Hyperspectral Imaging as a Tool to Study Optical Anisotropy in Lanthanide-Based Molecular Single Crystals

Summary

Here, we present a protocol to obtain luminescent hyperspectral imaging data and to analyze optical anisotropy features of lanthanide-based single crystals using a Hyperspectral Imaging System.

Abstract

In this work, we describe a protocol for a novel application of hyperspectral imaging (HSI) in the analysis of luminescent lanthanide (Ln³⁺)-based molecular single crystals. As representative example, we chose a single crystal of the heterodinuclear Ln-based complex [TbEu(bpm)(tfaa)₆] (bpm=2,2’-bipyrimidine, tfaa^– =1,1,1-trifluoroacetylacetonate) exhibiting bright visible emission under UV excitation. HSI is an emerging technique that combines 2-dimensional spatial imaging of a luminescent structure with spectral information from each pixel of the obtained image. Specifically, HSI on single crystals of the [Tb-Eu] complex provided local spectral information unveiling variation of the luminescence intensity at different points along the studied crystals. These changes were attributed to the optical anisotropy present in the crystal, which results from the different molecular packing of Ln³⁺ ions in each one of the directions of the crystal structure. The HSI herein described is an example of the suitability of such technique for spectro-spatial investigations of molecular materials. Yet, importantly, this protocol can be easily extended for other types of luminescent materials (such as micron-sized molecular crystals, inorganic microparticles, nanoparticles in biological tissues, or labelled cells, among others), opening many possibilities for deeper investigation of structure-property relationships. Ultimately, such investigations will provide knowledge to be leveraged into the engineering of advanced materials for a wide range of applications from bioimaging to technological applications, such as waveguides or optoelectronic devices.

Introduction

Hyperspectral Imaging (HSI) is a technique that generates a spatial map where each x-y coordinate contains a spectral information that could be based on any kind of spectroscopy, namely photoluminescence, absorption and scattering spectroscopies^1,2,3. As a result, a 3-dimensional set of data (also called “hyperspectral cube”) is obtained, where the x-y coordinates are the spatial axes and the z coordinate is the spectral information from the analyzed sample. Therefore, the hyperspectral cube contains both spatial and spectral information, providing a more detailed spectroscopic investigation of the sample than traditional spectroscopy. While HSI has been known for years in the field of remote sensing (e.g., geology, food industries⁴), it recently emerged as an innovative technique for the characterization of nanomaterials^2,5 or probes for biomedical applications^3,6,7,8. Generally speaking, it is not limited to the UV/visible/near-infrared (NIR) domain, but can also be extended using other radiation sources, such as X-rays – for instance in order to characterize elemental distribution in different materials⁹ – or Terahertz radiation, where HSI was used to perform thermal sensing in biological tissues⁸. Further, photoluminescence mapping has been combined with Raman mapping to probe the optical properties of monolayer MoS₂¹⁰. Yet, amongst the reported applications of optical HSI, there are still only a few examples on HSI of lanthanide-based materials^{11,12,13,14,15,16,17}. For instance, we can cite: detection of cancer in tissues⁶, analysis of the light penetration depth in biological tissues⁷, multiplexed biological imaging³, analysis of multicomponent energy transfer in hybrid systems¹¹, and investigation of aggregation-induced changes in spectroscopic properties of upconverting nanoparticles¹². Clearly, the attractiveness of HSI arises from its suitability for generating knowledge about environment-specific luminescence, providing simultaneous spatial and spectral information about the probe.

Taking advantage of this powerful technique we herein describe a protocol to investigate the optical anisotropy of the heterodinuclear Tb³⁺-Eu³⁺ single crystal [TbEu(bpm)(tfaa)₆] (Figure 1a)¹³. The optical anisotropy observed resulted from the different molecular packing of the Ln³⁺ ions in the different crystallographic directions (Figure 1b), resulting in some crystal faces showing brighter, others showing dimmer photoluminescence. It was suggested that the increased luminescence intensity at specific faces of the crystal was correlated with more efficient energy transfer along those crystallographic directions where the Ln³⁺···Ln³⁺ ion distances were the shortest¹³.

Motivated by these results, we propose the establishment of a detailed methodology to analyze optical anisotropy through HSI, opening the path for better understanding of ion-ion energy transfer processes and tunable luminescent properties stemming from specific molecular arrangement^18,19. These structure-properties relationships have been recognized as important aspects for innovative optical materials design including, but not limited to waveguide systems and opto-magnetic storage devices at nano and microscale – addressing the demand for more efficient and miniaturized optic systems²⁰.

Protocol

CAUTION: It is recommended to use safety goggles specific for the excitation wavelength being used at all times when operating the imager.

