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Ich finde die Frage-Überschriften struktur nicht gut! Zudem fehlen da noch ganz wichtige Abschnitte: Wie werden die iRIF bilder erzeugt (Quotzientenbilder)? Erzeugen von Bindekurven, wie auswerten? Wie verrechnet? Zeigt am besten zwei raw bilder, auf denen man nix sieht, dann ein quotientenbild, auf dem man dann die Spots sieht. Man muss an der stelle auch auf den Lookuptable zu sprechen kommen und in welchem Bereich die Intensitäten der Spots normalerweise liegen. (Stefan)

What is iRIf?

Imaging Reflectometric interference (iRIf) is an optical detection technology that can detect and visualize binding processes between biomolecules in real-time. For example the binding of antibodies to their corresponding antigens can be analyzed. The detection method is based on interference of light that is reflected at biolayers. A biolayer in this scenario would for example be antibodies that bind to immobilized antigens which cover the surface of a transparent material (see Figure 1). Adding such an additional biolayer to a surface changes the optical properties (optical thickness), resulting in an increased intensity of the reflected light. The intensity of the reflected light is detected by the CCD sensor of a camera and can be visualized with appropriate software.

Why use iRIf?

Labelfree: Many analyzing approaches to detect binding between molecules are dependent on labelling with a fluorophore or enzyme (ELISA, WB). Since iRIf is a labelfree optical method no expensive labeling reagents are required. Fast and simultenous: Within minutes it is possible to screen a complex sample for binding partners on a microarray. This for example enables us to screen a blood sample within 20 minutes for potentially hundreds of different pathogenic antigens or their corresponding antibodies, respectively. Real-time: With iRIf it is possible to observe binding events during your measurement in real-time! Small samples amounts: The iRIf detection method is very sensitive and can detect even tiny target molecule concentrations. Therefore, usually only small sample volumes (in µl range) are necessary to get reliable results. Hence, the iRIf detection is mostly coupled with a microfluidic device.

Applications

As large as the number of specific binding processes is the number of applications for labelfree reflectometric intereference techniques like iRIf. Applications include amongst others: drug discovery (Birkert & Gauglitz, 2001), kinetic interaction studies (Daaboul et al. 2011), food analysis (Rau et al., 2014), biomarker research (Ewald et al., 2015) and (serologic) diagnostics (Nagel et al., 2007). Usually, basis of each application is some kind of biomolecule microarray (often proteins) which are arranged in distinct spots on a transparent glass side. Protein microarrays facilitate high-throughput screening with a small quantity of sample (Lin et al., 2009).

Our focuses

(Serological) diagnostic of infectious diseases Concerning serological diagnostics iRIf can be used to simultaneously detect potential antibodies for hundreds of diseases from a few drops of blood. For that, one needs to arrange an antigen peptide array (or antibody array) and flood it with the blood of a patient (just like our Dia-Chip works). Different studies show successful detection of pathogenic antigens or the corresponding antibodies within the blood with this technique, e.g. for Tuberculosis (Nagel et al., 2007), Hughes-Syndrom (Bleher et al., 2014), Influenza (Schwarz et al., 2010) or Pox (Proll et al., 2014). Our Dia-chip shows promising results for the detection of Salmonella (Link zum Ergebnis) and Tetanus (Link zum Ergebnis) antibodies within a complex mixture like blood. Determining status of vaccinaton Another application is to examine wheter the vaccination of a patient is still up to date. Since iRIf measurements allow to quantify the amount of antibodies binding to the antigens, this provides a way of adequately quantifying the current vaccination statuses. Too little antibodies would indicate that a vaccination has to be refreshed (Link vergleich Vaccination before(after measurement).

What does an iRIf measurement look like?

[Video] The video shows an exemplary iRIf measurement. Three different proteins are arranged in 3 distinct spots on the glass slide. One after another, three different antibodies, which bind to the corresponding antigen spot, are flushed over the slide. Upon binding, the optical properties of the spots are changed, resulting in an increased intensity of the reflected light at this location of binding. Using a look-up-table(LUT) the amount of bound protein is visualized by different colors.

