Difference between revisions of "Team:Freiburg/Project/iRIf"
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+ | background-size: 10px 13px; | ||
+ | } | ||
+ | </style> | ||
− | + | <script type="text/javascript"> | |
+ | //===================BEGIN:Amazing Bubble Sidebar========================== | ||
− | + | $(document).ready(function(){ | |
+ | // CHANGE THE FOLLOWING ATTRIBUTES // | ||
+ | var href_text1='https://2015.igem.org/Team:Freiburg/Project/Overview', | ||
+ | // Text2 needs no href as it is the actual page // | ||
+ | img_url='https://static.igem.org/mediawiki/2015/7/76/Freiburg_icon_project_white_03.png', | ||
+ | href_text3='https://2015.igem.org/Team:Freiburg/Project/Future_Directions', | ||
+ | // Text1 needs no text as it is a pic // | ||
+ | text2='Optical Detection'; | ||
+ | // Text3 needs no text as its always 'next' // | ||
+ | // HOLD ON CHANGING THINGS --JABBERWOCK // | ||
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+ | //===================END:Amazing Bubble Sidebar========================== | ||
+ | </script> | ||
− | |||
− | |||
+ | <div class="content_box"> | ||
− | < | + | <h1>Imaging Reflectometric Interference (iRIf)</h1> |
− | + | <p> | |
− | + | <div class="horizontal_menu"> | |
− | < | + | <ul> |
+ | <li><a href="https://2015.igem.org/Team:Freiburg/Project/iRIf#iRIf_measurement_anchor" title="The iRIf measurement">Measurement</a></li> | ||
+ | <li><a href="https://2015.igem.org/Team:Freiburg/Project/iRIf#iRIf_detection_device_anchor" title="The iRIf detection device">Detection Device</a></li> | ||
+ | <li><a href="https://2015.igem.org/Team:Freiburg/Project/iRIf#physics_behind_anchor" title="The Physics behind">Physics Behind</a></li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </p> | ||
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− | < | + | <h2>Introduction to iRIf</h2> |
+ | <p> | ||
+ | Imaging Reflectometric Interference (iRIf) is a labelfree optical detection technology that can detect and visualize binding processes between biomolecules. For example, the binding of antibodies to their corresponding antigens can be analyzed. The detection method is based on the interference of light that is partially reflected at different (bio)layers. In this scenario, a biolayer would consist of antibodies that bind to antigens immobilized on the surface of a transparent material. Adding a further biolayer changes the optical properties, resulting in a changed interference pattern of the reflected light. The intensity of the reflected light for each location of the observed area is detected by the CCD sensor of a camera and can be visualized in real-time. | ||
+ | </p> | ||
− | + | ||
− | + | <h2>One Method, Many Advantages</h2> | |
− | + | ||
− | + | <div class="image_box left"> | |
− | + | <div class="thumb2 trien" style="width:90px"> | |
− | + | <img src="https://static.igem.org/mediawiki/2015/c/c1/Freiburg_labelfree.jpg" width="80px"> | |
− | + | <div class="thumbcaption"> | |
− | + | </div> | |
− | + | </div> | |
+ | </div> | ||
+ | <p> | ||
+ | <strong>Label-Free:</strong> A huge number of analytical procedures for detection of binding between molecules depend on labeling with a fluorophore or enzyme (ELISA, Western Blot). Since iRIf is a label-free optical method, no expensive labeling reagents are required. | ||
+ | </p> | ||
+ | |||
+ | <div class="image_box left"> | ||
+ | <div class="thumb2 trien" style="width:90px"> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/5/50/Freiburg_fast.jpg" width="70px"> | ||
+ | <div class="thumbcaption"> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | <p> | ||
+ | <strong>Fast and Simultaneous:</strong> It is possible to screen complex samples for several binding partners on a microarray within minutes. This, for example, allows us to screen a blood sample within 20 minutes for potentially hundreds of different pathogenic antigens or their corresponding antibodies. | ||
+ | </p> | ||
+ | |||
+ | <div class="image_box left"> | ||
+ | <div class="thumb2 trien" style="width:90px"> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/b/b5/Freiburg_realtime_RJ.jpg" width="70px"> | ||
+ | <div class="thumbcaption"> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
<br> | <br> | ||
+ | <p> | ||
+ | <strong>Real-Time:</strong> With iRIf it is possible to observe binding events during a measurement in real-time! | ||
+ | </p> | ||
− | ( | + | <div class="image_box left"> |
+ | <div class="thumb2 trien" style="width:90px"> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/6/62/Freiburg_smallsampleamount.jpg" width="70px"> | ||
+ | <div class="thumbcaption"> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | <p> | ||
+ | <b>Small Sample Amounts:</b> The iRIf detection method is very sensitive and can detect even tiny target molecule concentrations. Therefore, usually only small sample volumes (in the µl range) are necessary to obtain reliable results. Hence, the iRIf detection is often coupled to a microfluidic device. | ||
+ | </p> | ||
+ | |||
+ | <h2>Applications</h2> | ||
+ | <p> | ||
+ | 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 <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 (serological) diagnostics <sup><a class="fn_top" href="#fn__5" id="fnt__5" name="fnt__5">5)</a></sup>. Usually, the basis of each application is some type 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 Focus</h2> | ||
+ | |||
+ | <div class="floatbox left"> | ||
+ | <p> | ||
+ | <em>(Serological) diagnosis of infectious diseases</em> | ||
<br> | <br> | ||
+ | Concerning serological diagnostics, iRIf can be used to simultaneously detect potential antibodies for hundreds of diseases from one drop of blood. To this end an antigen peptide array (or antibody array) needs to be prepared and flooded with the blood of a patient (<a href="https://2015.igem.org/Team:Freiburg/Project/System"target="_blank">just like our DiaCHIP works</a>). Different studies show successful detection of pathogenic antigens or the corresponding antibodies in the blood with this technique, e.g. for tuberculosis <sup><a class="fn_top" href="#fn__5" id="fnt__5" name="fnt__5">5)</a></sup>, Hughes syndrome <sup><a class="fn_top" href="#fn__7" id="fnt__7" name="fnt__7">7)</a></sup>, influenza <sup><a class="fn_top" href="#fn__8" id="fnt__8" name="fnt__8">8)</a></sup> or pox <sup><a class="fn_top" href="#fn__9" id="fnt__9" name="fnt__9">9)</a></sup>. | ||
+ | <br> | ||
