Assembly, Tuning and Use of an Apertureless Near Field Infrared Microscope for Protein Imaging

Initial assembly of the optical setup is completed by setting up the multi-mode a FM and optical microscope. The alignment of the invisible infrared beam is made easier by first aligning with a visible red helium neon laser. The solid line to overlap the infrared, the dotted line, the objective being to observe the laser light striking the cantilever tip apex and infrared detector.

Final alignment of the infrared beam is made by adjusting the optical microscope position as well as the mirrors to obtain a one F signal with the right shape when the desired nearfield signal is obtained. Simultaneous a FM topography and nearfield images are collected using the nano scope software. Hi, I'm Professor Gilbert Walker from the Department of Chemistry at the University of Toronto.

Hi, I'm Melissa Paulite, also from the laboratory of Gilbert Walker. Today we will show you the procedure for the initial assembly and alignment of the aperture list, near field infrared scanning microscope. We use this procedure in our laboratory to study protein aggregates.

So let's get started. Before we describe the assembly and adjustment of the imaging system, let's first look at a brief overview of the complete system. The parts of the imaging system that we will describe in this video include the optical table, the base of the multimode atomic force microscope, or a FM, the scanner of the multi mode A FM, the elevated optical breadboard guiding mirrors the optical tube with iris and center.

Block the optical cube with the zinc selenide partial reflector, the optical tube used for securing the setup on the elevating platform. The infrared objective, the optical tube for collected back scattered light. The cube with an off axis paraboloid mirror the cube to hold the germanium plate at 45 degrees.

The microscope eyepiece the XY stage mounted pinhole, the mercury cadmium Telluride or MCT infrared detector. The infrared detector preamp the XY stage to move the beam position, the elevating table to focus the beam, the Z stage to position the infrared detector and the X stage to position the infrared detector. To begin assembly of the optical setup, use tuning mirrors to position the helium neon beam parallel to the optical table.

Next with no beam splitter in the cube and with the elongated optical tube at the other end of the cube, direct the helium neon beam through the irises attached to the ends of the tubes. Now remove the scanner and attach the elongated optical tube in place of the infrared objective. Insert the zinc selenide partial reflector to direct the beam toward the sample.

It should be mounted such that the helium neon beam hits the front surface of the reflector in the geometrical center of the optical cube. By rotating the partial reflector, direct the beam through the closed output. Iris, since the mount of the partial reflector does not hold it exactly perpendicular, we use two home installed adjustment screws to allow for vertical motion of the beam.

Next, use rubber O rings to mount the paraloid mirror by tightening the screws. The O-rings compress, allowing for mirror adjustment. Use additional mirrors to direct the helium neon beam in the opposite direction, adjusting it to pass through the previously used irises.

This beam will be used to adjust the paraboloid mirror. The paraloid mirror placed in the optical cube reflects the light toward the detector, which will be positioned after the pinhole. Adjustment screws allow the beam to be directed through the pinhole placed at the output of the cube.

After the beam is tuned through the pinhole, place the MCT detector close to the pinhole. Adjust the position of the infrared detector such that the helium neon beam is on the sensing element of the detector. Move the detector down by approximately two millimeters.

Now insert the mount with the germanian window into the optical cube located before the pinhole. The re reflection coated germanium window serves as an infrared filter and permits visual observations of the cantilever and the sample. Next, attach the eyepiece.

Twisting the angle of the cube allows the eyepiece to be pointed in the desired direction by rotating the mount of the germanium window. Direct the helium neon beam through the middle of the eyepiece. Connect the infrared objective and a FM scanner and attach the beam stop.

Position a sample on the scanner and direct the helium neon beam through the input optical tube. Make sure the beam is wide enough to spill over the beam. Stop by adjusting the thread of the eyepiece.

Obtain a sharp image of the beam focused on the sample. Next, attach the a FM head and engage the probe onto the sample. Focus the helium neon beam onto the end of the imaging cantilever.

If doing so, displaces the beam from the middle of the input optical tube. Repeat the tuning steps until the beam is centered. To begin the final adjustments of the peral mirror, replace the output pinhole with the iris at the focal position of the paraboloid mirror and remove the germanium window and infrared detector.

Remember to mark the position of the infrared detector. Attach an additional lens after the iris at the approximate focal length of the lens. The engaged cantilever should now be visible through the lens.

