Difference between revisions of "Team:Freiburg/Project/iRIf"
m |
|||
Line 48: | Line 48: | ||
<div class="kommentar_stefan"> 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) </div> | <div class="kommentar_stefan"> 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) </div> | ||
<h2>What is iRIf?</h2> | <h2>What is iRIf?</h2> | ||
+ | <p> | ||
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. | 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. | ||
− | + | </p> | |
<h2>Why use iRIf?</h2> | <h2>Why use iRIf?</h2> | ||
+ | <p> | ||
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. | 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. | 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! | 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. | 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. | ||
− | + | </p> | |
<h2>Applications</h2> | <h2>Applications</h2> | ||
+ | <p> | ||
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 <sup><a class="fn_top" href="#fn__1" id="fnt__1" name="fnt__1">1)</a></sup>, kinetic interaction studies <sup><a class="fn_top" href="#fn__2" id="fnt__2" name="fnt__2">2)</a></sup>, food analysis <sup><a class="fn_top" href="#fn__3" id="fnt__3" name="fnt__3">3)</a></sup>, biomarker research <sup><a class="fn_top" href="#fn__4" id="fnt__4" name="fnt__4">4)</a></sup> and (serologic) diagnostics <sup><a class="fn_top" href="#fn__5" id="fnt__5" name="fnt__5">5)</a></sup>. 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 <sup><a class="fn_top" href="#fn__6" id="fnt__6" name="fnt__6">6)</a></sup>. | 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 <sup><a class="fn_top" href="#fn__1" id="fnt__1" name="fnt__1">1)</a></sup>, kinetic interaction studies <sup><a class="fn_top" href="#fn__2" id="fnt__2" name="fnt__2">2)</a></sup>, food analysis <sup><a class="fn_top" href="#fn__3" id="fnt__3" name="fnt__3">3)</a></sup>, biomarker research <sup><a class="fn_top" href="#fn__4" id="fnt__4" name="fnt__4">4)</a></sup> and (serologic) diagnostics <sup><a class="fn_top" href="#fn__5" id="fnt__5" name="fnt__5">5)</a></sup>. 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 <sup><a class="fn_top" href="#fn__6" id="fnt__6" name="fnt__6">6)</a></sup>. | ||
− | + | </p> | |
<h2>Our focuses</h2> | <h2>Our focuses</h2> | ||
− | + | <p> | |
(Serological) diagnostic of infectious diseases | (Serological) diagnostic of infectious diseases | ||
Line 71: | Line 74: | ||
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). | 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). | ||
− | + | </p> | |
<h2>What does an iRIf measurement look like?</h2> | <h2>What does an iRIf measurement look like?</h2> | ||
+ | <p> | ||
[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. | [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. | ||
− | + | </p> | |
<h2>How it works</h2> | <h2>How it works</h2> | ||
− | + | <p> | |
To understand the basic physics of iRIf only three propositions of ideal wave optics have to be | To understand the basic physics of iRIf only three propositions of ideal wave optics have to be | ||
known: | known: | ||
Line 94: | Line 98: | ||
<br><br> | <br><br> | ||
− | + | <p> | |
What happens at the device is the following: A green ray of light (520 nm wavelength) | 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 | produced by an LED is directed onto the iRIf slide. What makes this slide special | ||
Line 105: | Line 109: | ||
(P2). | (P2). | ||
<br> | <br> | ||
− | + | </p> | |
(BILD?) | (BILD?) | ||
<br> | <br> | ||
− | + | <p> | |
Now what happens if antibodies are bound to a spot? Basically the same happens as if the | 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" | SiO2 layer would get thicker (optical thickness -> physics) which results in a "delayed" | ||
Line 123: | Line 127: | ||
eye (beyond 5% in relative intensity) but can be seen when comparing later pictures with | eye (beyond 5% in relative intensity) but can be seen when comparing later pictures with | ||
initial ones using picture division. | initial ones using picture division. | ||
− | + | </p> | |
<br><br> | <br><br> | ||
Line 129: | Line 133: | ||
<br><br> | <br><br> | ||
− | + | <p> | |
Even better, when observing the progression of relative intensity change (relative due to | Even better, when observing the progression of relative intensity change (relative due to | ||
picture division) "binding" curves" can be aquired making quantifications possible. | picture division) "binding" curves" can be aquired making quantifications possible. | ||
− | + | </p> | |
<br><br> | <br><br> | ||
Line 138: | Line 142: | ||
<br><br> | <br><br> | ||
− | + | </p> | |
<h2>Basic iRIf Setup</h2> | <h2>Basic iRIf Setup</h2> | ||
<div class="image_box right"> | <div class="image_box right"> | ||
Line 145: | Line 149: | ||
</div> | </div> | ||
</div> | </div> | ||
− | + | <p> | |
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. | 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. | ||
− | + | </p> | |
<h2>Building your own device</h2> | <h2>Building your own device</h2> | ||