+ | Our DiaCHIP shows promising results for the detection of <a href="https://2015.igem.org/Team:Freiburg/Results/Diagnostics#salmonella" title="Salmonella Measurement"><i>Salmonella</i></a> and <a href="https://2015.igem.org/Team:Freiburg/Results#anti_tetanus_results" title="Tetanus Measurement">Tetanus</a> antibodies within a complex mixture like blood. | ||
+ | </p> | ||
+ | </div> | ||
+ | |||
+ | <div class="floatbox right"> | ||
+ | <p> | ||
+ | <em>Determining the Status of Vaccinaton</em> | ||
+ | <br> | ||
+ | Another application is to decide whether the antibody levels of a patient still ensure sufficient immmunity or if a vaccination boost is needed. Since iRIf measurements allow to quantify the amount of antibodies binding to the antigens, they provide a way of adequately quantifying the current vaccination status. Detection of none or only a small amount of antibodies would indicate that the vaccination has to be refreshed. An <a href="https://2015.igem.org/Team:Freiburg/Results/Diagnostics#rabbit_serum_anchor" title="vaccination status">experiment comparing an immunized to a non-immunized blood sample</a> can be found on our Results Page. | ||
+ | </p> | ||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | |||
+ | <div class="content_box"> | ||
+ | <span class="anchor" id="iRIf_measurement_anchor"></span> | ||
+ | <h1>The iRIf Measurement</h1> | ||
+ | <h2>The iRIf Video</h2> | ||
− | + | <div class="image_box center"> | |
− | + | <div class="thumbinner"style="width:780px;"> | |
− | + | <meta http-equiv="X-UA-Compatible" content="IE=Edge"/> | |
− | + | <video width="780" height="200" poster="https://static.igem.org/mediawiki/2015/2/2a/Freiburg_20150911_PRESENTATION_ANTIGEN_123_00024.jpg" controls> | |
+ | <source src="https://static.igem.org/mediawiki/2015/d/db/Freiburg_20150911_PRESENTATION_ANTIGEN_123.mp4" type="video/mp4"> | ||
+ | Your browser does not support the HTML5 video tag. | ||
+ | </video> | ||
+ | <div class="thumbcaption">Video 1: Exemplary result of the binding of antibodies from three distinct solutions to the corresponding antigen spots. Antibody 1 and 2 bind their antigen specifically, antibody 3 shows slight cross-reactivity with antigen 2.</div> | ||
+ | </div> | ||
+ | </div> | ||
+ | <br> | ||
<br> | <br> | ||
− | Data aquirement | + | <div class="image_box right"> |
− | Consequently, if this is done while the slide stays in contact with | + | <div class="thumb2 trien" style="width:400px"> |
+ | <div class="thumbinner"> | ||
+ | <a href="https://static.igem.org/mediawiki/2015/d/de/Freiburg_LUT.png" class="lightbox_trigger"> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/d/de/Freiburg_LUT.png" width="400px"> | ||
+ | <div class="thumbcaption"> | ||
+ | </a> | ||
+ | <p><strong>Figure 1: Look-Up-Table (LUT) for the visual quantification of the relative intensity shift.</strong> To get a visual impression about the amounts of binding processes that take place during a real-time measurement LUTs are used. Shown here is just an exemplary LUT, which roughly fits to the images and videos shown on this page. A certain shift in intensity is encoded by a certain color. Therefore, the amount of binding can be estimated visually already during the real-time measurement.</p> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | <p> | ||
+ | Video 1 shows an exemplary iRIf measurement with the binding of proteins from the sample to proteins fixed on the glass slide. Three different antigens are arranged in 3 distinct spots on the glass slide. One after another, three different antibodies binding to the corresponding antigen spot are flushed over the slide. As the video shows, antibody 1 binds to antigen 1, antibody 2 to antigen 2 and antibody 3 to antigen 3 (with a slight cross-reactivity to antigen 2). Upon binding, the optical thickness changes, resulting in an increased intensity of the reflected light at the spot. Using a look-up-table (LUT) the amount of bound protein is visualized by different colors (figure 1). | ||
+ | </p> | ||
+ | |||
+ | <h2>From Reflected Light to Bright Spots</h2> | ||
+ | |||
+ | <div class="image_box right"> | ||
+ | <div class="thumb2 trien" style="width:310px"> | ||
+ | <div class="thumbinner"> | ||
+ | <a href="https://static.igem.org/mediawiki/2015/0/0e/Freiburg_Strahlengang.jpg" class="lightbox_trigger"> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/0/0e/Freiburg_Strahlengang.jpg" width="300px"> | ||
+ | <div class="thumbcaption"> | ||
+ | </a> | ||
+ | <p><strong>Figure 2: Light reflection and interference at iRIf.</strong> Each time the light of the LED travels from one medium to another, it is partially reflected. The reflected beams interfere and their amplitudes superimpose. | ||
+ | |||
+ | The optical properties of the Ta<sub>2</sub>O<sub>5</sub> layer are responsible for the generation of the first reflected beam R<sub>0</sub>. It interferes with the second reflected beam from the surface. In case of binding processes at the surface, the interference changes can be detected by the camera as an increase in intensity. </p> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <p> | ||
+ | To understand the basic physics of iRIf it is sufficient to be familiar with only three propositions (P1-3) of ideal wave optics: | ||
+ | <ul> | ||
+ | <li>(1) Everytime light passes from one medium to another (non-absorbing) medium, | ||
+ | it is partly reflected, while the rest is deflected to the next medium.</li> | ||
+ | <li>(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.</li> | ||
+ | <li>(3) The light intensity is proportional to the square of its amplitude. This also holds | ||
+ | true for waves generated by interference.</li> | ||
+ | </ul> | ||
+ | </p> | ||
+ | |||
+ | <p> | ||
+ | During a measurement the following processes take place: | ||
+ | <br style="line-height:1.5em"> | ||
+ | A green ray of light (wavelength: 520 nm) | ||
+ | produced by an LED is directed to the iRIf slide. <br> | ||
+ | The difference between an iRIf slide and a common microscopy glass slide is an additional layer of tantalum pentoxide (Ta<sub>2</sub>O<sub>5</sub>). This layer is located very close (nanometer scale) to the spotted slide | ||
+ | (More details can be found in the <a href="https://2015.igem.org/Team:Freiburg/Project/iRIf#physics_behind_anchor" title="Physics behind iRIf">physics section</a>). | ||
+ | <br style="line-height:1.5em"> | ||
+ | The ray of light will be reflected everytime the medium changes (P1): on the surface of the | ||
+ | slide, at the Ta<sub>2</sub>O<sub>5</sub>-layer and on the spotted backside of the slide (figure 2). <br> | ||
