Fluorescence microscope block diagram.
In this part of the lab, you will add epifluorescence imaging capability to your microscope, make images of fluorescent samples, and process the images to correct artifacts caused by nonuniform illumination. This week, you will test out your new fluorescence microscope by imaging fluorescent microspheres of several sizes. Next week you'll image mammalian cells stained with a fluorescent dye. To make the correction for nonuniform illumination, you will also make images of a uniform fluorescence reference slide and a dark image with the illuminator turned off.
||In this part of the lab, you will use a 5mW, 532 nm laser with focusing optics. You must attend the safety lecture before you work with the laser. See an instructor if you missed the lecture. Do not begin working with the laser until you thoroughly understand how to use it safely.
Stop working and ask an instructor immediately if you have any questions about working with lasers safely.
When you work with lasers, keep these laser safety best practices in mind:
- Remove reflective clothing items and jewelry when working with lasers.
- Use the lowest possible power during alignment or adjustment.
- Do not use reflective tools when the laser is on.
- Know the beam path at all times.
- Use a stop to prevent uncontained beams.
- Secure the stop so it will not move accidentally.
- Keep your eyes out of the plane of the beam, which his normally a plane just above the optical table.
- Disable lasers when they are not in use.
- Use the correct safety goggles.
- Always check the marking on the goggles to ensure they are appropriate for the laser you are working with.
- Laser safety goggles are a last line of defense, not an excuse for unsafe practices.
- Do not operate a laser unless the flashing laser warning sign is on.
Some specific ways to work safely with the 20.309 fluorescence microscope:
- Use an OD = 2 neutral density (ND) filter to attenuate the laser near the source.
- "OD" is an abbreviation for "optical density," which is log10 of the power transmitted by the filter divided by the incident power.
- "ND" means that the OD is equal for all wavelengths.
- The ND filter attenuates the power of the laser by a factor of 102, to about 50 μW, which is completely safe.
- Mount the ND filter securely so there is no chance that it will get knocked out of place accidentally.
- The inverted microscope design directs laser light upward, toward the ceiling. Do not lean over the objective mount. Use a stop to prevent the beam from propagating above your microscope.
- Confine beams inside lens tubes.
- Disable the laser when it is unattended or stored by removing a battery, disconnecting the power source, or removing the key.
- Safety goggles for the laser in this lab should have an OD of at least 2 at 532 nm.
- There are (at least) two different kinds of safety goggles in the lab that are appropriate for this lab. One has an OD of 2 at 532 nm and the other has an OD of 5. You can see the beam with the OD = 2 goggles, but the power is reduced to a safe level. This makes the OD 2 laser goggles very useful for making adjustments that must be done at full power.
Add laser illuminator, dichroic mirror, and barrier filter
Layout of fluorescence microscope.
- Go over your design with one of the instructors before you start building.
- Use cage rods to construct a cage of appropriate size for the beam expander and excitation tube lens (L3, L4, and L5).
- For maximum flexibility in positioning, removing, and reinstalling optics, use 3 cage rods instead of 4. Use a cage plate (CP02) for each lens. Mount the lens in a 1/2" lens tube (SM1L05) so it can be easily removed or installed.
- Add the two turning mirrors to the end of the cage.
- Use a thick cage plate (CP02T) only to join 2 cages. (You will use a total of two CP02T plates in your microscope.) Don't use CP02T to mount lenses — there are not enough of them in the lab to use this way. It is easy to tell the difference. CP02T has 8 set screws and is roughly twice as thick as the CP02; CP02 has 4 set screws.
- Use a cube optic mount (B5C) to mount dichroic mirror DM on a kinematic platform (B4C) . Place the kinematic platform in the cage cube (C6W).
- The first surface of the dichroic should face the laser.
- Some dichroics have an arrow indicating the first surface. If not, to ascertain which surface has the coating, watch the reflection of a corner of a piece of lens paper as it touches the mirror. On the first surface, the corner and its reflection will appear to touch. Held the other way, the corner and its image will appear a few millimeters apart.
- Mount the dichroic so that the first surface lies on exactly a diameter of the B5C mount.
- The mounting bracket should not stand in the way of the rotation of the kinematic stage.
- Use the clear plastic #4/40 screws affix the dichroic mirror without deforming nor scratching its surface.
- Hold the screwdriver at the tip to avoid slipping and scratching the dichroic. The screwdriver will instantly scratch the dichroic.
- Ask an instructor for help if you need to clean a dichroic mirror or barrier filter. Dichroics have delicate, exposed coatings and must be cleaned with extra care.
- Use four black, plastic #4/40 screws to hold the cube optic mount (B5C) on the cage cube (C6W).
- Tighten the screws enough so that the cube optic mount holds its adjustment, but can still be rotated.