1. Configuration of the hyperspectral microscope

NOTE: An overview of the hyperspectral imaging system is given in Figure 2a, with the main components of the imager being described. The imaging system can be used for the detection of the visible or the near-infrared (NIR) emission from a sample. Depending on which detection is desired (visible or NIR), the light goes through two different light paths (Figure 2e). A combination of different beam turning cubes and dichroic filter cubes (optical cubes) must be positioned at specific positions in the instrument to select the respective path.

1. Power on the computer which is connected to the imaging system. Turn on the computer’s monitor.
2. Set the appropriate optical cube configuration (Figure 2b,c).
   NOTE: Here, the imager configuration (optical cube configuration) for HSI mapping using UV excitation and visible emission detection is described. However, it is also possible to change it for NIR excitation and visible or NIR emission detection, depending on the sample analyzed. Refer to the section Representative Results for an example.
   1. Starting from the microscope stage (1 in Figure 2a) and following the emission beam pathway towards the detectors (3 in Figure 2a), leave the first position for an optical cube (4 in Figure 2b) vacant and place the confocal microscope optical cube (DFM1-P01) in the position indicated as 5 in Figure 2b, so that the emission from the sample is directed through the visible light path.
   2. Looking along the optical path towards the detector, place the visible optical cube (CM1-P01), which contains the dichroic mirror and the filters to direct the visible emission to the detection paths, in the position indicated as 6 in Figure 2b.
   3. Continuing the path towards the detector, place the confocal pinhole optical cube (DFM1-P01) in the position indicated as 7 in the Figure 2b to direct the light through the visible light detection path. Then, following the path, place the confocal spectrometer optical cube (DFM1-P01) in position 8 in Figure 2c so that the emitted light reaches the detector.
   4. For the HSI mapping, manually control the detector slit opening (9 in Figure 2c) in order to match with the size of the pinholes that are used (around 50 mm is optimal).
   5. In the PHySpec software, choose the aperture of the pinhole (5 in Figure 3).
      NOTE: The smaller the pinhole aperture, the better is the HSI resolution, at the cost of signal intensity.
3. Turn on the broadband lamp (Figure 2d, inset) by positioning the switch (10 in Figure 2d) into the ON position. To control the intensity of the excitation light, turn the knob indicated by 11 (Figure 2d) to higher (32 – lowest intensity) or lower values (1 – highest intensity). Keep the broadband lamp shutter (12 in Figure 2d) closed during the set-up.
   NOTE: The higher values correspond to higher attenuation of the power density emitted by the lamp, while lower values correspond to lower attenuation.
4. Turn on the following hardware in the order given below by setting their switches into the ON position:
   1. Turn on the ThorLabs motion controller.
   2. Turn on the Nikon power source.
   3. Turn on the ASI controller.
   4. Turn on the Galvo controller.
   5. Turn on the ProEm detector.
   6. Turn on the Bayspec detector.
5. At the computer, open the PHySpec software by double clicking on its icon.
   1. Press the F8 key on the keyboard in order to initialize the IMA Upconversion system and click the OK button on the Connect to System window.
      NOTE: Step 1.5.1. can also be performed by clicking on the System tab and then clicking on Connect to arrive to the Connect to System window. Then, the OK button can be clicked to connect the imaging system to the software.
   2. Ensure that all menus appear on the interface (Color camera, ProEm and Bayspec) as well as the instrument control panel, on the left side of the screen, as shown in Figure 3.