How it works

To understand the basic physics of iRIf only three propositions of ideal wave optics have to be known:

(P1)	Everytime light is confronting a (non-absorbing) different medium than the one it is propagating through it splits into two parts: one part is going to be reflected and one part is refracted to the next medium.
(P2)	If two waves of light are in the same place they interfere with each other: They are seen as a new wave resulting from summing up the original two.
(P3)	The light intensity is proportional to the square of its amplitude. This also holds up for waves due to interference.

What happens at the device is the following: A green ray of light (520 nm wavelength) produced by an LED is directed onto the iRIf slide. What makes this slide special is an additional layer made out of TaO5 very close (nanometer scale) to the spotted side (why this is necessary can be looked up in the physics section). The ray of light will be reflected everytime the medium changes (P1): On the surface of the slide, at the Ta2O5-layer and on the spotted backside of the slide. Finally the remaining ray is mostly absorbed by the flowcell. Neglecting the first reflection (coherence length, -> physics) we have two reflected waves which are interfering (P2).
(BILD?)
Now what happens if antibodies are bound to a spot? Basically the same happens as if the SiO2 layer would get thicker (optical thickness -> physics) which results in a "delayed" reflection of the ray. This delay especially changes the resulting interference waves' intensity.
Data aquirement at iRIf is done by constantly taking images of the reflected light. Consequently, if this is done while the slide stays in contact with a blood serum (or any solution containing antibodies) possible intensity changes (P3) due to antigen-antibody bindings at the spotted slide will be gathered too. The change is not visible by the naked eye (beyond 1% in relative intensity) but can be seen when comparing later pictures with initial ones using picture division.

(Bild -> Quotientenbild)

Even better, when observing the progression of relative intensity change (relative due to picture division) "binding" curves" can be aquired making quantifications possible.

(Bindekurven-Graph?)

Basic iRIf Setup

Homogenous illumination of the glass slide with monochromatic light source is necessary to archieve good iRIf results. This is best accomplished with a powerful LED light source shining into a lense which is positioned at the distance of one focal length, therefore parallelising the light rays. Since the idea of an iRIf measurement lies in the observation of the minute changes of light intensity of light reflected at the slide, using a sensitive camera with a color depth of 12 bit is advisable. Images in the camera have to be stored lossless, since compression methods such as JPEG or MPEG remove subtle changes in the picture, resulting in the removal of the binding signal. Although not mandatory, microfluidic systems are very well suited for use in conjunction with iRIf, since the iRIf measurement device and the microfluidic chamber can be positioned on opposite sites of the glass slide.

Building your own device

Since the physics behind iRIf is well characterized and the parts necessary for building such a device are easily obtainable, we took it on ourselves to build our very own, affordable iRIf device. The results of this endevour, including a detailed manual on how to build your own iRIf device can be found [here].

Physics behind iRIf (pictures coming soon...)

Next the physical theory behind iRIf will be explained in detail. After a short introduction to beam and lens optics wave optics will be introduced to illustrate effects not describable with the former model. Finally the manner of functioning of CCD chips will be explained to round up the theory.

1. Geometric optics

A very intuitive way to describe light is in form of thin beams. Using this approach effects around refraction can be illustrated by the following picture:



A beam (B1) propagating through a medium (M1) confronting the surface of a different medium (M2) is partially reflected (B2) at the surface and partially refracted (B3) into the medium. The rates of reflection and transmission, R and T, depend on the angle of incidence θ as well as the medias' refraction indices n1 and n2 and are described by the Fresnel equations:





These coefficients are depending on the lights' polarisation (linear in case of an LED). This polarisation can be seen as a combination of a perpendicular part (s) and a parallel one (p). And continuity demands that transmitted and reflected light together return the original beam.

