Comparing DIC and Phase Contrast images Lab Report
- Was this image taken with a DIC microscope or a Phase Contrast microscope?
- How can you tell?
DIC Worksheet Comparing DIC and Phase Contrast images The images below are of amoebas (Amoeba proteus). For each Figure (1-3): 1. Decide whether the image was taken with a DIC or Phase Contrast microscope 2. Explain how you can tell what microscope was used, based on how specific structures in the image look. A Figure 1. Figure 2. Figure 3. DIC: Differential Interference Contrast Microscopy Another form of Transmitted Light Microscopy Like Phase Contrast, it converts Phase Differences into Amplitude Differences • Polarization • Polarizing Filters • DIC Microscope • 4 components • Image Analysis • Pseudo 3D effect • Drawbacks • Advantages Remember Phase Objects? Phase Objects alter the phase of light as it passes through them. Objective lens 1 2 Cytosol Mitochondrion Cell Membrane Illuminating Light 3 The light that is collected by the lens has the same amplitude and wavelength, but the phase has changed, because… As the light passed through the mitochondrion, the high refractive index (n) of the organelle slowed the light down. Notice that the change in phase depends on the refractive index (n) AND ALSO on the thickness of the object. A THICKER object will slow light down more than a THINNER object, with the same n. Imagine you are running as fast as you can on grass. Suddenly you run through a pit of sand. It’s going to slow you down. How slow? It depends on how deep the sand is (n) and also how long the pit is (thickness) Phase Objects alter the phase of light based on their Refractive Index (n) and their thickness. D.I.C. microscopy • Converts differences in Phase to differences in Amplitude • Just like Phase Contrast Microscopy! • Another form of Transmitted Light Microscopy • A Brightfield microscope can be modified to perform D.I.C. • Historically, there were many ways to design a D.I.C. microscope • Today, the most common follows the 1952 design of G.Nomarski • D.I.C. microscopy is sometimes referred to as Nomarski Interference or N.I.C • One major difference from Phase Contrast Microscopy: D.I.C. relies on POLARIZED light… Polarization of light Human eyes have not evolved to detect changes in polarization so it is difficult to understand. Polarization describes the angle of the electric field as it travels through space. Polarization is NOT describing the angle the light ray is travelling (propagating)… These 2 rays are travelling in the same direction, at the same angle. But… The ray on the left is linearly polarized straight up and down. The electric field, shown in red, goes up and down parallel to the z-axis The ray on the right is linearly polarized at a slight angle. (the red curve is tilted back a little) A ray of light can be linearly polarized in an infinite number of angles (0-360°) Elliptical & Circular Polarization Instead of linear polarization, a ray of light can be elliptically or circularly polarized In these cases, the electric field doesn’t just bounce up and down in a flat plane. Instead, the electric field corkscrews down the path of light. The red line traces the electric field as it twists along the path of travel. If you look down the path, you see the red curves create a perfect circle Here, the red line isn’t tracing a perfectly circular profile, it’s a little oblong, or elliptical A ray of light can be linearly polarized (any angle), elliptically polarized (any “shape”), OR circularly polarized. To sum up… Most light in nature is UNPOLARIZED This doesn’t mean it has no polarization. EVERY ray of light has some kind of polarization. UNPOLARIZED light refers to a mixture of many rays of light, each with a random polarization (linear, circular, or elliptical). It would probably be clearer to say MIXED polarized instead of Unpolarized. But that’s the convention. Unpolarized light is often symbolized like this: It’s like you’re looking down the path of light and seeing many rays of linear polarized light (at many different angles) coming at you. I like to add a circle and ellipse to the symbol to remind us that there are also many rays of circular and elliptically polarized light in the mix Light from the sun and from lightbulbs (any kind) is unpolarized. POLARIZED light sources, like Lasers, are rare in the world. A Polarizing Filter controls the angle of light Polarizing filters only let light that is Linearly Polarized at ONE angle pass through. In this diagram, the light bulb emits Unpolarized light. The Polarizing filter is oriented straight up and down. So only linearly polarized light, at a straight up and down angle, passes through the filter Let’s take a closer look at what’s going on here… If a ray of light is linearly polarized at the same angle as the filter… 100% of the light passes through the filer. If a ray of light is linearly polarized Perpendicular (90° to) the filter… 0% of the light passes through the filer. These 2 situations are the simplest. What if we make things more complicated… More polarizing filter examples: If a ray of light is linearly polarized at some other angle, or elliptically, or circularly… A fraction of light in the direction of the filter will pass. (for example: 50%) Here, since the incoming light is at about a 45o angle, about half of it can pass through. The small blue arrows in the diagram show the horizontal and vertical components of the 45o light. Only the component that is vertical gets through the filter. This incoming ray is polarized at a steeper angle relative to the filter… A smaller fraction of light will pass. (for example: 25%) Here, since the incoming light is at about a 15o angle, a smaller component is pointing in the direction of the filter. So when it strikes the filter, a smaller amount of light passes through. Changing the angle of the polarizing filter changes the amount of light that passes In the case of circular or elipitically polarized light… Now the larger, horizontal component passes through Some component of the circularly polarized light is