A Multimodal Wide-Field Fourier-Transform Raman Microscope

We developed a multimodal hyperspectral microscope to acquire the Raman or photoluminescence spectrum from each pixel of an image, which reduces the measurement time. The conventional raster scaling approach relies on dispersive spectrometers. Advanced techniques enhance the Raman signal either using light at resonance or with synchronized short laser pulses which drive molecular vibrations.

The lower Raman scattering cross-section poses two main challenges. First, faint signal, which requires long acquisition times. Second, Raman signal may be overwhelmed by a strong photoluminescence background.

Fourier-transform spectroscopy enables parallel spectrum acquisition across all pixels for faster measurement, and the tunable sampling to access specific spectral information such as isolating weak Raman peaks from strong luminescence background. The Fourier transform approach enables smart sampling and data analysis that leverage information in temporal traces. We aim at tailoring undersampling strategies to further shorten the acquisition time.

To begin, use a scraper to deposit 20 milligrams of each pigment powder on a precision balance, obtaining a one-to-one-to-one weight proportion. Mix the powders with a mortar to remove clumps. Pour the resulting mixture onto a microscope slide.

Then, use the scraper tip to gently press it to obtain an almost uniform thickness of the layer. Apply nail polish on the edges of a microscope cover slip. Place it on the mixture with the nail polish facing down, and apply enough pressure to seal it.

Next, set the excitation wavelength and focus the excitation laser at the input of the large core multi-mode fiber. Put a test sample on the microscope stage and switch on the camera to view the laser-illuminated spot. Attach a middle section of the fiber to the vibrating membrane of a voice coil, which will remove speckle.

Mechanically scramble the fiber by tightly bending it to merge all its spatial modes. Insert a narrow bandpass filter at 532 nanometers with 2.0 nanometer bandwidth to clean the laser line and reject any unwanted pump spectral sidebands. Use a dichroic mirror to reflect the laser light toward the sample and transmit the red-shifted back-scattered radiation collected by the objective.

Insert a long pass filter at 532 nanometers to reject residual illumination light. Measure the power on the sample plane with a power meter. Adjust the pump beam to obtain an intensity at the sample which does not lead to damage.

Now, put the sample on the microscope stage and adjust the focus. Switch on the driver of the motor that performs the wedge translation and press the acquire option. Then, set the camera acquisition parameters like timeframe and hardware binning to optimize the signal intensity.

Now, set the step length, home position, and number of steps. Add the name of the directory and the file name. Move the cross point to the target position and click on measure to start the acquisition of a monochrome image for each wedge position.

Once the measurement is finished, switch off the laser before data analysis. Next, generate the spectral hypercube from the acquired dataset by loading the motor position's correction file specific to the stepper motor, and the frequency calibration file corresponding to the interferometer. Set the wavelength for the spectra to be computed by Fourier, transforming the interferograms of all pixels.

Apply the apodization function which offers a good trade-off between spectral broadening and artifact reduction, such as the Happ-Genzel window. Then, generate the spectral hypercube and save it in complex values. Launch the analysis software and open the spectral hypercube in complex values.

Generate a false color RGB image and obtain the average spectrum in selected areas to analyze the spectral hypercube. For the Raman measurement on the same field of view, add a bandpass filter in the detection path. Set the timeframe and hardware binning, then set the set length, home position, and number of steps.

Choose the file name and directory for saving before pressing Measure. Three pigments are clearly distinguished within the field of view based on their Raman spectral signatures, with characteristic peaks for rutile at 454 and 616 inverse centimeters, anatase at 396, 514, and 641 inverse centimeters, and cadmium yellow at 301 and 605 inverse centimeters. In the photoluminescence map instead, cadmium yellow is the only pigment visibly distributed across the field of view with strong emission dominating the entire image.

Indeed, the faint defect emission from rutile and anatase cannot be spectrally distinguished from the yellow powder.