+ | <p> | ||
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]. | 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]. | ||
+ | </p> | ||
<h2>Physics behind iRIf (pictures coming soon...)</h2> | <h2>Physics behind iRIf (pictures coming soon...)</h2> | ||
− | + | <p> | |
Next the physical theory behind iRIf will be explained in detail. After | 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 | 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 | effects not describable with the former model. Finally the manner of functioning of CCD | ||
chips will be explained to round up the theory. | chips will be explained to round up the theory. | ||
− | + | </p> | |
<br><br> | <br><br> | ||
Line 167: | Line 173: | ||
</div> | </div> | ||
</div> | </div> | ||
+ | <p> | ||
A very intuitive way to describe light is in form of thin beams. Using this approach effects around | 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: | refraction can be illustrated by the following picture: | ||
Line 175: | Line 182: | ||
− | + | </p> | |
<br><br> | <br><br> | ||
<div class="responsive_center_image"> | <div class="responsive_center_image"> | ||
Line 185: | Line 192: | ||
</div> | </div> | ||
<br><br> | <br><br> | ||
− | + | <p> | |
These coefficients are depending on the lights' polarisation (linear in case of an LED). This polarisation can | 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 | be seen as a combination of a perpendicular part (s) and a parallel one (p). And continuity demands that transmitted | ||
Line 196: | Line 203: | ||
convex lenses). Its origin lies in the idea of refraction on a curved surface. A simple illustration | convex lenses). Its origin lies in the idea of refraction on a curved surface. A simple illustration | ||
is the following: | is the following: | ||
− | + | </p> | |
<br><br> | <br><br> | ||
<div class="responsive_center_image"> | <div class="responsive_center_image"> | ||
Line 203: | Line 210: | ||
<br><br> | <br><br> | ||
− | + | <p> | |
An object in distance o from the lens and height O is given an image of distance j and height J. The | 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: | distance f describes the focal point of the lenses. To obtain a sharp image the lens equation must be obeyed: | ||
− | + | </p> | |
<br><br> | <br><br> | ||
<div class="responsive_center_image"> | <div class="responsive_center_image"> | ||
Line 213: | Line 220: | ||
<br><br> | <br><br> | ||
− | + | <p> | |
The resulting image ratio A can be obtained using the intercept theorem: | The resulting image ratio A can be obtained using the intercept theorem: | ||
− | + | </p> | |
<br><br> | <br><br> | ||
<div class="responsive_center_image"> | <div class="responsive_center_image"> | ||
Line 221: | Line 228: | ||
</div> | </div> | ||
<br><br> | <br><br> | ||
− | + | <p> | |
One case of interest is the one where the object is set at the focal point of the lens. This results in an | 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 | infinitely large image in an infinite distance i. Therefore the refracted beams behind the lens can be seen | ||
Line 230: | Line 237: | ||
<br><br> | <br><br> | ||
+ | </p> | ||
<h3>2. Introduction to wave optics</h3> | <h3>2. Introduction to wave optics</h3> | ||
− | + | <p> | |
Since the derivation of the Maxwell equations the wave character of light is accurately described. Light waves are propagating | 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 | 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: | to describe the waves' electric field at position r and time t by: | ||
− | + | </p> | |
<br><br> | <br><br> | ||
<div class="responsive_center_image"> | <div class="responsive_center_image"> | ||
Line 242: | Line 250: | ||
</div> | </div> | ||
<br><br> | <br><br> | ||
− | + | <p> | |
− | + | 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 | 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 | two waves from the same light source (LED), being in the same medium and the same position (fixed | ||
Line 250: | Line 258: | ||
gives the freedom of choosing one of the phases to be zero and the other one to be the phase shift between them | 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: | (Φ1 = 0, Φ2 = ΔΦ). Our system of interest therefore consists of two waves: | ||
− | + | </p> | |
<br><br> | <br><br> | ||
<div class="responsive_center_image"> | <div class="responsive_center_image"> | ||
Line 256: | Line 264: | ||
</div> | </div> | ||
<br><br> | <br><br> | ||
− | + | <p> | |
A property describing the power of light acting on a surface is its intensity. Physically the intensity is | A property describing the power of light acting on a surface is its intensity. Physically the intensity is | ||
described by | described by | ||
− | + | </p> | |
<br><br> | <br><br> | ||
<div class="responsive_center_image"> | <div class="responsive_center_image"> | ||
Line 265: | Line 273: | ||
</div> | </div> | ||
<br><br> | <br><br> | ||
− | + | <p> | |