+ | Finally, the remaining ray of light is mainly absorbed by the PDMS-flow cell on the opposite side of the flow chamber. <br> | ||
+ | Neglecting the first reflection (see <a href="https://2015.igem.org/Team:Freiburg/Project/iRIf#coherence_anchor" title="More on coherence length">coherence length</a>) there remain two reflected waves which are interfering (P2). | ||
+ | </p> | ||
+ | |||
+ | <p> | ||
+ | What happens if antibodies bind to a spot? Basically, the same processes take place as if the | ||
+ | SiO<sub>2</sub> layer (meaning the glass slide) would get thicker (see <a href="https://2015.igem.org/Team:Freiburg/Project/iRIf#optical_thickness_anchor" title="more on optical thickness">optical thickness</a>) which results in a "delayed" | ||
+ | reflection of the ray. This delay especially changes the resulting interfering waves' intensity. | ||
+ | </p> | ||
+ | |||
+ | <h3>Data Aquirement by Quotient Pictures</h3> | ||
+ | <p> | ||
+ | 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 | solution containing antibodies) possible intensity changes (P3) due to antigen-antibody | ||
− | + | binding event at the spotted slide will be gathered, too. The change is not visible by naked | |
− | eye (beyond | + | eye (beyond 5% in relative intensity) but can be visualized by comparing pictures taken at a later time to |
− | initial ones using picture division. | + | initial ones using picture division. So to any time during a real time measurement the actual picture you see is a quotient picture. This quotient picture is calculated |
+ | by dividing the grey values of your current picture (Pic1) by the grey values of an older reference picture (Pic2). | ||
+ | <br><br><strong>Quotient picture = Pic1 / Pic2</strong><br><br> | ||
+ | Since the grey values correspond to the amount of reflected light which is detected by the camera, changes due to binding processes can be visualized. | ||
+ | To illustrate how this works and looks like, four different quotient pictures (figure 3 to 6) are shown. All these quotient pictures were taken during a single iRIf measurement, but they differ due to different choices of either Pic1 or Pic2 that was taken to calculate the quotient picture. | ||
+ | During the illustrated measurement two solutions flush over the chip; the first solution contains the specific binding partner for the positive control. The second solution contains antibodies against GFP. For a proper understanding let us define three points in time: | ||
− | < | + | <ul> |
+ | <li>t = 0 ; no solution was flushed over the slide </li> | ||
+ | <li>t = 1 ; solution 1 flushed over the slide: this solution contains the binding partner for the positive control</li> | ||
+ | <li>t = 2 ; solution 2 flushed over the slide too: this solution contains GFP antibodies</li> | ||
+ | </ul> | ||
− | + | </p> | |
− | < | + | <div class="image_box left"> |
+ | <div class="thumb2 trien" style="width:400px"> | ||
+ | <div class="thumbinner"> | ||
+ | <a href="https://static.igem.org/mediawiki/2015/5/53/Freiburg_QP_0.png" class="lightbox_trigger"> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/d/d8/Freiburg_QP_0_preview.png" width="400px"> | ||
+ | <div class="thumbcaption"> | ||
+ | </a> | ||
+ | <p><strong>Figure 3: Quotient picture = Pic1 (t = 0) / Pic2 (t = 0) </strong> No binding events are visualized. The quotient picture is calculated by dividing the picture at timepoint "0" by the picture at timepoint "0" - so literally by itself. Therefore, no differences in intensity can be seen.</p> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
− | + | <div class="image_box right"> | |
− | picture | + | <div class="thumb2 trien" style="width:400px"> |
+ | <div class="thumbinner"> | ||
+ | <a href="https://static.igem.org/mediawiki/2015/a/a1/Freiburg_QP_1.png" class="lightbox_trigger"> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/3/34/Freiburg_QP_1_preview.png" width="400px"> | ||
+ | <div class="thumbcaption"> | ||
+ | </a> | ||
+ | <p><strong>Figure 4: Quotient picture = Pic1 (t = 1) / Pic2 (t = 0) </strong> The quotient picture is calculated by dividing the picture at timepoint "1" by the picture at timepoint "0" - every change in intensity that occurs during this period of time can be seen. During this period solution 1 flushed over the slide. Therefore, binding of the positive control binding partner is visualized </p> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
− | <br | + | <br style="clear:both"> |
− | ( | + | <div class="image_box left"> |
+ | <div class="thumb2 trien" style="width:400px"> | ||
+ | <div class="thumbinner"> | ||
+ | <a href="https://static.igem.org/mediawiki/2015/6/61/Freiburg_QP_2.png" class="lightbox_trigger"> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/7/71/Freiburg_QP_2_preview.png" width="400px"> | ||
+ | <div class="thumbcaption"> | ||
+ | </a> | ||
+ | <p><strong>Figure 5: Quotient picture = Pic1 (t = 2) / Pic2 (t = 0) </strong> The quotient picture is calculated by dividing the picture at timepoint "2" by the picture at timepoint "0" - every change in intensity that occurs during this period of time can be seen. During this period solution 1 and 2 flushed over the slide. Therefore, the binding of the positive control binding partner and anti-GFP to GFP is visualized.</p> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
− | < | + | <div class="image_box right"> |
+ | <div class="thumb2 trien" style="width:400px"> | ||
+ | <div class="thumbinner"> | ||
+ | <a href="https://static.igem.org/mediawiki/2015/f/fb/Freiburg_QP_3.png" class="lightbox_trigger"> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/8/8c/Freiburg_QP_3_preview.png" width="400px"> | ||
+ | <div class="thumbcaption"> | ||
+ | </a> | ||
+ | <p><strong>Figure 6: Quotient picture = Pic1 (t = 2) / Pic2 (t = 1) </strong> The quotient picture is calculated by dividing the picture at timepoint "2" by the picture at timepoint "1" - every change in intensity that occurs during this period of time can be seen. During this period solution 2 flushed over the slide. Therefore, the binding of anti-GFP to GFP is visualized. </p> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
− | < | + | <br style="clear:both"> |
− | < | + | <p></p> |
− | + | <h3>Quantifications Visualized by Binding Curves</h3> | |
+ | <p> | ||
