ECEN 4606, UNDERGRADUATE OPTICS LAB

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1 ECEN 4606, UNDERGRADUATE OPTICS LAB Lab 3: Imaging 2 the Microscope Original Version: Professor McLeod SUMMARY: In this lab you will become familiar with the use of one or more lenses to create highly magnified images of nearby objects. We will add a digitizing CMOS camera chip to this setup so that you can capture images. A quantity of specific interest in this lab is resolution and its various limits. PRELAB: The homework-style problems will give you data you will want for both the design and in the lab. The design problem is fairly realistic at this point and will use a good fraction of what we have learned so far. POSTLAB: There is some data analysis required after the lab. The matlab script or lab software ImageJ (available for download free of charge) should get you started, although you can use some other package if you wish. HOMEWORK PROBLEM 1: (A). Calculate a table of the diffraction-limited resolution (radius of the first null) of the objectives given in Table 1 of the first lab using violet (400 nm), green (525 nm) and red (650 nm) light. (B). For green (525 nm) light, calculate the size of the diffraction limited spot (radius of the first null) in the intermediate image plane for each of the objectives. (C). If you placed the CMOS camera chip at this intermediate focal plane, would the instrument resolution be diffraction or pixel limited? HOMEWORK PROBLEM 2: Figure 1 shows how depth of field depends on a finite pixel size which is much greater than the diffraction-limited spot size. Since a primary specification of a microscope is resolution, it is unusual to oversize the pixels and thus the equations given do not apply in this case. (A). Derive an equation for z 0, the half depth of field of a diffraction-limited microscope of given NA. Your equation for z o should be a function of r o and NA. Assume the camera (image) location is fixed which therefore fixes the plane of best-focus for the object. The diffraction-limited depth of field is the range of object positions in which the defocus blur (the diameter of the ray bundle) is less than or equal to the diffractionlimited spot diameter (2r 0 ). Hint use graphical ray tracing and work entirely on the object side of the lens (see Figure 2). Assume that the depth of field is small such that the Version 1.3, 8/15/12 McLeod and Gopinath 1

2 NA does not depend on object position. Your equation will look quite similar to that for r 0. (B). Calculate the depth of field for green light and the objectives from problem 1. Figure 1. Depth of field and finite pixel size, where p is the pixel size, D/2 is the radius of the lens, and the focal points are represented by the open circles. Figure 2. Beginning of graphical ray trace for HW2. DESIGN PROBLEM: Apply what you have learned so far to design an instrument that you will build in the lab with the following capabilities: 1. The object can be illuminated for transmission (like the objects used in the telescope lab) or in reflection (the light viewed by the microscope reflects off of the object and thus the light source and objective are on the same side of the object). You only have one lamp, so you will have to move it from one location to another. Reflection is the tricky case. Look at the equipment list and consider the best layout of components. Ask for help if you re stuck. 2. The microscope can be focused precisely and the impact of defocus can be quantified. Again, consider various choices here some are better than others. 3. The NA of the system can be adjusted. That is, the aperture stop has a variable diameter. 4. The microscope can be viewed by a human eye OR, possibly with a small adjustment, a real image can be captured by the CMOS camera chip. 5. The instrument is diffraction, not pixel limited. Since we will be quantifying how aberrations (specifically defocus) change the resolution, you want sufficient digital resolution to do this accurately. Design your system to give at least 10 pixels per diffraction-limited spot radius so that you can measure the spot size to an accuracy of ~10%. 6. The illumination wavelength can be changed to investigate the impact on resolution. Version 1.3, 8/15/12 McLeod and Gopinath 2

3 **Note specifically how you selected the objective lens. This should include comments on resolution and difficulty in achieving focus. You will be focusing the instrument precisely, so you really care about the latter. ***Make a careful drawing of your design including any important mechanics and any components that may be changed/moved. TECHNICAL RESOURCES: TEXTBOOK: Chapters 2 and 3 LECTURE NOTES: Lecture 3, the Microscope. EQUIPMENT AVAILABLE: 24 long optical rail and lens rail carriers for easy linear alignment of optics. A lamp with a fiber-bundle for illumination of your object. A diffuser (a non-glare page protector) to place in front of the light source in order to spread the illumination from the fiber- bundle more evenly. Microscope objectives: 5X, 10X, 20X (guaranteed). We also have 40 and 60X but not enough copies for all 3 groups, so if you design with these, be prepared with a back-up design using the 5, 10 or 20. Or bribe the TA to set them aside for you. Lenses: focal lengths of 50, 100 and 250 mm. Again, others are available, but those are guaranteed. Ask the TA during office hours about availability of other focal lengths. An alignment target printed on glass with ten micron ticks. Various lithographic masks with small, sharp-edged, opaque features printed on glass. Biological samples plus microscope slides and cover slips for preparation of biological samples. Translation stages with micrometers for quantitative, precision linear movement. A monochrome, digitizing USB CMOS camera. Specifications and Matlab routines to read and plot lines from the image files are at the back of the lab writeup. There is also free software in the lab that will perform these functions. A PC for viewing the images in real time and saving data files. Bring a USB stick to take the data away with you. Color filters glass containing dyes that absorb or transmit specific ranges of wavelengths. 50/50 beam splitters. These are partially-transmitting mirrors sandwiched in a glass cube such that half the light goes straight and half bounces at 90 degrees, as shown below. Version 1.3, 8/15/12 McLeod and Gopinath 3

