Rejection of Fluorescence Background in Resonance and Spontaneous Raman Microspectroscopy

We discuss the construction and operation of a complex nonlinear optical system that uses ultrafast all-optical switching to isolate Raman from fluorescence signals. Using this system we are able to successfully separate Raman and fluorescence signals utilizing pulse energies and average powers that remain biologically safe.

The aim of this procedure is to construct an all optical gait that passes Raman scattered light while rejecting fluorescent signals. This is accomplished by first exciting and polarizing the Raman scattering. The second step of the procedure is to prepare the pump beam to operate the gate.

Thirdly, the pump and ram and pulses must be adjusted so that they overlap. The final step of the procedure is to acquire time gated spectra. Ultimately, results can be obtained that show biochemical quantification and classification through analysis of ramen signals with high signal to noise ratios.

The main advantage of this technique over existing methods, such as software removal of the fluorescence background, is that the shot noise produced by the fluorescence is significantly reduced. This method can help answer key questions in the biological and biomedical fields, such as non-invasive characterization of chemical composition of endogenous fluor in bacteria cells and tissues, understanding cellular processes and diseases such as cancer, vascular, or neurodegenerative diseases by using intrinsic markers. This method may also help with the development of new probes that could be used both as fluorescence and RAM labels, as well as for non-invasive medical sensors for blood analysis.

Though this method can provide insight into biological systems for biomedical engineering. It can also be applied to other fields such as biofuel, research, or telecom industry. Biological samples are typically placed on a number one thickness cover slip mounted in an ATO Fluor cell chamber.

Liquid samples, particularly those toxic to humans, are placed in a small glass bottle with a cover slip cemented to the opening by means of silicone epoxy, which is then inverted for measurement place samples on the secondary stage mounted on top of the microscope stage that has its own independent focus control in order to take time gated ramen spectra, first, the excitation beam must be properly prepared. Start with light emerging from a 2.4 watt tunable pulsed GI sapphire laser. Each pulse in the 80 megahertz pulse train should have 30 nano joules of energy, a temporal width of 140 femtosecond and a spectrum centered at 808 nanometers with a spectral bandwidth of around six nanometers to prevent back reflections from reentering the laser cavity, the light should be passed through a Faraday isolator place, a half wave plate before the Faraday isolator to allow for continuous tuning of the total power sent into the system.

Because six nanometers is too broad a bandwidth to resolve most ramen modes, the beam is sent through a very narrow band pass filter. Send it at 808 nanometers. Next, use an aromatic doublet to focus the light onto a five millimeter beta barium bore.

Eight crystal to half the wavelength from 808 nanometers to 404 nanometers. Place the beta barium bate crystal in a mount with tip and tilt controls mounted on a translation stage. To maximize the efficiency of the wavelength conversion, the crystal must be placed precisely symmetric about the focus of the doublet and with its crystal axis aligned to the polarization of the incoming beam.

Because the efficiency of the wavelength conversion is polarization dependent control over the amount of light sent to the sample can be obtained by placing a second half wave plate after the faraday isolator. By rotating this wave plate, the intensity of light sent to the sample can be adjusted independently of the intensity sent in the pump beam. The wavelength converted light is then re collimated by a second aromatic doublet chosen such that the exciting beam is large enough to fill the back aperture of the microscope objective and directed into an inverted microscope by means of two steering mirrors.

The microscope objective defines the optic axis to align the excitation beam to this axis. Place a mirror in the sample plane of the microscope. The two steering mirrors are then iteratively tuned while observing the back reflected laser beam on a CCD camera attached to the imaging port of the microscope.

Assuming that the image on the camera is centered on the microscope's field of view, the beam is on axis. When the focal spot is centered on the microscope chip and translation of the objective along the Z axis does not change. The center point of the defocused beam ramen scattering occurs when the sample is placed in the sample plane and is irradiated with laser light.

A dichroic filter placed below the microscope objective separates the wave shifted. Ramen scattered light from the excitation beam directing the ramen scattered light to the side port of the microscope. The microscope has been modified to remove any lenses within this path, such that the signal light emerges from the microscope coated because the signal beam emerging from the microscope is larger than the clear aperture of the gland.

Thompson polarizers, a 0.47 times telescope constructed of two aromatic doublets is used to shrink the beam. The signal light is then polarized by a gland Thompson polarizer oriented at zero degrees with respect to vertical in the lab frame and directed to a dichroic mirror where it is recombined with the pump beam. The pump beam is sent into a delay line composed of two mirrors at right angles to each other, both placed on a linear translation stage that can be tuned to ensure temporal overlap of the pump and signal pulses.

After the delay line, the beam is sent through a halfway plate and polarizer oriented at 45 degrees with respect to vertical in the lab frame. This ensures the proper polarization state of the pump beam when it reaches the non-linear medium. The light is then reflected off of two steering mirrors, one with piezoelectric controls, which should be used to finally adjust the position of the pump beam such that it overlaps spatially with the signal beam.