+ | Moreover, by observing the progression of relative intensity change binding curves can be aquired, allowing quantification of the binding process. Figure 8 shows such a binding curve from a measurement during which two solutions were flushed over the slide successively. In the graph, the relative change of intensity (y-axis) at distinct spots (see figure 7) over time (x-axis) is shown. The lighter grey background indicates the time period in which a solution (that contains interaction partners for the immobilized proteins) is flushed over the slide. The darker grey background represents the washing steps between each binding step. When the first solution, which contains Streptavidin - the binding partner for the positive control (biotinylated BSA), is flushed over the slide, the relative intensity at the positive control spot increases. Therefore, the red line, representing the relative intensity at the bBSA spot, rises during this step of the measurement. During washing steps the relative light intensity remains the same. When the next antibody (anti-GFP) is flushed through, the corresponding GFP spot (blue line) shows an increase in relative intensity due to binding. <br> | ||
+ | The binding curves are generated as following: First you have to select regions of interests (ROIs) for which you want to plot the relative shift in intensity. Since it is a relative shift, one also needs to define reference spots (background - where no protein of interest is immobilized) to which the intensity values are normalized (see figure 7). Additionally, a reference point in time needs to be set: at this point in time the relative shift in intensity from your ROIs to the corresponding reference background spots is set to 1. Therefore, the relative light intensity at each following point in time is compared to its reference spot. If the intensity of your ROI increases more than the intensity of the reference spot the relative shift is greater than 1. If the intensity of your ROI decreases more than the intensity of the reference spot the relative shift is less than 1. According to this approach every picture of the measurement is evaluated. The resulting binding curve shows real-time binding kinetics for your marked regions (figure 8). | ||
+ | </p> | ||
− | < | + | <div class="image_box left"> |
− | + | <div class="thumb2 trien" style="width:400px"><div class="thumbinner"> | |
+ | <a href="https://static.igem.org/mediawiki/2015/3/3b/Freiburg_Binding_curve_ROI_selection.png" class="lightbox_trigger"> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/4/45/Freiburg_Binding_curve_ROI_selection_preview.png" width="400px"></img> | ||
+ | </a> | ||
+ | <div class="thumbcaption"> | ||
+ | <p> | ||
+ | <strong>Figure 7: Selecting regions of interest (ROIs) and reference spots for the calculation of binding curves.</strong> The shift in intensity for each ROI is normalized to its corresponding reference spot (background). Thus, ROI 1 is normalized to Ref 1, ROI 2 to Ref 2 and ROI 3 to Ref3. | ||
+ | </p> | ||
+ | </div></div> | ||
+ | </div> | ||
+ | </div> | ||
− | |||
− | + | <div class="image_box right"> | |
− | a | + | <div class="thumb2 trien" style="width:400px"><div class="thumbinner"> |
− | + | <a href="https://static.igem.org/mediawiki/2015/7/72/Freiburg_binding_curve_explanationnew.png" class="lightbox_trigger"> | |
− | + | <img src="https://static.igem.org/mediawiki/2015/3/36/Freiburg_binding_curve_explanationnew_preview.png" width="400px"></img> | |
+ | </a> | ||
+ | <div class="thumbcaption"> | ||
+ | <p> | ||
+ | <strong>Figure 8: Binding curves of the marked ROIs.</strong> The relative shift in intensity (y-axis) of the selected ROIs (figure 7) is plotted over time (x-Axis). The shift in intensity of the ROIs is normalized to their corresponding reference spots (Ref1-Ref3). As can be seen binding occurs during the Streptavidin flush at the positive control spot (red line) and during the anti-GFP flush at the GFP spot (blue line). No binding is detected at the negative control (grey line). | ||
+ | </p> | ||
+ | </div></div> | ||
+ | |||
+ | </div> | ||
− | |||
− | < | + | </div> |
+ | </div> | ||
− | + | <span class="anchor" id="iRIf_detection_device_anchor"></span> | |
− | + | <div class="content_box"> | |
+ | <h1>The Detection Device</h1> | ||
+ | <h2>The Basic iRIf Setup</h2> | ||
− | < | + | <div class="image_box right"> |
− | <img src="https://static.igem.org/mediawiki/2015/ | + | <div class="thumb2 trien" style="width:510px"> |
− | < | + | <div class="thumbinner"> |
+ | <a href="https://static.igem.org/mediawiki/2015/7/76/Freiburg_Projects_iRIf_basic_setup.png" class="lightbox_trigger"> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/7/76/Freiburg_Projects_iRIf_basic_setup.png" width="500px"> | ||
+ | <div class="thumbcaption"> | ||
+ | </a> | ||
+ | <p><strong>Figure 9: Schematic iRIf Setup.</strong> The PDMS flow cell and the glass slide form a chamber. Solutions are flushed through the chamber using a microfluidic system. Proteins (antigens) are fixed on the glass slide before. Light constantly illuminates the glass slide from the bottom. If other proteins (like antibodies) within the sample bind to the proteins (antigens) on the glass slide, the interference pattern changes. This change is detected by the CCD sensor of a camera and visualized by a software. </p> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | <p> | ||
+ | Homogenous illumination of the glass slide with monochromatic light is necessary to achieve good iRIf results. This is best accomplished with a powerful LED light source shining on a lense which is positioned at the distance of one focal length, therefore parallelizing the light rays. | ||
+ | <br> | ||
+ | Since the purpose of an iRIf measurement is 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 have to be stored lossless in the camera. Since compression methods such as JPEG or MPEG remove subtle changes in the picture, resulting the binding signal is lost otherwise. 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 (figure 9). | ||
+ | </p> | ||
+ | |||
+ | |||
+ | <h2>Building Our Device</h2> | ||
+ | <p> | ||
+ | Since the physics behind iRIf are 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 rebuild our iRIf device can be found on the Results Page. | ||
+ | |||
+ | <div class="link_button link_button_arrow"> | ||
+ | <p><a href="https://2015.igem.org/Team:Freiburg/Results/Own_Device" title="Our iRIf Device">Our iRIf Device</a></p> | ||
+ | </div> | ||
+ | |||
+ | </p> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | |||
+ | <div class="content_box"> | ||
+ | |||
+ | <span class="anchor" id="physics_behind_anchor"></span> | ||
