to be able to generate the frequency signals with great accuracy both in the movement of the laser light and in the evaluation of the signals of the optical measuring device.
  • the measurement signals are preferably evaluated via a lock-in amplifier to which a signal with twice the frequency is present as a reference signal.
  • the phase of the reference signal can be determined in a manner known per se. With the use of a lock-in amplifier, the occurring, very weak electrical signals of the optical measuring device can be amplified and evaluated.
  • a photodiode (PIN photodiode) is provided as the optical measuring device, which preferably has a quantum efficiency of over 50% and can therefore maintain the lower quantum noise of the squeezed laser light in the photovoltage.
  • a single PIN diode has no spatial resolution. It measures the power of the light beam over its entire area. A signal leads to reduced light output at double the scanning frequency.
  • a preferred embodiment uses a photodiode with a quantum efficiency of over 90%.
  • a preferred embodiment uses a balanced detector consisting of two photodiodes, which can read out the light at any phase of the optical oscillation.
  • a preferred embodiment uses a high quantum efficiency photodetector with additional spatial resolution, such as a quadrant photodiode having 4 segments.
  • a CMOS chip can also be used if its quantum efficiency is high enough.
  • conventional and squeezed laser light are used in combination for the measurement.
  • Conventional laser light is used to achieve the strongest possible signal for the measurement.
  • squeezed light with a vanishing or no portion of conventional laser light is used for objects that are particularly sensitive to light.
  • the signal corresponds to a decrease in the degree of squeezing. The more light the biomolecule scatters or diffracts, the more the breadth of the photon statistics approaches that of the conventional laser.
  • the device uses two light-collecting and focusing imaging optics with high transmission.
  • a first imaging optic focuses the laser light onto the object to be examined.
  • a second imaging optic focuses the transmitted laser light onto the optical measuring device.
  • the object according to the invention is also achieved by a method having the features of claim 14.
  • the method is a measuring method used for photometric mass determination.
  • laser light is directed onto an object to be examined, in particular onto individual biomolecules or viruses, and laser light scattered on the object to be examined is measured.
  • the scattered laser light that interferes with non-scattered laser light is preferably measured.
  • the method provides for the laser beam to have squeezed laser light.
  • the squeezed laser light has the advantage of having narrower photon count statistics and thus improving the signal-to-noise ratio.
  • the method according to the invention also provides that the scattered light is measured together with transmitted laser light.
  • the laser beam is guided over a region of the object to be measured at a predetermined frequency f.
  • the measurement signals are evaluated with twice the frequency 2f.
  • the frequency is above 200 Hz, preferably above 1 kHz. Higher frequencies have proven to be particularly advantageous.
  • Fig. 1 comparison of the widths of the photon count statistics of normal and squeezed laser light.
  • FIG. 3 shows an exemplary device for photometric mass determination with transmitted laser light and forward-scattered signal light
  • FIG. 4 shows a second exemplary embodiment for a photometric mass determination with transmitted laser light and forward-scattered signal light
  • 5 shows a third exemplary embodiment for a photometric mass determination with reflected laser light and backscattered signal light
  • 6 shows a fourth exemplary embodiment for a photometric mass determination with reflected laser light and backscattered signal light.
  • FIG. 1 shows in an exemplary representation the photon counting statistics of squeezed laser light in comparison to that of a conventional laser. If, for example, a light output and a measuring time are used that lead to an average of 10,000 registered photons, this number fluctuates with the best classic laser with repeated measurements with a standard deviation of 100 (root of 10,000). The standard deviation is half the width of the photon statistics and corresponds to the photon shot noise. It limits the measurement sensitivity. If squeezed light with a squeeze factor of lOdB is used, the standard deviation is reduced to approx. 32 (root of 1000). Each individual measurement is therefore just as good as an average of 10 measurements with a classic laser of the same power. The total measurement time is reduced by a factor of 10.
  • FIG. 2 The effect of using the squeezed laser light can be seen in FIG.
  • FIG. 2 On the left-hand side of FIG. 2 one can see how the use of squeezed light reduces the spectrally broken down shot noise of the laser and a significantly improved signal-to-shot noise is thus made possible.
  • the noise power is plotted against the frequency in the diagrams.
  • the noise power integrated over all measurement frequencies corresponds to the variance of the photon number uncertainty, i.e. the square of the standard deviation in Figure 1.
  • a possible embodiment of the measuring device is shown in FIG.
  • the optical elements relevant to the functional principle are shown in the figure and further elements have been left out for a better overview.
  • squeezed laser light 12 is superimposed and deflected by a scanning element 14 .
  • the value of 99% is only an approximation.
  • the scanning element performs a spatially limited movement with a frequency f.
  • the scan element 14 can be configured as a gal vo scanner, a micromirror, an acoustooptic deflector or as an electro optic deflector (EOD). Romer G. et al.
  • the lens 16 deflects the laser beam deflected by the scanning element onto the sample holder with the sample. Due to the movement of the scanning element, a finite area with the frequency f is swept on the sample holder 18, which also functions as a scanning frequency. As the schematic enlargement 20 shows, this means that an object to be examined, which can also be present in a solution on a slide, is scanned twice per scan period. The light scattered on the object interferes with the transmitted laser light and is focused by a second lens 22 onto a PIN photodiode. The measurement signal from the PIN photodiode 24 goes to a lock-in amplifier, which particularly amplifies signals at twice the frequency 2f.
