Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering (CARS)

This video demonstrates a procedure to conduct differential imaging of two ramen resonances associated with biological structures with coherent ramen scattering signals. First three synchronized short pulse lasers are overlapped in time and space. The overlapped pulses are then sent to a standard inverted microscope, which allows for the generation of coherent ramen signals from microscopic objects.

Signals are converted into images using commercial software in order to compare different ramen signals that are produced. Results indicate that the difference between images obtained from doubly and singly resonant ramen signals provide enhanced sensitivity for weak ramen signals. Hi, I'm Tyler Weeks from the Center for Bio Photonics at the University of California Davis.

I'm Thomas ER from the Center for Bio Photonics and the Department of Internal Medicine at the University of California Davis. Today we will show you a procedure for using doubly resonant coherent antis stokes ramen scattering for producing chemically specific images of cells. We use this procedure to detect signals from molecules that are too weak to be detected directly by coherent antis Stokes Ramon spectroscopy alone.

So let's get started In order to generate cars and Dr cars signal simultaneously three tunable and synchronized short pulse laser sources are required to obtain these synchronized pulses. Start with a single short pulse laser with an output power of 10 watts. A fixed wavelength of 1064 nanometers, a fixed pulse length of seven picoseconds, and a fixed repetition rate of 76 megahertz.

Using a series of half wave plates and polarizing beam splitter cubes, the beam is split into three parts. A halfway plate combined with a polarizing beam splitter cube allows adjustment of the amount of power in each component of the beam without changing its direction. Typically, two beams are adjusted to approximately 4.5 watts each, and the third beam contains the remaining one watt.

The two high power beams are then directed into two independent optical parametric oscillators or OPOs. OPOs use different frequency generation to convert a high energy photon into two lower energy photons with different wavelengths. By controlling the temperature of the crystal used to achieve this effect, the wavelengths of the resulting photons can be controlled to within 0.1 nanometers.

By frequency doubling these signals, a laser with a fixed wavelength of 1064 nanometers can now be transformed into a laser beam that can be tuned anywhere between 780 nanometers and 910 nanometers by pumping two separate OPOs with the same 1064 nanometer source. Two independently tunable laser sources are obtained that are automatically synchronized to our original pump laser. The third beam from the 1064 nanometer pump laser is directed around the OPOs by a combination of dielectric mirrors so that all three beams can be recombined later.

In order to efficiently produce coherent ramen signal photons, the recombined pulses must be overlapped both temporally and spatially because OPOs contain ring cavities allowing each laser pulse to pass through the crystal multiple times the beam sent through the OPOs travel an extra distance resulting in considerable delay relative to the original pump beam. This distance must be compensated for with a third beam by introducing extra mirrors that force this beam to travel the same distance as the other two before they're recombined using dichroic mirrors in order to provide fine adjustment of the length of the path. Each beam is sent through adjustable delay stages, which allow for adjustment of the temporal overlap of the different laser pulses.

Another set of halfway plates and polarizing beam splitter cubes are added to each beam to allow adjustment of the laser power of each beam independently. Dichroic mirrors are used to first combine the beams from the two OPOs, and then the 1064 nanometer beam care must be taken to ensure that the beams are precisely overlapped in space. This can be verified by comparing the overlap of the beams within a few centimeters of the dichroic mirror with those approximately one meter from the dichroic mirror.

Because exposure to high average power laser beams can damage the sample, the three combined beams are sent through an electric optic modulator functioning as a pulse picker, which allows adjustment of the repetition rate of the pulses that arrive at the sample, and therefore the average power. The combined laser beams are then coupled into an inverted microscope with an objective lens with high numerical aperture or na. The tight focusing generated by the high NA objective lens allows for the most efficient generation of coherent ramen signals on microscopic length scales.

The objective lens of the microscope is mounted on an X, Y, Z pizo stage, which allows images to be obtained by raster scanning the beams across the sample similar to commercial beam scanning, confocal microscopes. Now let's see how to generate cars and D-R-F-W-M signals the interaction of three short pulse laser beams at the sample results in the generation of several four wave mixing signals such as cars from the various combinations of two lasers, as well as three color cars and DR cars signals from the combination of all three lasers. When signals are relatively close in wavelength, it can be difficult to separate them for analysis.

For this reason, signals are passed into an imaging spectrometer that also serves as a monochromator or band pass filter to spatially separate signals at different wavelengths. An electronically actuated flip mirror within the spectrometer in the lowered position sends the signal to a back illuminated deep depletion charge coupled device or CCD camera, which provides spectroscopic information across the entire signal range, and allows us to identify and optimize the various coherent robin signals to select the signal to image. Simply rotate the grading within the spectrometer using the vendor supplied control and data acquisition software to center the peak of interest on the CCD camera.