+ | <h1>The Physics Behind iRIf</h1> | ||
+ | <p> | ||
+ | This chapter focuses on the physical theory behind iRIf 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. | ||
+ | </p> | ||
+ | |||
+ | <h2>1. Geometric optics</h2> | ||
+ | <div class="image_box right"> | ||
+ | <div class="thumb2 trien" style="width:400px"> | ||
+ | <img alt="Refraction iRIf" src="https://static.igem.org/mediawiki/2015/6/6b/Freiburg_IRIF_13_Refraction_PIC.png" width="400"/> | ||
+ | </div> | ||
+ | </div> | ||
+ | <p> | ||
+ | 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 | 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 | 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' | 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: | refraction indices n1 and n2 and are described by the Fresnel equations: | ||
+ | </p> | ||
+ | <p> | ||
+ | <div class="responsive_center_image"> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/a/a6/Freiburg_IRIF_01_Reflection_Coefficients.png" width="400"> | ||
+ | </div> | ||
<br><br> | <br><br> | ||
− | <img src="https://static.igem.org/mediawiki/2015/ | + | <div class="responsive_center_image"> |
− | < | + | <img src="https://static.igem.org/mediawiki/2015/b/b0/Freiburg_IRIF_02_Fresnel_Eq.png" width="400"> |
+ | </div> | ||
+ | </p> | ||
− | < | + | <p> |
− | + | These coefficients depend on the light's 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). Furthermore, continuity demands that transmitted | |
− | These coefficients | + | |
− | be seen as a combination of a perpendicular part (s) and a parallel one (p). | + | |
and reflected light together return the original beam. | and reflected light together return the original beam. | ||
+ | </p> | ||
− | < | + | <p> |
− | + | Geometric optics are also sufficient to describe the concept of lenses. A lens takes the light coming | |
− | Geometric optics are also | + | |
from an (illuminated) object and projects a sharp picture of it (here we will limit ourselves to only | 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 | convex lenses). Its origin lies in the idea of refraction on a curved surface. A simple illustration | ||
is the following: | is the following: | ||
+ | </p> | ||
− | < | + | <div class="responsive_center_image"> |
− | <img src="https://static.igem.org/mediawiki/2015/c/cd/Freiburg_IRIF_14_Lens_PIC.png"> | + | <img src="https://static.igem.org/mediawiki/2015/c/cd/Freiburg_IRIF_14_Lens_PIC.png" width="800"> |
− | < | + | </div> |
+ | <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 | + | distance f describes the focal point of the lenses. To obtain a sharp image the lens equation must be obeyed: |
+ | </p> | ||
− | < | + | <div class="responsive_center_image"> |
− | <img src="https://static.igem.org/mediawiki/2015/6/61/Freiburg_IRIF_03_Lens_Eq.png"> | + | <img src="https://static.igem.org/mediawiki/2015/6/61/Freiburg_IRIF_03_Lens_Eq.png"> |
− | < | + | </div> |
+ | <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> | ||
− | < | + | <div class="responsive_center_image"> |
− | <img src="https://static.igem.org/mediawiki/2015/2/22/Freiburg_IRIF_04_Image_Ratio.png"> | + | <img src="https://static.igem.org/mediawiki/2015/2/22/Freiburg_IRIF_04_Image_Ratio.png"> |
− | < | + | </div> |
− | One case of interest is the | + | <p> |
+ | One case of interest is the situation where the object is set at the focal point of the lense. 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 | ||
as parallel ones. This is done with the LED light at iRIf. | as parallel ones. This is done with the LED light at iRIf. | ||
<br> | <br> | ||
− | But not every effect can be described by a beam concept. | + | But not every effect can be described by a beam concept. In order to understand interference, light has to be described |
as a propagating wave which is done in the next chapter. | as a propagating wave which is done in the next chapter. | ||
+ | </p> | ||
− | |||
− | < | + | <h2>2. Introduction to Wave Optics</h2> |
− | + | <p> | |
− | Since the derivation of the Maxwell equations the wave character of light is | + | 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> | ||
− | < | + | <div class="responsive_center_image"> |
− | <img src="https://static.igem.org/mediawiki/2015/d/d6/Freiburg_IRIF_05_Electric_Field_General.png"> | + | <img src="https://static.igem.org/mediawiki/2015/d/d6/Freiburg_IRIF_05_Electric_Field_General.png" width="400"> |
− | < | + | </div> |
− | + | <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 | ||
− | camera sensor). | + | camera sensor). Therefore, we have the same angular frequency (ω1=ω2=ω) and can translate |
− | our coordinate | + | our coordinate system's 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 | 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> | ||
− | < | + | <div class="responsive_center_image"> |
− | <img src="https://static.igem.org/mediawiki/2015/8/85/Freiburg_IRIF_06_Electric_Fields_Simple.png"> | + | <img src="https://static.igem.org/mediawiki/2015/8/85/Freiburg_IRIF_06_Electric_Fields_Simple.png" width="400"> |
− | < | + | </div> |
+ | <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> | ||
− | < | + | <div class="responsive_center_image"> |
− | <img src="https://static.igem.org/mediawiki/2015/c/c6/Freiburg_IRIF_07_Intensity_Wave_Optics.png"> | + | <img src="https://static.igem.org/mediawiki/2015/c/c6/Freiburg_IRIF_07_Intensity_Wave_Optics.png"> |
− | < | + | </div> |
+ | <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 | ||
returning to a proper phaseshift-depending form of I. | returning to a proper phaseshift-depending form of I. | ||
+ | </p> | ||
− | |||
− | |||
− | |||
+ | <span class="anchor" id="optical_thickness_anchor"></span> | ||
+ | <h2>3. Interference of Two Waves</h2> | ||
+ | <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 | + | 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> | ||
− | < | + | <div class="responsive_center_image"> |
− | <img src="https://static.igem.org/mediawiki/2015/d/d3/Freiburg_IRIF_18_Electric_Field_Interference.png"> | + | <img src="https://static.igem.org/mediawiki/2015/d/d3/Freiburg_IRIF_18_Electric_Field_Interference.png" width="400"> |
− | <img src="https://static.igem.org/mediawiki/2015/1/13/Freiburg_IRIF_15_Interference_PIC.png"> | + | </div> |
− | < | + | |
+ | <div class="responsive_center_image"> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/1/13/Freiburg_IRIF_15_Interference_PIC.png" width="800"> | ||
+ | </div> | ||
