Optics Bootcamp

Imaging apparatus with illuminator, object, lens, and CCD camera mounted on an optical rail.

Overview

This lab exercise will introduce you to some of the optical components that you will use in the microscopy lab. You will build an apparatus that includes an LED illuminator, an object with precisely spaced markings, a lens, and a CCD camera. You will place the object at several distances from the lens and measure the corresponding image distance. Using the MATLAB Image Acquisition Tool, you will record images and use them to measure magnification. Finally, you will compare your measurements to the values given by the lens makers' formula that was derived in class.

Orientation

The 20.309 lab contains more than 15,000 optical, mechanical, and electronic components that you will use for your lab work throughout the semester. To the untrained eye, they all look pretty much alike. (For the final exam, you will be required to identify various components while blindfolded.)

You can waste a lot of time looking for things, so it's important to learn your way around. The floor plan below shows the layout of rooms 16-352 and 16-336. When you get to the lab, take a walk around. Go everywhere and check things out. Spend at least five minutes poking around. Read the time machine poster between the cable rake and the Nikon microscope. Discover the stunning Studley tool chest poster near the east parts cabinet.

See! Wasn't that a worthwhile journey?

Now go ahead and build your lens measuring apparatus. The first step is to gather the parts you will need.

Gather materials

Parts for the simple imaging apparatus.

The first step is to gather the materials required to build the lens measuring apparatus. The lists below include part numbers and descriptive names of all the components in the apparatus. It is likely that you will find some of the terms not-all-that-self-explanatory. Most of the parts are manufactured by a company called ThorLabs. If you have a question about any of the components, the ThorLabs website can be very helpful. For example, if the procedure calls for an SPW602 spanner wrench and you have no idea what such a thing might look like, try googling the term: "thorlabs SPW602". You will find your virtual self just a click or two away from a handsome photo and detailed specifications.

Optomechanics

These components are located in plastic bins on top of the center parts cabinet:

• 1 x RLA1800 dovetail optical rails
• 4 x RC1 rail carriers
• 1 x SM1L10 lens tube
• 1 x SM1RC lens tube slip ring
• 1 x CP02 cage plate
• 1 x LCP01 cage plate (looks like an "O" in a square)
• 1 x LCP02 cage plate adapter (looks like an "X")
• 2 x SM2RR retaining rings

These components are located on the counter above the west drawers.

• 4 x ER1 cage assembly rod (The last digit of the part number is the length in inches. Take a 1" rod. Lengths less than 1" have a part number that starts with a zero.)
• 6 x SM1RR retaining rings

Screws and posts

Stainless steel, ¼-20 size, socket head cap screws (SHCS), washers, posts, and post holders are located on top of the west parts cabinet. If you are unfamiliar with screw types, take a look at the main screw page on the McMaster-Carr website. Notice on the left side of the page that there are about ... links on the left side of the page. Click the links for more information about screw sizes and attributes. This link will take you to an awesome chart of SHCS sizes.

• 4 x PH2 post holders
• 4 x TR2 optical posts
• 4 x 8-32 set screws
• 6 x 1/4-20 x 5/16" socket cap screws
• 1 x 1/4-20 set screw

Optics

Lenses and microscope objectives are located in the west drawers.

• 1 x LA1951 plano-convex, f = 25 mm lens
• 1 x LB1811 biconvex, f = 35 mm lens

Object

Imaging targets are located in a plastic bin on top of the east cabinet.

• 1 x R1DS1N 1951 USAF test target

Optoelectronics

LEDs will be in a plastic bin on top of the center cabinet.

• 1 x red, super-bright LED (mounted)

Tools

Most of the tools are located in the drawers at your lab station. Be sure to put all of the tools you use back in their proper location.

Hex keys (also called Allen wrenches) are used to operate SHCSs. Some hex keys have a flat end and others have a ball on the end, called balldrivers. The ball makes it possible to use the driver at an angle to the screw axis, which is very useful in tight spaces. You can get things tighter (and tight things looser) with a flat driver.