  • the reference signals with frequency f and 2f come from a frequency generator 28.
  • the frequency generator provides a signal 30 with the frequency f to the scan element, which scans the area with this frequency, with one or more amplifiers being able to be connected between the frequency generator and the scan element, for example to provide the required voltage of some To provide 100 V for an EOD.
  • a second signal 32 with twice the frequency 2f is applied to the lock-in amplifier 26 as a reference signal. The phase angle between the measurement signal and the reference signal 26 can be adjusted.
  • FIG. 4 shows an alternative structure, identical elements being identified by identical reference symbols.
  • the squeezed laser light 12 and conventional laser light are superimposed via a 50/50 beam splitter 42 .
  • the beam splitter 42 is given here as 50/50 by way of example; however, any beam splitter ratio can be used, for example 30/70 or 80/20.
  • While one beam passes through the sample holder 18 analogously to FIG. 3 and is detected in the first PIN diode 24 , the second part of the radiator is detected in a second PIN diode 34 . Both measurement signals are subtracted from one another in a subtractor before they are amplified in a lock-in amplifier 40 .
  • variable amplifier or attenuator 36 is installed to balance the electrical signals from diodes 34 and 24, so that after the subtraction the noise, which is identical in both signals, is subtracted to zero as well as possible.
  • FIG. 5 shows the incident laser light 50, in which conventional and squeezed laser light are already superimposed. In principle, it is also possible to use only squeezed laser light in 50 Aii.
  • the total laser light is deflected at a scanning frequency in the scanning element, which is designed, for example, as an electro-optical deflector (EOD).
  • EOD electro-optical deflector
  • the deflected laser light is directed to a sample holder 56 via a lens 54 .
  • the lens 54 can have one or more lenses connected in front, which are also to be understood as part of the lens.
  • the object holder 56 has a cover glass 58 which is equipped with an anti-reflection layer.
  • the incident laser light is reflected on a substrate 60 with a highly reflective coating. This can be, for example, a highly reflective mirror.
  • the sample is located between the cover glass 58 and the substrate 60, with the object or objects 62 to be examined.
  • a Faraday isolator optical elements with an equivalent effect, e.g., a combination of a quarter-wave plate and a polarizing beam splitter cube (PBS) can be used.
  • the reflected beam 65 is separated from the original (incoming) beam path by the Faraday isolator and detected on a PIN photodiode 66 .
  • the signal component with double the scanning frequency 2f is particularly amplified via a lock-in amplifier 68, to which double the scanning frequency 2f is applied.
  • the frequencies f for the scan element 52 and 2f for the lock-in amplifier come from a frequency generator 70.
  • FIG. 6 shows the fourth exemplary embodiment, which works with two PIN photodiodes 72, 74.
  • the incident laser light 76 and the squeezed laser light 78 are superimposed on a 50/50 beam splitter 80 and passed to the scanning element 82 and the PIN photodiode 72 .
  • the laser light directed onto the object carrier 88 is deflected by the scanning element 82 with a frequency f and is directed onto the object carrier 88 via the lens 86, with one or more lenses being able to be arranged in front of the lens if required.
  • the slide 88 has an antireflection layer on the cover glass 90 and a substrate 92 with a highly reflective coating.
  • the signals of the two photodiodes 72, 74 are subtracted from one another 96.
  • the difference signal is present at the lock-in amplifier 98, which evaluates the signal with regard to double the scanning frequency 2f.
  • a variable amplifier or attenuator 100 is provided, which equalizes the electrical signals from the photodiode 72 with respect to those from the photodiode 74.
  • the use of squeezed light is associated with the following features.
  • Transmitted or reflected laser light is detected.
  • a modulation of the detected signal is generated by transversely scanning the laser beam relative to the sample.
  • the modulation in the laser light resulting from the scanning is used for the selective detection of the relevant measurement signals via a lock-in amplifier.
  • These features help shift the measurement signal into a shot-noise limited range.
  • the frequency range relevant to the modulation is above 200 Hz, preferably above 1 kHz, preferably even higher. Avoiding optical losses due to weak reflection avoids reducing the squeeze factor.
  • the squeezed laser light on the photoelectric detector is amplitude-modulated by the scattering at the biomolecule at twice the scanning frequency, or amplitude-modulated at two frequencies, which add up to double the scanning frequency.
  • the fact that the scanning frequency is high means that disruptive influences, such as scattered light from unwanted sources, are suppressed.
  • the transmitted light is coupled into a second imaging optics with the highest possible numerical aperture and little optical loss. A high numerical aperture is important in order to be able to strongly focus the light.
  • an imaging optics is required, whereby the Effort for the measuring device reduced. With both approaches, the scattered light is focused onto the detection diode. This measures the modulated light output.
  • the modulation depth i.e.
  • the difference between the maximum and minimum detected intensity varies with the size of the molecule, because larger molecules scatter more light.
  • the measurable contrast as a photometric variable and the molecular mass in this structure.
  • the modulation depth of the laser light output is also low.
  • the intensity maximum and minimum therefore differ only slightly from the average power measured at the diode.
  • the modulation frequency is known, the signal can be filtered with a lock-in amplifier and thus visualized with a good signal-to-noise ratio.

