1. Introduction

Hyperspectral imaging is a technique where spectral information is recorded over many wavelengths for each pixel of a high-resolution microscopic image [1,2]. It plays an essential role in multi-biomarker based imaging such as immunoassay-based disease analysis, where tumor originated cells are isolated [3] and identified [4] though labeling of multiple disease specific proteins. A swept light source is a key element in hyperspectral microscopy. It usually consists of a wide band light source coupled with mechanically motorized color wheels or gratings coupled with mechanically scanned stages [1,2], which make the setup complicated and expensive (see Fig. 1(a)). In addition, the emission spectra of conventional white or wide band light sources, such as white LEDs, mercury vapor lamps, fluorescent tubes, usually contains material-specific non-uniform peaks, which make them difficult to be used for a swept light source. In this paper, we introduce a method to utilize colloidal quantum dots (QDs) as a light source for hyperspectral microscopy. The emission spectrum of QDs is mainly decided by their size-dependent band gap. The use of QDs allows for easy construction of a compact swept light source that uniformly covers visible-NIR wavelength range. Another unique advantage of the QD based swept light source is that it does not contain any moving parts. Elimination of mechanically moving parts is a recent trend in developing compact, fast, robust and low cost devices. Solid-state disks (SSDs) and solid state relays (SSRs) are examples of commercial success of such schemes. Our light source takes advantage of the simple UVLED-based excitation and the wide variety of emission wavelengths of colloidal QDs. Emission wavelengths of the light source can be easily changed by digital on-off control of solid state LEDs.

 

Fig. 1 Schematic of QD excitation setup. (a) Typical hyperspectral microscopes consisting of a color wheel (top) or a mechanical stage (bottom). The figures are adapted from [2]. (b) The light source using colloidal QDs. Emission of 520 nm-800 nm can be chosen by digital on-off control of the excitation UVLEDs. (c) Emission spectra of the QD light source3. Gold nanorods and nanospheres.

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2. QD light source

Figure 1(a) shows schematics of typical hyperspectral microscopes which employ mechanically controlled color wheels (top) or mechanical stages (bottom). Figure 1(b) shows the digitally switchable QD excitation setup used in this study. The QDs are packed in a polydimethylsiloxane (PDMS) enclosure that is replicated from a plastic mold fabricated by rapid 3D printing. It includes an array of 8 miniature wells, each of which contains a single color of QDs (see the top view). QDs in a well are excited by a UV-LED with the emission peak at 380 nm. The enclosure also serves as a circular light guide plate that transmits emission from the QDs (see the side view of Fig. 1(b)). The emission is coupled into a circular plate that has a concave surface designed to diffuse the emission from the QDs uniformly onto the top plate. Multiple dimples are created on the concave surface to scatter the light. On top of the concave surface is a top plate consisting of a diffuser and a 400 nm long-pass filter to cut off excitation UV lights. Figure 1(c) shows the normalized emission spectra from the swept light source. We used 8 colors of CdSe/ZnS and CdTe/ZnS colloidal quantum dots with the peak emission wavelengths ranging from 520 nm to 800 nm. The emission intensity for each color can be controlled by adjusting that of its respective excitation UVLED, which is driven by a transistor array connected to a LabVIEW based digital controller. Since each LED is operated independently, any linear combination of the 8 colors can be created by the light source.

3. Gold nanorods and nanospheres

Due to the effect of localized surface plasmon resonance, gold nanoparticles have size and shape-dependent characteristic absorption spectra. Gold nanoparticles with different absorption peaks can be used for optical labeling of multiple biomarkers used in hyperspectral absorption spectroscopy [5]. Our QD light source, which allows for simple spectral measurements, gives a clear edge to such nanoparticle based imaging schemes. Here we image cells labeled with gold nanorod and nanosphere to test our custom-made light source.

Gold nanospheres typically show one absorption peak, while nanorods have two characteristic absorption peaks. Gold nanorods can be used for multiplex imaging, as longitudinal absorption peak of gold nanorods is tunable to the aspect ratio of nanorods (length-to-diameter ratio). However, the nanorods show plasmon coupling and broadening of the optical spectra. For multiplex imaging, it is crucial to reduce this plasmon coupling between neighboring nanoparticles so that the optical characteristics of individual nanoparticles are preserved. We have recently demonstrated that the plasmon coupling of gold nanoparticles can be reduced by coating silica-shell around the nanoparticles [6]. In this study we have coated gold nanospheres and nanorods with silica-shell to enable multiplex imaging. The gold nanospheres (20 nm diameter) were synthesized via citrate reduction of chloroauric acid under reflux. Cetrimonium bromide (CTAB)-stabilized gold nanorods were first synthesized using a seed-mediated reaction described by Jana et al. [7] and Nikoobakht et al. [8]. The aspect ratio of the gold nanorods can be controlled by changing the amount of AgNO₃ added to the reaction mixture. Gold nanospheres and nanorods were PEGylated before silica-coating them using a modified Stöber method [9, 10]. TEM photograph in Fig. 2 shows the gold (a) nanorods and (b) nanospheres we used for the measurement. Figure 2(c) show the transmission spectra of (top) nanorods and (bottom) nanospheres suspended in buffer solution measured with UV-VIS spectrometer.