Then change the position of the flip mirror to redirect the signal to a second exit port to which a single photon counting avalanche photo DDE or a PD is attached.Raster. Scan the objective lens and then use the signals recorded on the A PD to generate an image by displaying the photon count rate for each pixel with the data acquisition software. Repeat this imaging procedure for each desired coherent ramen signal so that signals can be compared during post-processing.

Now let's see how to properly prepare a sample in order to obtain clear reproducible images. Some care must be taken in preparing the samples. Samples are typically prepared on approximately 150 micron thick glass cover slips.

These cover slips are thin enough to allow for high resolution imaging with the high NA water or oil immersion objective lenses that are used in these experiments. To demonstrate the preparation of a typical sample, we prepare approximately 20 micron thick sections of mouse muscle tissue that are fixed to a microscope slide. A 20 microliter droplet of five molar deuterated glucose solution is added to the sample.

The deuterated glucose solution provides a unique and strong ramen background signature. A glass cover slip is placed on top of the sample and fixed in place with nail polish for cells and culture. Glass bottom culture dishes can be used that allow for imaging without the need to detach the cells from their cell culture growth dishes.

Now let's see how to determine the proper rum and peaks for Dr cars. In order to properly take advantage of the doubly resonant enhancement effect, the ramen spectra of both ramen resonant substances must be known. Typically, these are 2, 845 inverse centimeters associated with lipids and 2, 121 inverse centimeters for the CD stretch mode associated with the deuterated glucose.

Coherent ramen scattering is achieved when the frequency difference between two lasers matches the frequency of a molecular vibration. By tuning one OPO to 817 nanometers, it will probe the 2, 845 inverse centimeters C mode when combined with the 1064 nanometer laser beam, and by tuning the other OPO to 868 nanometers, it will probe the 2, 121 inverse centimeter when combined with the 1064 nanometer beam. In spectroscopic mode, the car's microscope allows us to observe three coherent ramen signals of interest, a car signal probing the CH stretching vibration, a car signal probing the CD stretch mode, and a DR car signal probing both in the diagram shown here, dashed arrows indicate photons from the laser in the OPOs and solid arrows indicate the resulting signal.

The solid horizontal lines indicate the energy of the ramen vibration and give a visual representation that in DR cars mixing the same three input photons simultaneously probes two different ramen vibrations. The signal strength for each peak will need to be optimized by tuning in fine steps around these peak locations. Here we select each peak and take images of sea elgan worms in a solution of derated glucose, extracting additional information based on these three individually obtained images requires some fairly simple image processing.

First, the images must be normalized. A practical method for normalization relies on the fact that lipids are hydrophobic. This means that in regions of high density lipids, the CD resonance from the glucose should contribute very little to the doubly resonance signal.

Conversely, in regions of pure glucose solution, the CH resonances from the lipids should also contribute very little. With this in mind, normalize the DR car's image and the car's image obtained on the CD resonance of the Deuterated glucose solution to a region well outside the CL egan's worm that should exhibit no CH resonance. Next, identify a region within the worm in the CH resonant car image that is rich in lipids and normalize it to the corresponding region within the normalized DR car image.

For this method to work properly, assume that deep within this region no deuterated glucose is present. This is a safe assumption since the lipids are hydrophobic and will not mix with the solution. Now by subtracting the normalized CH resonant car image from the normalized DR car image, just the amplified CD resonant signal remains similarly by subtracting the normalized CD resonant car image from the normalized Dr.FWM image.

Just the amplified ch resonant signal remains shown here is the ramen spectrum for a modified oleic acid that includes an alkin modification, the strong CH resonances at 2, 845 inverse centimeters, and the alpine resonance at 2, 121 inverse centimeters are both well isolated from the fingerprint region, which is the region of densely packed peaks, making them ideal markers for coherent ramen imaging. This is a typical spectrum of coherent ramen signals generated when three short pulse lasers are overlapped within the sample. The arrows point to the processes responsible for each signal as represented by energy diagrams.

These are typical results from imaging C Elgin's worms using DR cars and cars. The top row used the three signals just shown to image a worm in a solution of derated glucose. In the second row, the images were appropriately normalized and in the third row the difference images were produced By subtracting each of the car's images from the DR car's image, We have just shown you how to perform Raman difference imaging based on doubly resonant coherent antis Raman microscopy.

When doing this procedure, remember, it's important to make sure the beams are overlapped and synchronized properly. Also, when switching between different coherent signals, make sure not to bump or move the sample as this will affect analysis. So that's it.

Thanks for watching and good luck with your experiments.