+ | <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 | + | them being the amplitude. In our case this amplitude change is mainly depending on the phase |
− | difference between the waves. Using trigonometric identities [1] | + | difference between the waves. Using trigonometric identities [1] the resulting light intensity can be found: |
− | light intensity: | + | </p> |
− | < | + | <div class="responsive_center_image"> |
− | <img src="https://static.igem.org/mediawiki/2015/e/e1/Freiburg_IRIF_08_Intensity_Phase_Dependent.png"> | + | <img src="https://static.igem.org/mediawiki/2015/e/e1/Freiburg_IRIF_08_Intensity_Phase_Dependent.png" width="800"> |
− | < | + | </div> |
+ | <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> | ||
− | < | + | <div class="responsive_center_image"> |
− | <img src="https://static.igem.org/mediawiki/2015/0/03/Freiburg_IRIF_16_Phaseshift_PIC.png"> | + | <img src="https://static.igem.org/mediawiki/2015/0/03/Freiburg_IRIF_16_Phaseshift_PIC.png" width="800"> |
− | < | + | </div> |
+ | <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 | ||
− | maxima would be problematic yet are easily preventable by a clever multilayer system). Why is this important to | + | 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 | 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: | + | distance of the second wave to return to the first one. Due to the described interference 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> | ||
− | < | + | <div class="responsive_center_image"> |
− | <img src="https://static.igem.org/mediawiki/2015/3/3a/Freiburg_IRIF_17_Slide_PIC.png"> | + | <img width="400" src="https://static.igem.org/mediawiki/2015/3/3a/Freiburg_IRIF_17_Slide_PIC.png"> |
− | < | + | </div> |
− | Ray 1 (R1) is the reference ray (the one set to have no phase shift), R2 the one reflected at the unbound side of | + | <p> |
− | the slide and R3 one that is reflected from the artificially longer end at bound spots. The binding can be seen as | + | Ray 1 (R1) is the reference ray (the one set to have no phase shift), R2 is the one reflected at the unbound side of |
+ | the slide and R3 is the 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) | 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. | and is physically described by the optical thickness, the product of refraction index and medium thickness. | ||
− | < | + | <7p> |
− | How do we obtain the phase shift between R1 and R2/R3? By comparing the distances light has to overcome to return | + | <p> |
− | from the backside of the slide to the tantalum pentoxide layer. We assume the light to have zero phase at this layer, | + | How do we obtain the phase shift between R1 and R2/R3? By comparing the distances light has to overcome in order to return |
+ | 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> | ||
− | < | + | <div class="responsive_center_image"> |
− | <img src="https://static.igem.org/mediawiki/2015/c/c4/Freiburg_IRIF_09_Phase_Shift.png"> | + | <img width="400" src="https://static.igem.org/mediawiki/2015/c/c4/Freiburg_IRIF_09_Phase_Shift.png"> |
− | < | + | </div> |
+ | <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> | ||
− | < | + | <span class="anchor" id="coherence_anchor"></span> |
− | Let us | + | <h2>4. Coherence</h2> |
+ | <p> | ||
+ | Let us consider 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 | ||
(after being reflected on the next layer) still interferes with the other packet and not a new reflected one. The | (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 | + | concept behind this condition is called coherence. If it was not for coherence windows would be as colored as soap |
bubbles. | bubbles. | ||
<br> | <br> | ||
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> | ||
− | < | + | <div class="responsive_center_image"> |
− | <img src="https://static.igem.org/mediawiki/2015/0/0e/Freiburg_IRIF_10_Intensity_Add_Thickness_Dependency.png"> | + | <img width="400" src="https://static.igem.org/mediawiki/2015/0/0e/Freiburg_IRIF_10_Intensity_Add_Thickness_Dependency.png"> |
− | < | + | </div> |
− | < | + | <div class="responsive_center_image"> |
− | <img src="https://static.igem.org/mediawiki/2015/5/5e/Freiburg_IRIF_11_Coherence_Length.png"> | + | <img width="400" src="https://static.igem.org/mediawiki/2015/5/5e/Freiburg_IRIF_11_Coherence_Length.png"> |
− | < | + | </div> |
− | Fortunately this is a very high limit as the tantalum pentoxid layer lies in nanometer distance to the last layer and | + | <p> |
+ | 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 | ||
with iRIf. | with iRIf. | ||
+ | </p> | ||
− | |||
− | |||
− | |||
+ | <h2>5. Technical Details: CCD Sensors</h2> | ||
+ | <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 the 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) | 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 | 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 | + | 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 | 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. | intensity. Intensity generally is defined as power acting on a surface. The power is the work done in a specific time. | ||
Line 326: | Line 667: | ||
carrying. If the light is monochromatic the photons are all quanta of the same energy. Therefore: | carrying. If the light is monochromatic the photons are all quanta of the same energy. Therefore: | ||
− | < | + | <div class="responsive_center_image"> |
− | <img src="https://static.igem.org/mediawiki/2015/4/42/Freiburg_IRIF_12_Intensity_Photons.png"> | + | <img width="400" src="https://static.igem.org/mediawiki/2015/4/42/Freiburg_IRIF_12_Intensity_Photons.png"> |
− | < | + | </div> |
+ | <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 | + | to the number of gaps generated due to the 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. | ray. Or in a short form: The registered light strength of the CCD chip is linearly dependent on the lights' intensity. | ||
+ | </p> | ||
− | |||
− | < | + | <h2>6. Summary</h2> |
− | + | <p> | |
− | + | In order 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 | ||
− | interfering waves as proteins | + | interfering waves as proteins bind 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 | 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 | + | LED light while the other makes a sharp 1:1 image from the flow cell to the CCD sensor. |
− | + | </p> | |
+ | </div> | ||
− | < | + | <div class="content_box"> |
− | + | <h1>Legal Notice</h1> | |
− | + | ||
<p> | <p> | ||
+ | The iRIf detection method is patented. | ||