2. Hyperspectral imaging of a [TbEu(bpm)(tfaa)₆] single crystal

1. In order to prepare the sample, place the crystal on a microscopy glass slide. In case it is needed to use higher magnification, cover the crystal with a thin cover glass and secure it with tape, so that the sample can be placed with the thin cover glass facing toward the objective lens.
2. Place the glass slide on which the sample has been prepared on the microscope stage and secure it using the metal arms (Figure 4a,b).
3. Move the sample using the joystick (Figure 4c) of the ASI controller in order to position the sample over the objectives in use.
4. Manually position the right filter cube in the wheel underneath the objectives (3 in Figure 5) to select the UV excitation of the lamp and to let pass the visible emission towards the detector.
   NOTE: Additional filter cubes are available to use either green or blue light excitation, thus, having the right filter cube in place is important for the appropriate excitation wavelength.
5. Manually position the 20X objective (indicated by 5 in Figure 5) under the sample and press the white button (6 in Figure 5) on the left side of the microscope to turn on the white light.
   1. Adjust the brightness by turning the knob underneath the white light power button (7 in Figure 5).
6. In the PHySpec software, press the Play (video) button on the color camera window, which will cause the acquisition of a live scan.
   1. If the color camera window shows a black image, increase the Exposure Time (2 in Figure 3) and/or the Gain Value (3 in Figure 3) found in the instrument control panel, under the Color Camera tab. If the image viewed is too bright, decrease the exposure time and/or gain value.
   2. Ensure the forward knob on the right side of the microscope (2 in Figure 5) is set to R in order to send 20% of the signal to the camera/binoculars and 80% of the signal to the detector.
7. Focus on the sample by adjusting the distance between the objective and the stage (Figure 4b). This is done by turning the knobs shown in Figure 4d on the right side of the microscope.
   NOTE: The larger knob is used for coarse adjustments, while the smaller knob is for more delicate and small focus changes.
8. Ensure that the manually chosen objective is also selected at the software. First, click on the View button in the top menu bar and then click one Show/hide scale bar to display the scale bar on the image (1 in Figure 3). Then, go to the Galvanometer tab in the instruction control panel and select the Objective used (4 in Figure 3). Ensure that the displayed scale bar is correct by selecting the proper objective in the software.
9. In the software, select the appropriate detector by going to the tab Diverter (ProEM – 6 in Figure 3) and gratings by going to the tab Filter (1200 gr/mm in case of ProEM – 7 in Figure 3) under the SpectraPro SP-2300 tab.
10. Open the broadband lamp shutter (12 in Figure 2) to allow the UV excitation of the sample to take place. Turn the intensity knob (11 in Figure 2d) to the desired position (e.g., 8 – intermediate intensity) to control the intensity of the broadband lamp (UV) excitation.
    1. To choose between wide field illumination (open aperture) or a smaller spot illumination (more closed aperture), control the size of the UV lamp field aperture using the stick and knobs shown in 4 in Figure 5.
11. Under the SpectraPro SP-2300 tab, select a wavelength to observe the sample emission.
12. If the emission wavelengths of the sample are unknown, acquire an emission spectrum.
    1. In the sequencer, click on the + sign to add a new sequence (“node”) for the acquisition of an emission spectrum.
       1. Click on Spectrometer and then Spectrum Acquisition (with spectral scan).
       2. Input a Minimum Wavelength (i.e., 400 nm) and a Maximum Wavelength (i.e., 700 nm) and click OK to set the spectral range in which the spectrum will be recorded.
       3. Choose the adequate Exposure Time in the left side menu of the software. Choose shorter times (e.g., 0.1 s) for very bright samples and longer times (e.g., 2 s) for dim emitters.
       4. Adjust the excitation power in case of the broadband lamp (UV) excitation (see step 1.3 above).
          NOTE: In case of NIR diode excitation, it can be adjusted from the Neutral Density drop-down menu at the left side of the PHySpec software.
    2. On the sequencer, click on the double play button to run the entire sequence. Once the spectrum is shown, note the regions of interest for the detection of the sample emission (e.g. 580 to 640 nm in case of Tb³⁺- and Eu³⁺-based samples).
    3. As needed, optimize signal detection by either changing the focus of the sample or by adjusting the Exposure Time in the PHySpec software. Achieve further optimization of signal detection through the increase of the sample emission intensity by changing the power of the excitation source (broadband lamp), as described above.
13. Adjust the Exposure Time (e.g., 0.5 s – 2 in Figure 3) and Gain (3 in Figure 3) of the Color Camera accordingly, to obtain a good quality image. If needed, add the scale bar to the image by clicking at the button Show/hide scale bar at the second row of the menu in the top of the PHySpec software window.
14. Recommendation: Prior to acquiring the hyperspectral cube, record a bright field optical microscopy image of the crystal under white light (Figure 6a) and/or UV full (Figure 6b) or confined (Figure 6c) illumination (UV illumination controlled by the shutter aperture, shown as 4 in Figure 5). To do so, with the sample in focus, click on the play button of the color camera.
15. Click on File and then Export Window View, choose the desired format to export the obtained image, and save the file with the desired extension (.h5, .JPEG).
16. Before acquiring the hyperspectral image, turn off the white light illumination as well as the room light.
17. To obtain the hyperspectral cube, write a new sequence. Therefore, in the sequencer, click on the + sign to add a new node.
    1. Click on Confocal Imager.
       1. Click on Multi-Spectrum Acquisition. Here, the desired field of view is defined by the number of points to acquire in the x and y directions and the step size. For example, use 100 points in x and 100 points in y with a 5 µm step size to obtain an image of 500 by 500 µm.
          Note: The total number of acquisition points and the integration time at each point will directly affect the total acquisition time of the hyperspectral cube.
          1. Input the desired X Position (e.g., 100) and Y Position (e.g., 100) counts as well as the desired Step Size (e.g., 5 µm). Select the Hardware option for the camera sync, for visible emission mapping (and Software option in case of NIR detection). Click OK.
18. In the sequencer, click on the newly added Multi-Spectrum Acquisition line to highlight the node.
19. Click the Play button to run the selected node.
    NOTE: The remaining time that the acquisition will take will appear next to the node (in minutes, e.g., 28 min).
20. Once the acquisition is complete, save the hyperspectral cube in the appropriate file format (.h5).