Geometric optics are also enough to describe the concept of lenses. A lens takes the light coming from an (illuminated) object and projects a sharp picture of it (here we will limit ourselves to only convex lenses). Its origin lies in the idea of refraction on a curved surface. A simple illustration is the following:



An object in distance o from the lens and height O is given an image of distance j and height J. The distance f describes the lenses focal point. To obtain a sharp image the lens equation must be obeyed:



The resulting image ratio A can be obtained using the intercept theorem:



One case of interest is the one where the object is set at the focal point of the lens. This results in an infinitely large image in an infinite distance i. Therefore the refracted beams behind the lens can be seen as parallel ones. This is done with the LED light at iRIf.
But not every effect can be described by a beam concept. To understand interference light has to be described as a propagating wave which is done in the next chapter.

2. Introduction to wave optics

Since the derivation of the Maxwell equations the wave character of light is known. Light waves are propagating electric and magnetic fields and therefore can be described as such. For the sake of simplicity it's sufficient to describe the waves' electric field at position r and time t by:



where E is the wave amplitude, k is the wave vector, ω the angular frequency and φ the wave phase. Regarding the given iRIf system this form can further be simplified: We only have to consider two waves from the same light source (LED), being in the same medium and the same position (fixed camera sensor). We therefore have the same angular frequency (ω1=ω2=ω) and can translate our coordinate systems' origin unto the position of interest (where r=0). Also the periodicity of the two waves gives the freedom of choosing one of the phases to be zero and the other one to be the phase shift between them (Φ1 = 0, Φ2 = ΔΦ). Our system of interest therefore consists of two waves:



A property describing the power of light acting on a surface is its intensity. Physically the intensity is described by



where Z is a proportional constant and brakets mean the average over one period. The idea behind iRIf is the measurement of changes in the intensity of an interfered wave. What interference is will be explained now before returning to a proper phaseshift-depending form of I.

3. Interference of two waves

When two forces are acting on the same body the body will move as if only one force being the sum of those two would act on it. This principle of superpositioning is applicable to light waves too: If two waves are in the same place they can be summed up to one.



This effect is called interference and it leads to new waves with different properties, one of them being the amplitude. In our case this amplitude change will be mostly depending on the phase difference between the waves. Using trigonometric identities [1] one can find for the resulting light intensity:



Expecting the single wave amplitudes as well as Z to remain constant over the whole process (all are set to "1" in the following chart) makes the intensity a function depending solely on the phase shift between the interfering waves:



This function is not a monotoneous one which theoretically could lead to a problem if checking for changes. Yet iRIf is very sensitive and works in very small intervals in which monotony is given (only areas around minima and maxima would be problematic yet are easily preventable by a clever multilayer system). Why is this important to know? Because the phase shift will change if antibodies bind to the surface. The phase shift depends on the additional distance of the second wave to return to the first one. The following sketch visualises the system:



Ray 1 (R1) is the reference ray (the one set to have no phase shift), R2 the one reflected at the unbound side of the slide and R3 one that is reflected from the artificially longer end at bound spots. The binding can be seen as if the slide is actually thicker in this area (thanks to antibodies having almost the same refraction index as glass) and is physically described by the optical thickness, the product of refraction index and medium thickness.
How do we obtain the phase shift between R1 and R2/R3? By comparing the distances light has to overcome to return from the backside of the slide to the tantalum pentoxide layer. We assume the light to have zero phase at this layer, so what phase does it have after returning? We only need the angular frequency to solve this:



The additional phase shift therefore depends only on the wavelength of the light as well as the thickness of the bound spot.

4. Coherence

Let us see light as a statistical stream of wave packets. If one wave packet confronts a different medium, two new wave packets are created: a reflected and a transmitted one. Interference is only possible if the returning transmitted packet (after being reflected on the next layer) still interferes with the other packet and not a new reflected one. The concept behind this condition is called coherence. If it weren't for coherence windows would be as colored as soap bubbles.
As long as the path between the two reflections is shorter than the coherence length l interference will occur. The coherence length depends on the spectral range of the light source and, for a 520nm LED, can be approximated by:







Fortunately this is a very high limit as the tantalum pentoxid layer lies in nanometer distance to the last layer and proteins have a similar scale. At the same time this shows that standard glass slides could not be used for detection with iRIf.

