Team:Freiburg/Project/iRIf
Introduction to 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. In this scenario, a biolayer would 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 a changed interference pattern of the reflected light. The intensity of the reflected light for each location on the observed area is detected by the CCD sensor of a camera and can be visualized with appropriate software.
One Method, Many Advantages
Label-Free: A huge number of analytical procedures for detection of binding between molecules depend on labelling with a fluorophore or enzyme (ELISA, Western Blot). Since iRIf is a label-free optical method, no expensive labelling reagents are required.
Fast and Simultaneous: It is possible to screen complex samples for several binding partners on a microarray within minutes. 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 a measurement in real-time!
Small Sample 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 often coupled with a microfluidic device.
Applications
The number of applications for label-free reflectometric interference techniques like iRIf is as large as the number of specific binding processes. Applications include amongst others: drug discovery 1), kinetic interaction studies 2), food analysis 3), biomarker research 4) and (serologic) diagnostics 5). 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 6).
Our Focus
(Serological) diagnosis of infectious diseases
Concerning serological diagnostics, iRIf can be used to simultaneously detect potential antibodies for hundreds of diseases from one drop of blood. For that, an antigen peptide array (or antibody array) needs to be prepared and flooded with the blood of a patient (just like our DiaCHIP works). Different studies show successful detection of pathogenic antigens or the corresponding antibodies within the blood with this technique, e.g. for Tuberculosis 5), Hughes-Syndrom 7), Influenza 8) or Pox 9).
Our DiaCHIP shows promising results for the detection of Salmonella and Tetanus antibodies within a complex mixture like blood.
Determining the Status of Vaccinaton
Another application is to examine whether the antibody levels of a patient still show successful immmunity or a vaccination boost is needed. Since iRIf measurements allow to quantify the amount of antibodies binding to the antigens, this provides a way of adequately quantifying the current vaccination status. Detection of no or only a small amount of antibodies would indicate that the vaccination has to be refreshed. An experiment comparing an immunized to a non immunized blood sample can be found on our Results Page.
The iRIf Measurement
The iRIf Video
After each measurement, a video visualizing the binding of proteins from the sample to proteins fixed on the glass slide can be made.
From Reflected Light to Bright Spots
To understand the basic physics of iRIf it is sufficient to be familiar with only three propositions (P1-3) of ideal wave optics:
- (1) Everytime light passes from one medium to another (non-absorbing) medium, one part is going to be reflected and one part is deflected to the next medium.
- (2) If two waves of light superimpose they interfere with each other: They are considered as a new wave resulting from adding up the original two.
- (3) The light intensity is proportional to the square of its amplitude. This also holds true for waves due to interference.
During a measurement the following processes take place:
A green ray of light (wavelength: 520 nm)
produced by an LED is directed to the iRIf slide.
The difference between an iRIf slide and a common microscopy glass slide is an additional layer of tantal oxide (Ta2O5). This layer is located very close (nanometer scale) to the spotted slide
(More details can be found 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 of light is mainly absorbed by the PDMS-flowcell on the opposite side of the flowchamber.
Neglecting the first reflection (coherence length,
-> physics)there remain two reflected waves which are interfering (P2).
What happens if antibodies bind to a spot? Basically, the same processes take place as if the SiO2 layer (meaning the glass slide) would get thicker (optical thickness -> physics) which results in a "delayed" reflection of the ray. This delay especially changes the resulting interfering waves' intensity.
Data Aquirement by Quotient Pictures
Data aquirement in iRIf is done by constantly taking images of the reflected light. Consequently, if this is done while the slide stays in contact with 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 5% in relative intensity) but can be seen when comparing pictures taken at a later time to initial ones using picture division.
Quantifications Visualized by Binding Curves
Even better, when observing the progression of relative intensity change (relative due to picture division) "binding" curves" can be aquired making quantifications possible.
The Detection Device
The Basic iRIf Setup
Homogenous illumination of the glass slide with monochromatic light source is necessary to achieve 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 parallelizing 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 endeavour, 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 focal point of the lenses. 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 accurately described. 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 superposition 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 is mainly depending on the phase difference between the waves. Using trigonometric identities [1] the resulting light intensity can be found:
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. Due to the described intereference conditions it is necessary to select an LED with a suitable wavelength. The selected wavelength in combination with the optical properties of the used substrate determine the signal dynamics. For this project a substrate with a tantalum pentoxide layer in nanometer range was selected. 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 (Ta2O5). 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 is 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 photoelectric 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 interfering 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
The iRIf detection method is patented. Biametrics and associated persons own patents concerning the detection principle. These patents are:
- Method for examining physical, chemical and biochemical interactions (DE102004051098.9, DE102005015030.6, EP05797776.1)
- Study of molecular interactions on and / or in thin layers (DE102007038797.2)
- Method and apparatus for determining reflection coefficients to filter arrangement with thin layers (DE102009019711.7)
- Again Detectable support for optical measuring methods (DE102009019476.2)
References