+ | <a href="http://biametrics.com/en/" target="_blank"> Biametrics </a> | ||
+ | and associated persons own patents concerning the detection principle. | ||
+ | These patents are: | ||
− | < | + | <ul> |
− | + | <li> | |
+ | <a href="http://www.google.com/patents/EP1805502A1?cl=en&hl=de" target="_blank"> Method for examining physical, chemical and biochemical interactions </a> (DE102004051098.9, DE102005015030.6, EP05797776.1) | ||
+ | </li> | ||
− | + | <li> | |
+ | <a href="http://www.google.com/patents/DE102007038797A1?cl=en&hl=de" target="_blank"> Study of molecular interactions on and / or in thin layers </a> (DE102007038797.2) | ||
+ | </li> | ||
− | + | <li> | |
+ | <a href="http://www.google.com/patents/DE102009019711A1?cl=en&hl=de" target="_blank"> Method and apparatus for determining reflection coefficients to filter arrangement with thin layers </a> (DE102009019711.7) | ||
+ | </li> | ||
− | + | <li> | |
+ | <a href="http://www.google.com/patents/DE102009019476A1?cl=en&hl=de" target="_blank"> Again Detectable support for optical measuring methods </a> (DE102009019476.2) | ||
+ | </li> | ||
+ | </ul> | ||
− | + | </p> | |
− | |||
− | |||
− | |||
− | + | <!-- EDIT6 SECTION "Outlook" [17910-] --> | |
+ | <div class="footnotes"> | ||
+ | <h3>References</h3> | ||
+ | <div class="fn"><sup><a class="fn_bot" href="#fnt__1" id="fn__1" name="fn__1">1)</a></sup> | ||
+ | <a target="_Blank" href="http://www.ncbi.nlm.nih.gov/pubmed/11939184">Birkert & Gauglitz, 2001. Development of an assay for label-free high-throughput screening of thrombin inhibitors by use of reflectometric interference spectroscopy. Anal Bioanal Chem.</a> | ||
</div> | </div> | ||
+ | |||
+ | |||
+ | <div class="fn"><sup><a class="fn_bot" href="#fnt__2" id="fn__2" name="fn__2">2)</a></sup> | ||
+ | <a target="_Blank" href="http://www.researchgate.net/publication/47566135_LED-based_Interferometric_Reflectance_Imaging_Sensor_for_quantitative_dynamic_monitoring_of_biomolecular_interactions">Daaboul <i>et al</i>., 2011. LED-based Interferometric Reflectance Imaging Sensor for quantitative dynamic monitoring of biomolecular interactions. Biosensors and Bioelectronics.</a> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | <div class="fn"><sup><a class="fn_bot" href="#fnt__3" id="fn__3" name="fn__3">3)</a></sup> | ||
+ | <a target="_Blank" href="http://www.ncbi.nlm.nih.gov/pubmed/24658575">Rau <i>et al</i>., 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.</a> | ||
+ | </div> | ||
+ | |||
+ | <div class="fn"><sup><a class="fn_bot" href="#fnt__4" id="fn__4" name="fn__4">4)</a></sup> | ||
+ | <a target="_Blank" href="http://www.ncbi.nlm.nih.gov/pubmed/25772557">Ewald <i>et al</i>., 2015. A multi-analyte biosensor for the simultaneous label-free detection of pathogens and biomarkers in point-of-need animal testing. Anal Bioanal Chem.</a> | ||
+ | </div> | ||
+ | |||
+ | <div class="fn"><sup><a class="fn_bot" href="#fnt__5" id="fn__5" name="fn__5">5)</a></sup> | ||
+ | <a target="_Blank" href="http://www.sciencedirect.com/science/article/pii/S0925400507008180">Nagel <i>et al</i>., 2007. Direct detection of tuberculosis infection in blood serum using three optical label-free approaches. Sensors and Actuators.</a> | ||
+ | </div> | ||
+ | |||
+ | <div class="fn"><sup><a class="fn_bot" href="#fnt__6" id="fn__6" name="fn__6">6)</a></sup> | ||
+ | <a target="_Blank" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2757036/">Lin <i>et al</i>., 2009. Development of a novel peptide microarray for large-scale epitope mapping of food allergens. Journal of Allergy and Clinical Immunology.</a> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | <div class="fn"><sup><a class="fn_bot" href="#fnt__7" id="fn__7" name="fn__7">7)</a></sup> | ||
+ | <a target="_Blank" href="http://www.ncbi.nlm.nih.gov/pubmed/24281326">Bleher <i>et al</i>., 2014. Development of a new parallelized, optical biosensor platform for label-free detection of autoimmunity-related antibodies. Anal Bioanal Chem.</a> | ||
+ | </div> | ||
+ | |||
+ | <div class="fn"><sup><a class="fn_bot" href="#fnt__8" id="fn__8" name="fn__8">8)</a></sup> | ||
+ | <a target="_Blank" href="http://www.researchgate.net/publication/241123800_Label-free_detection_of_H1N1_virus_for_point_of_care_testing">Schwarz <i>et al</i>., 2010. Label-free detection of H1N1 virus for point of care testing. Procedia Engineering.</a> | ||
+ | </div> | ||
+ | |||
+ | <div class="fn"><sup><a class="fn_bot" href="#fnt__9" id="fn__9" name="fn__9">9)</a></sup> | ||
+ | <a target="_Blank" href="http://spie.org/Publications/Proceedings/Paper/10.1117/12.2073841">Proll <i>et al</i>., 2014. Optical biosensor system for the quick and reliable detection of virus infections – VIROSENS. SPIE Proceedings.</a> | ||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | |||
</html> | </html> | ||
{{Freiburg/wiki_content_end}} | {{Freiburg/wiki_content_end}} |
Latest revision as of 22:40, 18 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)
Imaging Reflectometric Interference (iRIf)
Introduction to iRIf
Imaging Reflectometric Interference (iRIf) is a labelfree optical detection technology that can detect and visualize binding processes between biomolecules. For example, the binding of antibodies to their corresponding antigens can be analyzed. The detection method is based on the interference of light that is partially reflected at different (bio)layers. In this scenario, a biolayer would consist of antibodies that bind to antigens immobilized on the surface of a transparent material. Adding a further biolayer changes the optical properties, resulting in a changed interference pattern of the reflected light. The intensity of the reflected light for each location of the observed area is detected by the CCD sensor of a camera and can be visualized in real-time.
One Method, Many Advantages
![](https://static.igem.org/mediawiki/2015/c/c1/Freiburg_labelfree.jpg)
Label-Free: A huge number of analytical procedures for detection of binding between molecules depend on labeling with a fluorophore or enzyme (ELISA, Western Blot). Since iRIf is a label-free optical method, no expensive labeling reagents are required.
![](https://static.igem.org/mediawiki/2015/5/50/Freiburg_fast.jpg)
Fast and Simultaneous: It is possible to screen complex samples for several binding partners on a microarray within minutes. This, for example, allows us to screen a blood sample within 20 minutes for potentially hundreds of different pathogenic antigens or their corresponding antibodies.