• 1 x 3/16 hex balldriver for 1/4-20 cap screws
• 1 x 9/64 hex balldriver
• 1 x 0.050" hex balldriver for 4-40 set screws (tiny)
• 1 x SPW602 spanner wrench

You will also need to use an adjustable spanner wrench. The adjustable spanner resides at the lens cleaning station. There is only one of these in the lab. It is likely that one of your classmates neglected to return it to the proper place. This situation can frequently be remedied by yelling, "who has the adjustable spanner wrench?" at the top of your lungs. Try not to use any expletives. And please return the adjustable spanner wrench to the lens cleaning station when you are done.

• 1 x SPW801 adjustable spanner wrench

Things that should already be (and stay at) your lab station

• 1 x Manta CCD camera
• 1 x Calrad 45-601 power adapter for CCD
• 1 x ethernet cable connected to the lab station computer

Build the apparatus

		Optical rails Optical rails are useful for arranging components in a line that require variable separation. Sliding clamps sit on the rail. The clamps have a thumbscrew that locks them in position. Secure the optical rail on the optical table using two 1/4-20 x 5/16 cap screws and the 3/16 hex balldriver. Prepare four sliding posts, each by attaching one RC1 rail carrier to one PH2-ST post holder with one 1/4-20 x 5/16 cap screws.
		Mount the LED light source: In the LCP01 cage plate, the LED will get sandwiched in-between two SM2RR retaining rings. First screw in one SM2RR only 1 mm deep. Next place the LED above it. Finally tighten down the second SM2RR using the SPW801 adjustable spanner wrench. The SPW801 can be opened until its width matches the SM2RR diameter, the separation between the ring's notches. In the LCP02 cage plate adapter, screw in on SM1RR 3 mm deep. Carefully (use lens paper unsparingly to protect the lens surface) place the 25 mm plano-convex lens above it, with the hemisphere facing down yet not touching any potentially damaging surface. Tighten down a second SM1RR using the SPW602 spanner wrench, whose guide flanges sit in the ring's notches to prevent any scratching of the lens's optical surface. Attach the LCP01 cage plate (holding the LED) to the LCP02 adapter (holding the 25 mm condenser lens), using the 0.050" hex balldriver to secure four ER1 rods with eight 4-40 set screws. Affix a TR2 optical post to the LCP01 cage plate (holding the LED). Slide in the LED assembly along the optical rail. Power the LED light source: The red LED will be connected to a DC power supply. Ensure that the current limit on the power supply (CH1) is set to a value below 0.5 A. Connect channel CH1 to the red and black threads of the LED, using alligator clip cables. Turn on the power supply, and press its Output button to light the LED. Adjust the LED brightness using the power supply's Voltage knob.
	Mount the object (US Air Force target 1951): Tighten the R1DS1N 1951 USAF test target in-between two SM1RR retaining rings inside the SM1L10 lens tube, using the SPW602 spanner wrench. (This procedure should be reminiscent of the insertion of the 25 mm hemispherical lens in the cage plate adapter.) Slide in the lens tube through the SM1RC slip ring. By rotating the lens tube, you will be able to modify orientation of the object. Lock the lens tube in place using the 9/64 hex balldriver. Affix a TR2 optical post to the SM1RC slip ring (holding the USAF target). Slide in the object assembly along the optical rail. Mount the lens (f = 35 mm): Tighten the LB1811 biconvex f = 35 mm lens in-between two SM1RR retaining rings inside the CP02 cage plate. Affix a TR2 optical post to the CP02 cage plate (holding the lens). Slide in the lens assembly along the optical rail.
		Mount the CCD camera: Affix a TR2 optical post directly to the CCD camera plate using the 1/4-20 set screw. Slide in the camera assembly along the optical rail. Connect the CCD to the computer using a red ethernet cable. Power up the CCD using the Calrad 45-601 power adapter.
Vertically align the LED, object, lens, and camera assemblies.