To obtain this overlap, observe the pump and signal beams at two locations, one close and one far from the dichroic mirror where the beams are combined by using the first steering mirror to overlap the two beams at the near point and the pizo mirror to overlap the beams at the far point, the pump beam can be made exactly coline with the signal beams. Next, the corrugate and collection system should be set up to maximize the collected time gated signal. To do this, the pump and signal beams are first passed through a diic filter that has an OD of six at 404 nanometers to prevent any residual excitation light from exciting and scattering within the non-linear medium.

The pump and signal beams are then focused by an aromatic doublet into a one centimeter path length quartz s vete containing the non-linear material, any non-linear material, having a suitably high non-linear index and suitably short temporal response can be utilized.Here. For these experiments, we use carbon di sulfide. The light is then re collimated by a second doublet with a focal length identical to the first.

The beams are then passed through a gland Thompson analyzer on a rotating mount, and then through a set of absorption and interference filters that combined have an OD of 10 at 808 nanometers. Finally, the signal light is focused by an aromatic doublet into a 50 micron multimode optical fiber, where the fiber is mounted in a stage that allows translation in X, Y, and Z.The fiber is then coupled to a commercial imaging spectrograph with attached CCD camera to align the collection system to maximize collected signal, set the analyzer to zero degrees and place a test sample of toluene in the sample plane, optimize the collected Raman signal by adjusting the X, Y, and Z controls of the fiber mount to ensure proper spatial and temporal overlap of the pump and signal beams. Place a mirror in the sample plane of the microscope.

Then remove the 404 nanometer filter from the system. Rotate the analyzer to 90 degrees so that the retro reflected 404 nanometer beam is sent to the spectrograph with the intensity adjusted such that it does not saturate the camera. Now with the pump beam off, rotate the analyzer to minimize the transmitted 404 nanometer signal.

Then turn the pump beam back on and slowly adjust the delay stage until the transmission of the 404 nanometer light begins to increase. Next iteratively turn, the delay stage, the piezoelectric mirror and the X, y, and Z controls of the fiber to maximize the signal because the retro reflected 404 nanometer beam and the Raman scattered light may take slightly different paths through the system. Make final adjustments by placing a strong Raman Scatterer such as toluene on the sample stage, replacing the Raman filter and slightly tweaking the alignment by changing the delay stage pizo electric mirror, and X, Y, and Z controls of the fiber to optimize the Raman signal.

Now the system is ready to collect spectra. This first requires the acquisition of several background curves to correct for system artifacts. First, with the analyzer set to zero degrees, the excitation beam on and the pump beam off.

Obtain an ungated spectrum, then set the analyzer to 90 degrees and collect a background spectrum representing stray light leaking through the polarizers. Next, with the analyzer remaining at 90 degrees, turn the ramen beam off and pump beam on. Collect a second background spectrum representing the amount of pump light leaking through the dichroic filters.

Finally, with all lasers off, collect a baseline dark spectrum representing the dark current level of the camera and electronics. Finally turn all beams on and collect a gated spectrum. To get the true spectrum of just the light through the gate, the two background spectrum and the dark spectrum must be subtracted from this gated spectrum.

Here we see a schematic diagram of the corrugating system. The pump beam path is shown as a solid red line while the SHG path is shown as a solid navy line. The path where ramen and fluorescence are overlapped is shown in green.

While the path where the fluorescence has been temporarily filtered out is shown in yellow. Here we see the raw spectra of coumarin dissolved in immersion oil. The red curve shows the spectrum taken with the gate held open with the black curve shows the spectrum taken with the analyzer aligned for minimum transmission and a pump beam applied.

The blue curve shows the spectrum taken with the analyzer aligned for minimum transmission and no pump beam applied, and the green curve shows the spectrum taken with only the pump beam applied. All spectra have been smooth with an 11 point third order KY gole filter. The dashed magenta lines indicate the spectral region shown in the following graph.

Here we see spectra of coumarin dissolved in immersion oil after fluorescence background subtraction. The red curve is the spectrum with the gate held open, and the blue curve is the gated spectrum. The gated spectrum clearly shows the convoluted high wave number, peak characteristic of oils.

While attempting this procedure, it is important to remember that the laser must first have enough pulse energy to drive the ga, and that the system requires exquisite spatial and temporal overlap of the two pulses. After watching this video and reading the attached protocol, you should have a good understanding of how to separate the laser beam into an excitation and curve beam. How to overlap those two beams, both spatially and temporally.

How to record a ramen signal via spectrograph and C, c, D, and also how to visualize and analyze the ramen signal. Don't forget that working with lasers can be extremely dangerous, and precautions such as wearing laser goggles should always be taken while attempting this procedure. Additional safety rules apply for the use of the nonlinear material and for the use of your specific sample.