![](https://static.igem.org/mediawiki/2015/b/b5/Freiburg_realtime_RJ.jpg)
Real-Time: With iRIf it is possible to observe binding events during a measurement in real-time!
![](https://static.igem.org/mediawiki/2015/6/62/Freiburg_smallsampleamount.jpg)
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 the µl range) are necessary to obtain reliable results. Hence, the iRIf detection is often coupled to 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 (serological) diagnostics 5). Usually, the basis of each application is some type 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. To this end 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 in the blood with this technique, e.g. for tuberculosis 5), Hughes syndrome 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 decide whether the antibody levels of a patient still ensure sufficient immmunity or if a vaccination boost is needed. Since iRIf measurements allow to quantify the amount of antibodies binding to the antigens, they provide a way of adequately quantifying the current vaccination status. Detection of none 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
Video 1 shows an exemplary iRIf measurement with the binding of proteins from the sample to proteins fixed on the glass slide. Three different antigens are arranged in 3 distinct spots on the glass slide. One after another, three different antibodies binding to the corresponding antigen spot are flushed over the slide. As the video shows, antibody 1 binds to antigen 1, antibody 2 to antigen 2 and antibody 3 to antigen 3 (with a slight cross-reactivity to antigen 2). Upon binding, the optical thickness changes, resulting in an increased intensity of the reflected light at the spot. Using a look-up-table (LUT) the amount of bound protein is visualized by different colors (figure 1).
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, it is partly reflected, while the rest 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 generated by 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 tantalum pentoxide (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 (figure 2).
Finally, the remaining ray of light is mainly absorbed by the PDMS-flow cell on the opposite side of the flow chamber.
Neglecting the first reflection (see coherence length) 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 (see optical thickness) 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
binding event at the spotted slide will be gathered, too. The change is not visible by naked
eye (beyond 5% in relative intensity) but can be visualized by comparing pictures taken at a later time to
initial ones using picture division. So to any time during a real time measurement the actual picture you see is a quotient picture. This quotient picture is calculated
by dividing the grey values of your current picture (Pic1) by the grey values of an older reference picture (Pic2).
Quotient picture = Pic1 / Pic2
Since the grey values correspond to the amount of reflected light which is detected by the camera, changes due to binding processes can be visualized.
To illustrate how this works and looks like, four different quotient pictures (figure 3 to 6) are shown. All these quotient pictures were taken during a single iRIf measurement, but they differ due to different choices of either Pic1 or Pic2 that was taken to calculate the quotient picture.
During the illustrated measurement two solutions flush over the chip; the first solution contains the specific binding partner for the positive control. The second solution contains antibodies against GFP. For a proper understanding let us define three points in time:
- t = 0 ; no solution was flushed over the slide
- t = 1 ; solution 1 flushed over the slide: this solution contains the binding partner for the positive control
- t = 2 ; solution 2 flushed over the slide too: this solution contains GFP antibodies
Quantifications Visualized by Binding Curves
Moreover, by observing the progression of relative intensity change binding curves can be aquired, allowing quantification of the binding process. Figure 8 shows such a binding curve from a measurement during which two solutions were flushed over the slide successively. In the graph, the relative change of intensity (y-axis) at distinct spots (see figure 7) over time (x-axis) is shown. The lighter grey background indicates the time period in which a solution (that contains interaction partners for the immobilized proteins) is flushed over the slide. The darker grey background represents the washing steps between each binding step. When the first solution, which contains Streptavidin - the binding partner for the positive control (biotinylated BSA), is flushed over the slide, the relative intensity at the positive control spot increases. Therefore, the red line, representing the relative intensity at the bBSA spot, rises during this step of the measurement. During washing steps the relative light intensity remains the same. When the next antibody (anti-GFP) is flushed through, the corresponding GFP spot (blue line) shows an increase in relative intensity due to binding.
The binding curves are generated as following: First you have to select regions of interests (ROIs) for which you want to plot the relative shift in intensity. Since it is a relative shift, one also needs to define reference spots (background - where no protein of interest is immobilized) to which the intensity values are normalized (see figure 7). Additionally, a reference point in time needs to be set: at this point in time the relative shift in intensity from your ROIs to the corresponding reference background spots is set to 1. Therefore, the relative light intensity at each following point in time is compared to its reference spot. If the intensity of your ROI increases more than the intensity of the reference spot the relative shift is greater than 1. If the intensity of your ROI decreases more than the intensity of the reference spot the relative shift is less than 1. According to this approach every picture of the measurement is evaluated. The resulting binding curve shows real-time binding kinetics for your marked regions (figure 8).
![](https://static.igem.org/mediawiki/2015/3/36/Freiburg_binding_curve_explanationnew_preview.png)
Figure 8: Binding curves of the marked ROIs. The relative shift in intensity (y-axis) of the selected ROIs (figure 7) is plotted over time (x-Axis). The shift in intensity of the ROIs is normalized to their corresponding reference spots (Ref1-Ref3). As can be seen binding occurs during the Streptavidin flush at the positive control spot (red line) and during the anti-GFP flush at the GFP spot (blue line). No binding is detected at the negative control (grey line).
The Detection Device
The Basic iRIf Setup
Homogenous illumination of the glass slide with monochromatic light is necessary to achieve good iRIf results. This is best accomplished with a powerful LED light source shining on a lense which is positioned at the distance of one focal length, therefore parallelizing the light rays.
Since the purpose of an iRIf measurement is 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 have to be stored lossless in the camera. Since compression methods such as JPEG or MPEG remove subtle changes in the picture, resulting the binding signal is lost otherwise. 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 (figure 9).
Building Our Device
Since the physics behind iRIf are 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 rebuild our iRIf device can be found on the Results Page.
The Physics Behind iRIf
This chapter focuses on the physical theory behind iRIf 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 depend on the light's 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). Furthermore, continuity demands that transmitted and reflected light together return the original beam.
Geometric optics are also sufficient 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 situation where the object is set at the focal point of the lense. 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. In order 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). Therefore, we have the same angular frequency (ω1=ω2=ω) and can translate our coordinate system's 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 interference 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 is the one reflected at the unbound side of the slide and R3 is the 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. <7p>
How do we obtain the phase shift between R1 and R2/R3? By comparing the distances light has to overcome in order 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 consider 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 was not 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 the 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 generated due to the 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
In order 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 bind 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 flow cell 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