Visualize, capture, and save images in Matlab

Now that you've learned the basics of mounting, aligning and adjusting optical components, you will through this lab exercise

• verify the lens maker and the magnification formulae:

$ {1 \over S_o} + {1 \over S_i} = {1 \over f} $

$ M = {h_i \over h_o} = {S_i \over S_o} $

• become familiar with image acquisition and distance measurement using the Matlab software.

Log on to the computer, launch Matlab, and run imaqtool. Select the Manta GigE Mono 12" hardware in the left bar. The Start Preview button will bring up a window of the live image from the CCD camera. Move the lens and USAF 1951 target object to produce a focused image. Under the Device Properties tab, optimize the Exposure Time Abs field for good contrast without pixel saturation. Measure the distance $ S_o $ from the target object to the lens and the distance $ S_i $ from the lens to the CCD active imaging plane. Does the lens maker formula $ {1 \over S_o} + {1 \over S_i} = {1 \over f} $ apply as it should when the image focus is optimized?

Save images in Matlab: Make sure you limit to 1 the number of Frames Per Trigger in the General tab of the Acquisition Parameters; Use the Start Acquisition and Export Data buttons; Navigate to the Student Data\ Spring 2015\ directory accessible from the computer desktop to save your data files remotely on a server you'll be able to browse from your home computer. The file extension will be .MAT (e.g. 1951target_01.mat). The variable within this file (e.g. im01) will represent the image as a 492x656 matrix of 16-bit integers.

Examine images in Matlab

	To display the image in Matlab, use the imshow command: Change your Matlab path to the Student Data\ Fall 2014\ directory. Add the saved images to your current workspace by typing load( '1951target_01.mat' ); When the 12-bit numbers from the camera get transferred to the computer, they are converted to 16-bit numbers. 16-bit numbers can represent a range of values from 0-65535. This leaves a considerable portion of the number range unoccupied. Because of this, if you type imshow( im01 ), you will see an image that looks almost completely black. Adjust the image to fill the full range by typing imshow( 16.0039 * im01 ). Note: 16.0039 equals 16 + 1/256. This factor maps values in the range 0-4095 to 0-65535.
	Determine the distance (in pixels) between two specific points in the image: Either use the Data Cursor Tool to display the X and Y coordinate of your mouse pointer, or use the interactive improfile function from the Matlab command window, which lets you trace a segment across the active figure (visualized as a dotted line) and generates a plot of pixel intensity vs. pixel position along the segment in a new figure. This manipulation allows you to calculate the image size $ h_i $, taking into account the CCD pixel size: 7.4 μm x 7.4 μm. Confirm the corresponding object size $ h_o $: Refer to the | specification sheet of the USAF 1951 test target, pages 5 and 8 in particular. The 1" R1DS1N 1951 USAF test target includes elements of groups 2 and 3. Example: Element 2 in Group 2 has 4.49 cycles (= line pairs) per millimeter. So 2 line pairs from Element 2 in Group 2 span 2 ÷ 4.49 = 0.4454 mm = 445.4 μm. Do both magnification relationships $ M = {h_i \over h_o} = {S_i \over S_o} $ match ?

Plot and discuss your results

• Repeat these measurements of $ S_o $, $ S_i $, $ h_o $, and $ h_i $ for several values of $ S_o $.
• Plot $ {1 \over S_i} $ as a function of $ = {1 \over f} - {1 \over S_o} $.
• Plot $ {h_i \over h_o} $ as a function of $ {S_i \over S_o} $.
• What sources of error affect your measurements?
• What is the uncertainty of your magnification measurement?

Optical microscopy lab

Code examples and simulations

• Converting Gaussian fit to Rayleigh resolution
• MATLAB: Estimating resolution from a PSF slide image
• Matlab: Scalebars
• Calculating MSD and Diffusion Coefficients

Background reading

• Geometrical optics and ray tracing
• Physical optics and resolution
• Optical aberrations
• Aperture and field stops
• Optical detectors, noise, and the limit of detection
• Manta G032 camera measurements
• Understanding log plots