3. Hyperspectral data analysis

1. Right after the acquisition, if the saved hyperspectral cube does not open automatically in the software, recover the hyperspectral cube, which was saved as .h5 file, by clicking on File in the top menu bar and then hovering the cursor and click at Open File…. When the window titled Select data(s) to open pops up, choose the folder where the .h5 file is saved and double click on the file to open it.
2. Once the hyperspectral cube file is imported, modify the displayed hyperspectral cube image to show the intensity of a specific spectral wavelength by moving the bar on the top of the cube image to the left (lower wavelength, e.g., 580 nm) or right (higher wavelength, e.g., 638 nm).
   NOTE: The selected wavelength is displayed in the left side of this upper bar (1 in Figure 7).
3. After choosing the wavelength of interest for the analysis (e.g., the maximum intensity, which in the case of [TbEu(bpm)(tfaa)₆] is 613.26 nm), make one (or all) of the three possible types of spectral analysis: (A) the spectral distribution in form of an image (2 in Figure 7); (B) an emission intensity profile across a region of interest (3 in Figure 7); (C) extraction of a spectrum at a specific point or region of interest (4 in Figure 7).
   1. In case of the spectral distribution from an image, use the Crop and Bin function to increase the signal-to-noise ratio in the image. In order to do that, click in the top menu Processing then choose Data and then the option Crop and Bin.
   2. For an emission intensity profile, on the cube image, right click and select the Create Target or Create X profile or Create Y profile depending if only one point (Target - 5 and 6 in Figure 7) or a line (Profile - 7 and 8 in Figure 7) needs to be analyzed. Select the area of analysis by dragging the target, the horizontal or the vertical line profile with the cursor and move it across the cube.
      1. Once the profile has been properly selected, right click on the region and select the Add Target to Graph. Chose the option to create a new graph to display the emission intensity (y axis) as a function of the physical position of the target (x axis). The spectrum will appear on the new graph which was inserted (6 and 7 in Figure 7).
         NOTE: Multiple targets can be created, and these will show up as different colored emission profiles (5 and 6 in Figure 7).
   3. Alternatively, obtain an emission spectrum of a specific area of the sample (9 in Figure 7). To begin with, hover the cursor over the cube image and right click. Click on the Rectangle Selection or Ellipse Selection options on the tab that pops-up.
      1. Draw the selection shape (e.g., a rectangle) over the desired region by clicking and dragging the cursor across the cube. Once the area has been properly selected, right click on the region and select the Add Selection to Graph.
      2. At the appearing window Add to Graph, select Create a New Graph to display the emission spectra of the target and click OK.
         NOTE: A new colored line (8 in Figure 7) will appear on the graph in which the target emission is being shown, with the emission intensity as the y axis and the wavelength at the x axis. This spectrum corresponds to the averaged intensity of the selected area for each wavelength.
4. Once the spectrum is obtained, save it before selecting a new region because only one region can be selected at a time. To do so, select the window in which contains the graph. In the File menu, select Save as and choose to save the graph in the folder of choice, using the name of choice, either in .h5 format, which can be opened in the PHySpec software, or in .csv format, which can be imported in Excel.

Results

To illustrate the configuration of the hyperspectral microscope for the data acquisition on a Ln-based, molecular single crystal (i.e., [TbEu(bpm)(tfaa)₆], Figure 1a), Figure 2 shows an overview of the system as well as the right placement of the optical cubes in the setup. Figure 3 shows a screen shot of the PHySpec software containing the menus used during the HSI acquisition. Figure 4 and ...

Discussion

The hyperspectral imaging protocol here described provides a straightforward approach that allows to obtain spectroscopic information at precise locations of the sample. Using the described setup, the spatial resolution (x and y mapping) can reach down to 0.5 µm while the spectral resolution can be of 0.2 nm for the mapping at the visible range and 0.6 nm for the NIR range.

In order to conduct hyperspectral mapping on a single crystal, sample preparation follows an easy ...

Materials

Name	Company	Catalog Number	Comments
Microscope glass slides	FisherBrand	12-550-15	Glass slides used for sample preparation
Visible and Near Infrared Hyperspectral Confocal Imager	PhotonETC		Microscope used for the analysis, builted according to the user needs, therefore it is no catalog number