oriented horizontally. That component will pass What about 2 filters? When elliptically polarized light strikes a polarizing filter, some component is oriented in the same direction as the filter… The component that passes the 1st filter is straight up and down When the vertical light strikes the angled 2nd filter, only the component going the same direction as the filter can pass. If you take 2 polarizing filters and turn them 90° to each other, then NO light will pass… The first filter only lets linearly polarized, straight up and down light pass through. Because the second filter is turned 90°, There is no component of the light that can pass through. How do polarized sunglasses work? Light can become “naturally” polarized when it reflects off a flat surface. The light is linearly polarized at the same angle as the surface. When this happens off water, or snow, or the road or cars in front of you, we call this reflected light GLARE. Polarized sunglasses have their polarizing filters oriented to block GLARE. Can you see which direction the polarizing filters should be oriented for this to work? The image on the left was taken through a polarizing filter oriented vertically to block the horizontally reflected light that creates glare. You can see the effect of glare in the image on the right. This improved visibility explains why polarized sunglasses are helpful when driving (glare from the road and other cars) and around water. OK, back to DIC… A good Transmitted Light, Brightfield Microscope can be converted to a D.I.C. Microscope by adding FOUR components: 1 The Polarizer is a polarizing filter that sits between the microscope’s light source and the specimen. This filter controls the light that illuminates the specimen by forcing it all to be linearly polarized in one angle. 1 Polarizer OK, back to DIC… A good Transmitted Light, Brightfield Microscope can be converted to a D.I.C. Microscope by adding FOUR components: 2 The Beam Splitter is a thin slice of crystal (all natural!) that takes each ray of light and splits it into 2. It sits below the specimen These 2 rays serve as “sampling points” that probe the specimen for changes in phase. 2 Beam Splitter 1 Polarizer Early in the development, there were different ways to create these beam splitters. Nomarski’s design is the one that became most popular. OK, back to DIC… A good Transmitted Light, Brightfield Microscope can be converted to a D.I.C. Microscope by adding FOUR components: 3 The Beam Recombiner is just like the Beam splitter, but instead of splitting light rays, it brings the 2 sampling rays BACK TOGETHER. It sits above the specimen and operates on light after it has passed through the specimen The “recombination” of the 2 sampling rays occurs by the phenomenon of INTERFERENCE. 3 Beam Recombiner 2 Beam Splitter 1 Polarizer OK, back to DIC… A good Transmitted Light, Brightfield Microscope can be converted to a D.I.C. Microscope by adding FOUR components: 4 The Analyzer is just like the Polarizer: it’s another polarizing filter. But it’s oriented perpendicular (90°) to the Polarizer. This filter sits just below the eyepieces and determines what light reaches your eyes (or the camera) 4 Analyzer 3 Beam Recombiner 2 Beam Splitter 1 Polarizer So how do these 4 components work? 6 The Analyzer determines what light passes up to the eyepieces. It is rotated perpendicular (-45°) to the Polarizer (+45°). In this example, since the light coming from the Beam Recombiner is +45°, NONE of this light will get through. The result: areas of the sample with NO phase difference appear completely black. +45° 5 0° Beam Recombiner The Beam Recombiner puts each set of sampling rays back together. The result is a single ray with a certain angle of polarization. In this example, there is no phase difference between the 2 sampling rays, so when they recombine, they go back to their original +45° angle 4 Pairs of sampling rays strike ALL OVER the slide. They are “looking” for phase changes in the specimen. 3 The Beam Splitter takes every ray of 45° light and splits into 2 new sampling rays. These rays are linearly polarized at 0° and 90°. (Because you can “decompose” a 45° into ½ 90° and ½ 0°) 90° +45° 2 The Polarizer polarizes all the light to a specific angle. For this example: +45° 1 Unpolarized light leaves the light source Beam Splitter How do we make a complete image? 6 When both sampling beams pass through the background, there is no phase difference between them. So when recombined, they reform the +45° light. This is completely blocked by the Analyzer 7 When both sampling beams pass through the cell, there is no phase difference between them. So when recombined, they reform the +45° light. This is completely blocked by the Analyzer 6 The Beam Recombiner puts each set of 5 sampling rays back together. The result is a single ray with a certain angle of polarization. Pairs of sampling rays strike ALL OVER the slide. They are “looking” for phase changes in the specimen. The Beam Splitter takes every ray of 45° light and splits it into 2 new sampling rays. These rays are linearly polarized at 0° and 90°. 3 The Polarizer polarizes all the light rays to a specific angle. For this example: +45° 2 +45° 1 No phase difference 4 0° Unpolarized light leaves the light source An infinite number of light rays rises up. +45° No phase difference 4 8 7 +45° 8 -15° Phase difference! Cell 90° 0° +45° 90° 0° +45° 90° When one sampling beam passes through the cell and the other passes through the background, there is a BIG phase difference between them. When they recombine, they create light at a NEW angle of polarization (-15° in this example). SOME of this light will be able to pass through the Analyzer and create a bright spot (on the dark background) The big picture The DIC microscope works by creating pairs of sampling rays that “probe” the specimen for phase differences. (Stick out your index and middle fingers with a tiny space between them. Now poke them at a picture. Poke them all over the picture so you cover its entire surface. Anytime there’s a difference