5. Technical details: CCD sensors

Modern cameras use CCD chips to take images. A CCD sensor is a 2D-array made out of photo diodes. In these diodes an electric potential is induced by incoming light due to photoelectric effect: Electrons in the diode material are excited and pushed out of the material leaving behind gaps. By closing a circuit at specific times (periodically) a current (due to the potential difference) can be measured. The more gaps were produced in a time interval the stronger the resulting current (direct proportionality). Consequently the sensored light strength is depending on the number of photons the light ray is transporting per time interval. Here we return to the definition of intensity. Intensity generally is defined as power acting on a surface. The power is the work done in a specific time. The work of light (seen as a ray of photons instead of waves) can be described as the total photon energy it is carrying. If the light is monochromatic the photons are all quanta of the same energy. Therefore:



The lights intensity is proportional to the average number of photons it carries, and the resulting current is proportional to the number of gaps through photoelectrical effect which obviously is proportional to the number of photons of the light ray. Or in a short form: The registered light strength of the CCD chip is linearly dependent on the lights' intensity.

6. Summary

To understand iRIf one needs to know that the change in intensity of two interferencing waves is measured by this system. The measuring tool is a CCD chip inside a camera. The intensity changes due to a lengthened distance between the two interfering waves as proteins bound to the glass slide make it virtually thicker. Other possibly disturbing reflections can be neglected due to incoherence. Lastly our iRIf system uses two lenses from which one is needed to produce parallel LED light while the other makes a sharp 1:1 image from the flowcell to the CCD sensor

Legal notice

Johannes Landgraf (Karlsruhe, DE) Günther Proll (Denkendorf, DE) and Florian Pröll (Mannheim, DE) from Biametrics own a patent ( Patent: US 20120058569 A1 ) for the iRIf detection method (“Method and device for determining reflection coefficients on filter arrangements having thin layers”).

References

Birkert & Gaulitz, 2001. Development of an assay for label-free high-throughput screening of thrombin inhibitors by use of reflectometric interference spectroscopy. Anal Bioanal Chem Vol. 372, doi: 10.1007/s00216-001-1196-4 Bleher et al., 2014. Development of a new parallelized, optical biosensor platform for label-free detection of autoimmunity-related antibodies. Anal Bioanal Chem Vol. 406, doi: 10.1007/s00216-013-7504-y Daaboul et al., 2011. LED-based Interferometric Reflectance Imaging Sensor for quantitative dynamic monitoring of biomolecular interactions. Biosensors and Bioelectronics Vol. 26, doi: 10.1016/j.bios.2010.09.038 Ewald et al., 2015. A multi-analyte biosensor for the simultaneous label-free detection of pathogens and biomarkers in point-of-need animal testing. Anal Bioanal Chem Vol. 407, doi: 10.1007/s00216-015-8562-0 Lin et al., 2009. Development of a novel peptide microarray for large-scale epitope mapping of food allergens. Journal of Allergy and Clinical Immunology Vol. 124, doi: 10.1016/j.jaci.2009.05.024 Nagel et al., 2007. Direct detection of tuberculosis infection in blood serum using three optical label-free approaches. Sensors and Actuators B Vol. 129, doi: 10.1016/j.snb.2007.10.009 Proll et al., 2014. Optical biosensor system for the quick and reliable detection of virus infections – VIROSENS. SPIE Proceedings Vol. 9253, doi: 10.1117/12.2073841 Rau et al., 2014. Label-free optical biosensor for detection and quantification of the non-steroidal anti-inflammatory drug diclofenac in milk without any sample pretreatment. Anal Bioanal Chem Vol. 406, doi: 10.1007/s00216-014-7755-2 Schwarz et al., 2010. Label-free detection of H1N1 virus for point of care testing. Procedia Engineering Vol. 5, doi: 10.1016/j.proeng.2010.09.256