Adjust the position of the paraboloid mirror to center to the end of the tip through the closed iris. Note that when the helium neon beam is correctly focused onto the tip, there is a bright sparkle between the tip and its reflection in the samples surface. Reattach the germanium window and adjust it for convenient visual observation of the cantilever.

Replace the iris with the pinhole. Remember that the pinhole used for infrared detection should be shifted off-center due to the displacement of the infrared beam by the germanium window. Place the infrared detector at the previously marked position and direct the infrared beam to travel together with the visible beam used for the tuning.

We are now ready to look at the routine adjustment procedure. The setup is easier to align with the visible helium neon beam than with the invisible infrared beam. The path of the helium neon beam and the infrared beam come together at the tiltable mirror.

If this mirror is tilted downward, the helium neon can pass. If the mirror is in position, the infrared goes to the near field. Setup for alignment of one of the beams.

Use two mirrors that are not in common with the other path. If you look at the beam in the homo dyne arm, you should see a corona like beam profile. The a FM is now engaged with force feedback for fine alignment with the near field stage.

Move the reflecting microscope objective to focus the light on the tip where it is scattered. Place the germanium window into the optical cube. After the paraloid mirror, the beam is focused on this point by a paraloid mirror.

Move the stage backward or forward until you can see a sharp image of the tip and its reflection on the sample surface using the germanium window. Now look through the telescope where you can see the cantilever and the tip without the helium neon beam. You can still see some red light, but this is internal light from the A FM distance control.

Move the stage in the right, left and up down directions until you get a bright red sparkle on the tip. To overlap the ho dyne field with scattered light first open the homo dyne arm. Then look through the eyepiece.

You will see three or more spots in a line with decreasing intensity. Move the mirror in the homo dyne arm so that the second spot overlaps with the image of the tip where the tip and its reflection are coming together. To position the detector, remove the additional mirror.

The helium neon beam will now go in the direction of the detector behind the tube. You can see at least two ring-like spots. The second spot from the right should go through the hole in the heat protection foil in front of the detector and should hit the sensing element.

The spot with the highest intensity should be seen on the of the hole with routine adjustment complete. Let's look at alignment of the infrared beam. To align the infrared beam, use a carbon monoxide laser line with an intensity of at least 100 milliwatts.

This will make the alignment easier. To begin, fill the detector with liquid nitrogen. Flip up the mirror mounted on a kinematic flip mount so the infrared beam is directed towards the microscope.

Put a power meter behind the first iris and adjust the mirror located before the mirror mounted on a kinematic flip mount. In order to get the highest power, hold the power meter behind the iris closest to the near field stage and move the tiltable mirror until you get maximum power. Here during the alignment process, you'll repeat these steps a few times while watching the one F signal.

To check the one F signal, switch the reference of the lock in the amplifier to one f equals a FM oscillation frequency and go to a three meter Zs scan size in force plot mode. If you have bad alignment, you will see a broad bump on the three meter scale. To fix this, you will need to continually repeat the earlier steps until the alignment improves.

If you have medium alignment, the curvature of the first bump will look slightly concave, though the second bump will be bigger than the first bump. Here you can try to fix the alignment by adjusting one or both of the alignment mirrors. If your alignment is good, you will see just two bumps and the first will be higher than the second.

The curvature on the right side of the first bump will be negative. Once you have good alignment, you can move the mirrors to increase the maximum of the first bump. The one F signal should lie somewhere between five and 12 volts, though the shape of the first bump is more critical.

Finally, switch over to two F and try to improve the signal a bit more. By turning the mirrors slightly changing the phase, the two F signal should lie somewhere between 0.6 and two volts. Experimental topography and near field.

Images of 21 to 31 peptide fibrils are shown. The upper scale shows height in nanometers and the lower scale shows nearfield contrast in lock-in voltage shown here is a topography image collected simultaneously with the nearfield image obtained using 1, 631 inverse centimeters.Radiation. A topography image collected simultaneously with the near field image obtained using 1, 691 inverse centimeters.

Radiation is shown. The labels represent particular fis and are used to highlight the type of secondary confirmation each FI has. We've just shown you how to assemble a line and use an aperture list near field scanning infrared microscope.

Remember the steps undertaken during alignment so that you can retrace your steps if your alignment is wrong, and most of all be patient. That's it. Thanks for watching.

Good luck with your experiments.