where Z is a proportional constant and brakets mean the average over one period. The idea behind iRIf is the | 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 | measurement of changes in the intensity of an interfered wave. What interference is will be explained now before | ||
Line 271: | Line 279: | ||
<br><br> | <br><br> | ||
− | + | </p> | |
<h3>3. Interference of two waves</h3> | <h3>3. Interference of two waves</h3> | ||
− | + | <p> | |
When two forces are acting on the same body the body will move as if only one force being | 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 | 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. | light waves too: If two waves are in the same place they can be summed up to one. | ||
− | + | </p> | |
<br><br> | <br><br> | ||
<div class="responsive_center_image"> | <div class="responsive_center_image"> | ||
Line 287: | Line 295: | ||
</div> | </div> | ||
<br><br> | <br><br> | ||
− | + | <p> | |
This effect is called interference and it leads to new waves with different properties, one of | 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 | 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: | difference between the waves. Using trigonometric identities [1] the resulting light intensity can be found: | ||
− | + | </p> | |
<br><br> | <br><br> | ||
Line 299: | Line 307: | ||
<br><br> | <br><br> | ||
− | + | <p> | |
Expecting the single wave amplitudes as well as Z to remain constant over the whole process (all are set | 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 | to "1" in the following chart) makes the intensity a function depending solely on the phase shift between | ||
the interfering waves: | the interfering waves: | ||
− | + | </p> | |
<br><br> | <br><br> | ||
<div class="responsive_center_image"> | <div class="responsive_center_image"> | ||
Line 309: | Line 317: | ||
</div> | </div> | ||
<br><br> | <br><br> | ||
− | + | <p> | |
This function is not a monotoneous one which theoretically could lead to a problem if checking for changes. Yet | 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 | iRIf is very sensitive and works in very small intervals in which monotony is given (only areas around minima and | ||
Line 315: | Line 323: | ||
know? Because the phase shift will change if antibodies bind to the surface. The phase shift depends on the additional | 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: | 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: | ||
− | + | </p> | |
<br><br> | <br><br> | ||
<div class="responsive_center_image"> | <div class="responsive_center_image"> | ||
Line 322: | Line 330: | ||
<br><br> | <br><br> | ||
− | + | <p> | |
Ray 1 (R1) is the reference ray (the one set to have no phase shift), R2 the one reflected at the unbound side of | 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 | the slide and R3 one that is reflected from the artificially longer end at bound spots. The binding can be seen as | ||
Line 331: | Line 339: | ||
from the backside of the slide to the tantalum pentoxide layer (Ta<sub>2</sub>O<sub>5</sub>). We assume the light to have zero phase at this layer, | from the backside of the slide to the tantalum pentoxide layer (Ta<sub>2</sub>O<sub>5</sub>). 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: | so what phase does it have after returning? We only need the angular frequency to solve this: | ||
− | + | </p> | |
<br><br> | <br><br> | ||
<div class="responsive_center_image"> | <div class="responsive_center_image"> | ||
Line 337: | Line 345: | ||
</div> | </div> | ||
<br><br> | <br><br> | ||
− | + | <p> | |
The additional phase shift therefore depends only on the wavelength of the light as well as the thickness of the bound | The additional phase shift therefore depends only on the wavelength of the light as well as the thickness of the bound | ||
spot. | spot. | ||
+ | </p> | ||
<h3>4. Coherence</h3> | <h3>4. Coherence</h3> | ||
+ | <p> | ||
Let us see light as a statistical stream of wave packets. If one wave packet confronts a different medium, two new wave | 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 | packets are created: a reflected and a transmitted one. Interference is only possible if the returning transmitted packet | ||
Line 350: | Line 360: | ||
As long as the path between the two reflections is shorter than the coherence length l interference will occur. The | 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: | coherence length depends on the spectral range of the light source and, for a 520nm LED, can be approximated by: | ||
− | + | </p> | |
<br><br> | <br><br> | ||
<div class="responsive_center_image"> | <div class="responsive_center_image"> | ||
Line 362: | Line 372: | ||
</div> | </div> | ||
<br><br> | <br><br> | ||
− | + | <p> | |
Fortunately, this is a very high limit as the tantalum pentoxid layer lies in nanometer distance to the last layer and | 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 | proteins have a similar scale. At the same time this shows that standard glass slides could not be used for detection | ||
Line 368: | Line 378: | ||
<br><br> | <br><br> | ||
− | + | </p> | |
<h3>5. Technical details: CCD sensors</h3> | <h3>5. Technical details: CCD sensors</h3> | ||