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  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)

Abstract

Dispositif de mesure photométrique en masse comportant au moins une source laser et une unité de mesure optique. Au moins une source laser est dirigée vers l'objet à examiner, en particulier vers des biomolécules ou virus d'un individu, et l'unité de mesure optique détecte la lumière diffusée sur l'objet à examiner, le faisceau laser comprenant de la lumière comprimée.
EP22713512.6A 2021-02-04 2022-02-04 Dispositif et méthode de mesure photométrique en masse Pending EP4288762A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE102021102634.2A DE102021102634A1 (de) 2021-02-04 2021-02-04 Vorrichtung und Verfahren zur photometrischen Massenbestimmung
DE102021127117.7A DE102021127117A1 (de) 2021-10-19 2021-10-19 Vorrichtung und Verfahren zur photometrischen Massenbestimmung
PCT/EP2022/052752 WO2022167601A1 (fr) 2021-02-04 2022-02-04 Dispositif et méthode de mesure photométrique en masse

Publications (1)

Publication Number Publication Date
EP4288762A1 true EP4288762A1 (fr) 2023-12-13

Family

ID=80979121

Family Applications (1)

Application Number Title Priority Date Filing Date
EP22713512.6A Pending EP4288762A1 (fr) 2021-02-04 2022-02-04 Dispositif et méthode de mesure photométrique en masse

Country Status (2)

Country Link
EP (1) EP4288762A1 (fr)
WO (1) WO2022167601A1 (fr)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017041809A1 (fr) 2015-09-07 2017-03-16 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Procédé et appareil pour détecter des particules, comme des macromolécules ou des nanoparticules biologiques
GB2552195A (en) 2016-07-13 2018-01-17 Univ Oxford Innovation Ltd Interferometric scattering microscopy
GB201720162D0 (en) 2017-12-04 2018-01-17 Univ Oxford Innovation Ltd Method

Also Published As

Publication number Publication date
WO2022167601A1 (fr) 2022-08-11

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