- Make sure to block the (small percentage of) excitation laser light that will be transmitted through the dichroic mirror with some black lab tape affixed to the B5C cube optic mount.
Align the laser illumination path
- Remove L3, L4, L5, and the objective lens L1.
- Mount the laser.
- Insert a neutral density filter (ND filter) between the laser and M2 to reduce the laser power to a safe level for adjustment.
- Remove jewelry. Turn of the laser before using reflective tools.
- Turn on the laser. Use a beam stop until the laser position is set. Adjust the laser position so that the laser shines near the middle of M2.
- You can use a DG10 mounted frosted-glass alignment disk with a pinhole to best center the laser beam onto M2.
- Adjust M2 and M3 to center the laser light in the cage.
- Use two CPA1 alignment targets to gauge beam alignment.
Using pinholes and irises to optimize initial microscope alignment. Notice that this person is breaking one of the laser safety rules. Which one?
- Adjust the dichroic mirror DM so that the beam will enter the middle of the objective lens.
- Use a DG10 mounted frosted-glass alignment disk with a pinhole to show the optical center of the vertical beam path.
- Verify that the centered beam is perpendicular to the floor. If the beam is at an angle, verify that the dichroic mirror DM is mounted on a diameter of the rotating mount.
- Insert the beam expander lenses, L3 and L4. Adjust the separation between L3 and L4 to achieve a collimated beam.
- It may be necessary to make small adjustments to M2 and M3 to recenter the beam.
- Replace the excitation tube lens L5 and the objective lens L1. Adjust the position of L5 for best beam collimation (you can try to focus it far away).
- The product of beam divergence and diameter is constant. L5 and L1 shrink the beam, causing increased divergence. The beam emerging from the objective will likely appear to grow in size as it propagates, even when the lenses are in their optimal positions.
- It may be necessary to make small adjustments to M2 and M3 to recenter the beam.
- Use a fluorescence reference slide to center the field of view and to optimize the uniformity of illumination.
- Make sure to use an ND filter when you use the fluorescence reference slide. The slide bleaches quickly at high intensities and you will not get a good image.
- Put a barrier filter in the afocal part of the imaging path.
- Center the camera's FOV in the objective's FOV.
- If the laser light emerges from the objective at an angle, double-check your alignment and dichroic mounting.
- The barrier filter has delicate, exposed metal coatings. Ask an instructor for help if you need to clean it.
Example images and histogram. Top: fluorescent beads; center: reference slide, bottom: image histogram.
In this part of the lab, you will make images of fluorescent microspheres plus your dishes of cells, and correct them for nonuniform illumination. In order to do the correction, you will need a reference image and dark images in addition to the image of the sample. See this page for more information about flat-field correction.
- Reference Images: Take the reference image as close as possible in time to the sample images. Don't make any adjustments to your microscope between capturing the reference image and the sample image. For example, every time you move a mirror or re-align the laser, you will change the illumination profile and you must take a new reference. Adjusting the camera exposure and gain between recording the reference and sample images is okay.
- Dark images: Each time you record an image (reference or sample), make sure to take a corresponding dark image using the EXACT SAME camera settings (i.e. use the exact same Exposure Time and Gain settings you had chosen for your reference/sample image). This is the only valid way to subtract the correct dark value from your reference/sample image. Use 12-bit imaging mode to get the best results.
- Saving: Remember to save your images in a format that preserves all 12 bits. We recommend using the MATLAB save command to save data in a .mat file so you can reanalyze it later if necessary. You could also save in an image file format with imwrite. Convert the image to 16-bit, unsigned integer format with the correct range before saving. Read this section of Part 1 if you need to refresh your memory.
- Record a reference image
- Put in an ND filter and take an image of the reference slide with the 40X objective.
- Turn off the laser and record a dark image (without changing any camera settings!)
- Use MATLAB to generate a histogram of to be certain that the image is exposed correctly.
- Remove the ND filter and image the samples:
- 0.84 μm beads and a corresponding dark image
- 3.26 μm beads and a corresponding dark image
- For each of these samples remember to make sure the image is exposed correctly, and to take a corresponding dark image
- Adjust the gain (to zero) and exposure of the camera to get the best picture.
Flat field correction
- Perform flat-field correction on the images.
- Divide the image by a normalized version of your reference image minus the dark image (see this page for more detail).
- Tip: If all your images are appearing dark in MATLAB you probably have not rescaled your image properly. Read through the displaying images section of this page if you need a refresher.
- For each bead sample, include the original, reference, and flat-field corrected images in your lab report. In the caption note the exposure and gain settings used for each image.
- For one set of images (either the 0.84 or 3.6 μm beads and their corresponding dark and reference images), include the MATLAB code you used to calculate the flat-field correction.
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