− | + | <p> | |
Modern cameras use CCD chips to take images. A CCD sensor is a 2D-array made out of photo diodes. In these diodes an | 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 | electric potential is induced by incoming light due to photoelectric effect: Electrons in the diode material are | ||
Line 386: | Line 396: | ||
</div> | </div> | ||
<br><br> | <br><br> | ||
− | + | <p> | |
The lights intensity is proportional to the average number of photons it carries, and the resulting current is proportional | 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 | to the number of gaps through photoelectric effect which obviously is proportional to the number of photons of the light | ||
Line 392: | Line 402: | ||
<br><br> | <br><br> | ||
− | + | </p> | |
<h3>6. Summary</h3> | <h3>6. Summary</h3> | ||
− | + | <p> | |
To understand iRIf one needs to know that the change in intensity of two interfering waves is measured by this system. | 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 | The measuring tool is a CCD chip inside a camera. The intensity changes due to a lengthened distance between the two | ||
Line 401: | Line 411: | ||
LED light while the other makes a sharp 1:1 image from the flowcell to the CCD sensor. | LED light while the other makes a sharp 1:1 image from the flowcell to the CCD sensor. | ||
− | + | </p> | |
<h2>Legal notice</h2> | <h2>Legal notice</h2> | ||
<p> | <p> |
Revision as of 08:51, 16 September 2015
![](https://static.igem.org/mediawiki/2015/8/85/Freiburg_wiki_background_2_parted_left.png)
![](https://static.igem.org/mediawiki/2015/f/f4/Freiburg_wiki_background_2_parted_right.png)
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 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 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 5), Hughes-Syndrom 7), Influenza 8) or Pox 9). 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 transits from one medium to another (non-absorbing) medium, 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 true 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 Ta2O5. This layer is located 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 PDMS-flowcell. Neglecting the first reflection (coherence length,
-> physics) we have two reflected waves which are interfering
(P2).
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 5% 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
![](https://static.igem.org/mediawiki/2015/7/76/Freiburg_Projects_iRIf_basic_setup.png)
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
![Refraction iRIf](https://static.igem.org/mediawiki/2015/6/6b/Freiburg_IRIF_13_Refraction_PIC.png)
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:
![](https://static.igem.org/mediawiki/2015/a/a6/Freiburg_IRIF_01_Reflection_Coefficients.png)
![](https://static.igem.org/mediawiki/2015/b/b0/Freiburg_IRIF_02_Fresnel_Eq.png)
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:
![](https://static.igem.org/mediawiki/2015/c/cd/Freiburg_IRIF_14_Lens_PIC.png)
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:
![](https://static.igem.org/mediawiki/2015/6/61/Freiburg_IRIF_03_Lens_Eq.png)
The resulting image ratio A can be obtained using the intercept theorem:
![](https://static.igem.org/mediawiki/2015/2/22/Freiburg_IRIF_04_Image_Ratio.png)
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:
![](https://static.igem.org/mediawiki/2015/d/d6/Freiburg_IRIF_05_Electric_Field_General.png)
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:
![](https://static.igem.org/mediawiki/2015/8/85/Freiburg_IRIF_06_Electric_Fields_Simple.png)
A property describing the power of light acting on a surface is its intensity. Physically the intensity is described by
![](https://static.igem.org/mediawiki/2015/c/c6/Freiburg_IRIF_07_Intensity_Wave_Optics.png)
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.
![](https://static.igem.org/mediawiki/2015/d/d3/Freiburg_IRIF_18_Electric_Field_Interference.png)
![](https://static.igem.org/mediawiki/2015/1/13/Freiburg_IRIF_15_Interference_PIC.png)
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:
![](https://static.igem.org/mediawiki/2015/e/e1/Freiburg_IRIF_08_Intensity_Phase_Dependent.png)
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:
![](https://static.igem.org/mediawiki/2015/0/03/Freiburg_IRIF_16_Phaseshift_PIC.png)
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:
![](https://static.igem.org/mediawiki/2015/3/3a/Freiburg_IRIF_17_Slide_PIC.png)
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:
![](https://static.igem.org/mediawiki/2015/c/c4/Freiburg_IRIF_09_Phase_Shift.png)
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:
![](https://static.igem.org/mediawiki/2015/0/0e/Freiburg_IRIF_10_Intensity_Add_Thickness_Dependency.png)
![](https://static.igem.org/mediawiki/2015/5/5e/Freiburg_IRIF_11_Coherence_Length.png)
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:
![](https://static.igem.org/mediawiki/2015/4/42/Freiburg_IRIF_12_Intensity_Photons.png)
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