Assignment 2: epi illuminator for fluorescence microscopy

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20.309: Biological Instrumentation and Measurement

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Laser safety

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.

Design your beam expander

Fluorescence microscope block diagram.

Design a beam expander to illuminate your sample with a 40x objective. You will be using a 532nm laser with a beam diameter of roughly 1mm.


a block diagram of your microscope, including your choice of focal lengths for lenses L3, L4 and L5. Label all components and and important distances. In one or two sentences, justify your choice of values for L3, L4 and L5.

Add laser illuminator, dichroic mirror, and barrier filter

Layout of fluorescence microscope.
  1. Go over your design with one of the instructors before you start building.
  2. 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.
  3. 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.
  4. 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.
    • Dichroic mirror mount
    • 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.
  5. 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

  1. Remove L3, L4, L5, and the objective lens L1.
  2. Mount the laser.
  3. 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.
  4. 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.
  5. 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?
  6. 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.
  7. 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.
  8. 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.
  9. 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.