4 100% 50% 50% Figure 1. A 50/50 beam splitter showing how optical power is divided. Note that they work the same way if light is introduced into any of the four faces. LAB PROCEDURE: STEP 1: 1:1 ALIGNMENT AND MAGNIFICATION ADJUSTMENT IN TRANSMISSION. Set up your transmission design using the alignment target as an object and broad spectrum (white light) illumination. Place the CMOS camera at the intermediate image plane and adjust for the design magnification (e.g 10X) and best focus. Use a translation stage for a focus adjust. Hint: it is easier to move the camera than the object. Be precise. Make sure that you are looking a printed spot on the target, not a blank spot. Remove the camera and add the eyepiece for viewing with your eye. Insure the optics are all centered. Consider (and test if you wish) the orientation of the eyepiece lens. Adjust the eyepiece location on the rail and note the apparent changes you observe. Adjust for a real image and replace the camera. Again precisely adjust to match your design magnification and best focus. In your lab book: Document the set up and the performance including any non-idealities you observe. How large is your field of view? Now consider the image on the computer screen. If the CMOS pixels are displayed 1:1 on the monitor pixels, what total magnification have you obtained considering this additional electronic magnification? STEP 2: DIFFRACTION-LIMITED RESOLUTION AND DEPTH-OF-FIELD In this section, you will test the resolution and depth of field to verify you calculated performance. Your data will be in the form of images of a knife edge at various defocus positions. Carefully record file names in your notebook as you proceed. 1. Illuminate a sharp edge such as a lithographic mask feature using white light filtered to a known color. Place an edge of the object in the center of your field with the edge running vertically and carefully focus your microscope by moving the focus knob in just one direction. That is, defocus the image and, noting which direction you are moving, bring the image back into focus by moving in the opposite direction. If you overshoot, start over. This procedure avoids backlash in the micrometer. Note the reading on the micrometer. Record an image with the digital camera. 2. Now, continuing to move in the same direction that you last moved the focus knob, move the object by your calculated half depth of field, z 0. If this is too small a distance to move accurately with the micrometer, pick a small multiple of Version 1.3, 8/15/12 McLeod and Gopinath 4

5 z 0 instead. Be as precise as you can. Record an image of the (presumably) slightly blurry edge. Move this same distance again and record an image. Continue this process so that you have 5 images of increasing defocus. In your lab book: Record the measured and calculated depth of field of the system. If they are different, explain why. Use to extract plots of the step response of your system. Label the x-axis of these plots in object coordinates (e.g. microns). If needed, normalize the intensity data (the y axis) so the data is easier to compare. By hand (or by Matlab if you re feeling spunky), extract the width of the blurred edge and plot this vs. the amount of defocus. Near perfect focus ( z < z 0 ), diffraction should dominate and the edge width should be similar to r 0. For large defocus ( z > z 0 ), the geometric expansion of the ray bundle should dominate and the edge width should grow like z times the numerical aperture. At best focus, how many resolvable spots (the field of view area divided by the smallest spot area) are captured by your microscope? How does this compare to a typical camera that might be used to capture the image? STEP 3: BIOLOGICAL SAMPLES Place some of the biological samples in your microscope and view them either by eye or with your camera system. Note the impact of the depth of field in limiting your ability to see the entire sample depth. Describe what you see. STEP 4: REFLECTION MICROSCOPE Switch to your reflection configuration and observe the change in the image of the lithographic target. If you have time, capture some images and compare them to the transmission implementation. Look at some of the random microchips for more interesting images. Compare this mode to the transmission mode. Version 1.3, 8/15/12 McLeod and Gopinath 5

6 Grading Expectations Lab Report 3: Imaging 2: the Microscope (100 total points) Name Name and group members. Abstract (10 points). Introduction (10 points) Methods (35 points) Alignment and magnification adjustment in transmission (11 points 5 for figures, 5 for description of how microscope works include equations and justification for design) Diffraction-limited resolution and depth of field vs. NA and wavelength (15 points 7 for figure or figures, 8 for description) Biological samples (5 points for description of preparation) Reflection microscope (4 points 2 for figure, 2 for description) Results and Analysis (35 points) Alignment and magnification adjustment in transmission (10 points 3 - description/analysis of physical magnification, 2 - description/analysis of electronic magnification, 3 - measurement of field of view, 2 -description of any nonidealities) Diffraction limited resolution and depth of field vs NA and wavelength (15 points comparison of measured (10 points) and calculated depth of field (5 points) w and w/o iris for both red and blue illumination (time permitting)) *note that your measured depth of field should include two methods measurement by moving object and measurement by moving the camera on the rail. Do this for both wavelengths of illumination. Biological samples (5 points description of what is seen) Reflection microscope (5 points comparison with transmission microscope, images if time, or description of what expect) e. Conclusion (10 points) Summary of lab report f. References Include any references that you used. Version 1.3, 8/15/12 McLeod and Gopinath 6