in phase between what your index finger and your middle finger touches, you’ll get a little bright spot.) The separation between the sampling rays is TINY. Ideally less than the resolution of the microscope, so in the hundreds of nanometers. The DIC microscope is great at finding CHANGES in refractive index and CHANGES in thickness. It highlights the BORDERS between structures. This is another big difference from Phase Contrast microscopy. Phase contrast looks for phase changes between objects and the background. DIC looks for phase changes between adjacent points. DIC Images Unstained human cheek epithelial cells Notice the dark grey background Notice that there isn’t a big difference in the brightness of the background, cytosol, or nucleus. That’s because in the center of these objects, there are no phase differences. But the BORDERS between the background, cytosol, and nucleus are either very bright or very dark! The borders are where the big phase changes are (because of a difference in refractive index and/or thickness)! Some Complications: Unstained human cheek epithelial cells The background is grey, not completely black. If the Polarizer and Analyzer are perpendicular, then the background should be completely black. If we really setup the DIC microscope as described in the previous slides, we’d have a black background and we’d only see bright highlights for the borders between objects. But that would mean we’d lose all the dark borders in this image. (Because pixels can’t get darker than black.) Instead, we adjust the microscope so that the background is grey, instead of black. That way, we can see the bright highlights and the dark highlights. (Remember that the beam recombiner puts the sampling rays back together by Interference. If you’ve heard of interference before you may remember that interference can be constructive (brighter) or destructive (darker). We want to be able to see both kinds of interference.) Adjusting Bias in D.I.C. To make the background grey, not black, and to allow us to see both bright and dark highlights in our image, we shift the position of the Beam Recombiner. On a DIC microscope, there is usually a small metal knob that you twist to shift the Beam Recombiner. Twisting that knob is called adjusting the Bias. This shows the effect of adjusting the Bias in different directions on the same image. (b) shows what happens when the background is set to black. (a) and (c) show bias shifted in opposite (but equal) directions Pseudo-3D images Let’s take a closer look at those Bias adjustment images. Image (a) looks like a fried egg to me. Do you see the same thing? Or do you see the opposite? Now try this… Carefully lift your screen up and rotate it 180° so it’s upside down. Now how do they look? Did they switch? What’s going on here? Image (c) looks like a crater on the moon to me. (You can get the same effect if you turn your chair around so you’re facing away from the screen and then bend over backwards to look at it upside down.) ((Don’t die)) Pseudo-3D images DIC creates images that look 3-Dimensional. But they’re NOT. This DIC image of a human cheek cell looks like the nucleus is projecting out of the cell. It’s like there’s a light source at the upper left corner lighting up the left side of the nucleus. The right side of the nucleus is in shadow. This is the same cheek cell on the same microscope, just with a different bias setting. It looks like a crater dug deep into the surface. Here the light source is lighting up the right side while the left side is in shadow. Same cell. Same microscope. Completely different interpretations of structure! What?! Pseudo-3D images DIC creates images that look 3-Dimensional. But they’re NOT. So don’t interpret what you see in a DIC image as 3D information. Don’t think egg or crater. A The nucleus in this image looks like it’s sticking out of the cell. But you KNOW that the nucleus of a cell doesn’t project out of it like an egg yolk; it’s usually somewhere deep in the cytoplasm. B The bright and dark borders of DIC give the appearance of depth. But don’t be fooled. Those dark and bright borders are really the changes in phase created by differences in refractive index (n) and/or thickness. Why is one side bright and the other dark? In the image above, if we look at the region labeled (A), the plasma membrane is bright white. This is because one sampling ray (white arrow) was in the background and one was in the cytoplasm (black arrow). There was a big phase difference, going from water to cytoplasm. At region (B) the plasma membrane is dark grey. Here, one sampling ray (white arrow) was in the cytoplasm and the other was in the background (back arrow). Because the order was reversed, we see the same phase change, but in the opposite direction. You can interpret it as A= less dense to more dense = bright. B= more dense to less dense = dark. Pros & Cons of DIC DIC microscopy is a nice alternative to Phase Contrast • It’s more convenient than Phase Contrast because it doesn’t require a dedicated set of objective lenses. • The 4 DIC components are pretty expensive, so the $ is almost even. • There are no HALOs! But DIC does have its drawbacks • The pseudo-3D appearance can mislead anyone who doesn’t know better • But you are a DIC-master now, so you’ll never make that mistake • Because it is detecting phase differences, it will be susceptible to invisible and identical objects just like Phase Contrast. • Because it relies on Polarized Light, you cannot use any materials (slides/coverslips/culture vessels) that might alter the polarization of light. • This is usually not a problem except with Plastic Culture bottles and plates. Plastic is notorious for messing with polarization, which will screw up DIC. • Plastic culture vessels are commonly used when growing human and other mammalian cells, so this is a bummer. • The glass in your objective lenses should also be “strain-free” so that they don’t alter the polarization of light. But good quality objective lenses are normally strain-free, so that’s not too big a deal.