Hyperspectral imaging allows for the acquisition of spectroscopic information at precise locations within a sample. For example, for the unveiling of optical anisotropy at a single crystal level. The generation of both spectral and spatial data of a sample allows for a more detailed investigation of the sample than is possible by either spectroscopy or fluorescence microscopy alone.
A large number of adjustable parameters which affect the resolution can seem overwhelming at first. However, making a checklist may help with familiarizing yourself with the system. This technique requires manual tuning of the imaging hardware as well as software manipulation.
It is essential to illustrate both aspects visually to showcase how both methods complement each other. Demonstrating the procedure with Nelson Rutajoga will be Emily Rodrigues, post-doctorate fellow at my laboratory. To set up the imager configuration for hyperspectral imaging mapping, starting from the microscope stage and following the emission beam pathway toward the detectors, leave the position for an optical cube right next to the optical microscope vacant and place the confocal microscope optical cube in that position that directs the emission from the sample through the visible light path.
Looking along the optical path toward the detector, place the visible optical cube containing the dichroic mirror and filters for directing the visible emission to the detection path in its position. Continuing the path toward the detector, place the confocal pinhole optical cube in the right position to direct the light through the visible light detection path and following this path, place the confocal spectrometer optical cube in the appropriate position so that the emitted light reaches the detector. When all of the cubes are in place, manually adjust the detector's slit opening to match the size of the pinhole selected.
Then, in the PHySpec software, select the aperture of the pinhole. For hyperspectral imaging mapping of terbium europium bi-pyrimidine trifluoroacetylacetonate single crystal, manually position the 20 times objective of the optical microscope under the sample and press the white button on the left side of the microscope to turn on the white light. Turn the knob under the button to adjust the brightness.
Set the forward knob on the right side of the microscope to R to send 20%of the signal to the camera and 80%of the signal to the detector. Press play in the color camera window at the PHySpec software to initiate a live scan. If the color camera window shows a too dark or black image, increase the exposure time and/or the gain value under the color camera tab.
If the image is too bright, decrease the exposure time and/or gain value. To focus on the sample, turn the knobs of the microscope to adjust the distance between the objective in the stage and open the broadband lamp shutter to allow UV excitation of the sample. Then turn the intensity knob to the desired position to control the intensity of the broadband lamp excitation.
Click Show/Hide scale bar to add a scale bar to the bright-field optical microscopy image of the crystal and observe an image of the crystal under UV full or confined illumination. To select between the confined illumination or a wide field illumination, use the stick and knobs to adjust the size of the UV lamp field aperture. Under the SpectraPro SP 2300 tab, select a wavelength to observe the sample emission and adjust the exposure time of the detector.
To obtain the hyperspectral cube in the sequencer, click plus to add a new node and click Confocal Imager. Click Multi-Spectrum Acquisition and input the desired X and Y position counts, as well as the desired step size. Select the hardware option for the camera sync and for visible emission mapping, and click okay.
In the sequencer, click the newly added multi-spectrum acquisition line to highlight the node and click Play to run the selected node. For analysis of the captured hyperspectral imaging data, for example, for a spectral distribution of an image, in the Processing menu, select Data and Crop and Bend to increase the signal to noise ratio of the image. For an emission intensity profile, right-click on the cube image and select Create X-Profile for the analysis of one line.
Drag to select the area and right-click on the region of interest. To select Add to Graph. To display the emission intensity as a function of the physical position of the target.
The intensity profile will appear in the new graph. To obtain an emission spectrum of a specific area of the sample, hover the cursor over the cube image, right click, and click Rectangle Selection. Drag and click to draw the selection shape over the desired region.
Then right click on the region of interest and select Add Selection to Graph. In the Add to Graph window, select Create a New Graph to display the emission spectra of the target, and click okay. Then save the acquired spectrum before selecting a new region.
Here, a bright-field image of a crystal recorded after adjusting the sample in the proper focus is shown. The needle-like morphology of the crystal can be clearly observed. Here, images of the same crystal under UV excitation with either full illumination or locally confined illumination can be observed.
The confined illumination can be used to investigate the effects of energy or light transfer within the crystal that may trigger wave guide-like behavior. For example, in this image, a strong emission is detected in a point not directly under excitation, suggesting that efficient energy migration takes place through the crystal. From the acquired hyperspectral cube, it is also possible to obtain the spectral distribution in the form of an image, representing a specific wavelength.
The intensity profile of a specific emission wavelength and the emission spectra at any pixel or area of the acquired hyperspectral cube. For example, the emission spectra for this analysis shows the most characteristic emission bands of the europium ion. Moreover, the spatial profile along the different crystal faces indicates a brighter emission at the tip and side faces and can be correlated with the lanthanide/lanthanide ion distances in the three spatial directions.
To obtain a good signal in a reasonable recording time of the hyperspectral cube, it is important to adjust the microscope configurations, focus, and detector exposure time. Emissions UV excitation and visible emission, this technique can be performed using near infrared excitation and near infrared emission detection. This allows it to be applicable for a large array of luminescent materials.
Correlation of optical signals with an array of dependent features makes this technique very interesting for the investigation of structure/property relationships, including in-vitro assessment of nano-bio interactions.