 

Fig. 2 TEM micrographs of silica coated (a) nanorods and (b) nanosphere. (c) Transmission spectra of (top) nanorods and (bottom) nanospheres suspended in buffer solutions.

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4. Theoretical analysis

The emission peak of synthesized QDs is often slightly shifted from the nominal or designed value by about ± 5 nm, which may result in uneven spectral coverage. In order to assess the impact of such errors in the peak emission wavelengths, we built analytical models of two different QD light sources.

Figure 3(a) shows the two models. One is composed of Gaussian curves which peak at nominal values of a commercially available product (Life Technologies QDot®) as shown in the uniform coverage of QD spectra 1. The other, shown as QD spectra 2, is built by using peak values shown in the measurement of Fig. 1(c). Figure 3(b) shows the procedure of the theoretical analysis. For each model of nanorod suspension (see the left panel), transmitted intensities of multicolor QDs can be found by calculating products of QD spectra and the transmission spectrum (see the middle panel). The nanorod transmission spectrum can be reconstructed by curve fitting. Figure 3(c) shows the result of analytical experiments. Transmission curves of model nanorods are built by fitting Gaussian functions to the curves shown in Nikoobakht et al. [8]. Each curve is modeled as a combination of a fixed peak at 520 nm and an additional peak ranging from 608 nm to 829 nm depending on the aspect ratio of the nanorods (see the left panel). Transmission curves are reconstructed based on the transmitted intensities calculated with QD spectra 1 and QD spectra 2, respectively. The graphs shown in the middle and the right panels of Fig. 3(c) demonstrate that both light source models (QD spectra 1 and 2) can resolve five different types of nanorods.

 

Fig. 3 Theoretical analysis of spectral resolution. (a) Emission spectra of two models of the QD light source. QD spectra 1 uniformly cover the spectral range of 520 – 800 nm, while QD spectra 2 are based on the measured value shown in Fig. 1(c). (b) Procedure of the theoretical analysis. A transmission spectrum can be reconstructed from the values measured with the multicolor QDs. (c) Transmission spectra of different types of gold nanorods are reconstructed using QD spectra 1 and QD spectra 2.

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5. Experimental results

5.1 Preparation of macrophage cells

We performed hyperspectral transmission imaging of macrophage cells labeled with nanorods and nanospheres. To load cells, macrophage cells were incubated with nanorod or nanosphere containing media for 18 hours, allowing the cells to uptake nanoparticles. Cells labeled with gold nanorods and nanospheres are collected as a pellet after centrifugation as shown in Fig. 4(a). Cells are then washed with buffer solution before being fixed on a glass slide using fluoramount. Figure 4(b) show microscopic RGB color images of macrophage cells labeled with nanorods and nanospheres, respectively. The cells are fixed on glass slides, and the images were taken with a 12-bit color mosaic CCD camera (SPOT Pursuit XS, Diagnostic Instruments). They show different colors (pink for nanospheres and blue for nanorods) according to the absorption spectra. Figure 4(c) shows hyperspectral images of the same regions as 4 (b) with color coded peak wavelengths in the 500−800 nm spectral region. They were taken using a PARISS spectral imager (Lightform Inc.). For all imaging, 20 × 0.5 NA objective and 100 W halogen light source were used. The hyperspectral system was calibrated using a standard mercury lamp (Lightform, Inc.). The hyperspectral images were normalized by the lamp spectrum obtained from the part of the microscope slide that did not have cells.

 

Fig. 4 Macrophage cells stained with nanorods (top images) and nanospheres (bottom images). (a) Cell suspensions. (b) Microscopic RGB images. (c) Hyperspectral images. The peak wavelength for each pixel is shown with a color indicated in the color bar.

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5.2 Hyperspectral imaging

The cell samples were imaged with eight colors of the QD light source (see Fig. 1(c) for the spectra). A monochromatic cooled CCD (Pixis 400, Princeton instruments) with the wavelength sensitivity range of 450 nm-1000 nm was used for imaging. Figure 5(a) and 5(b) show imaging results with nanorods and nanospheres, respectively. For each of Fig. 5(a) and 5(b), 8 images taken with the eight colors the QD light source are shown in the top panels. The values of absorbance at the spot indicated by the circle in the middle panel were calculated from the eight images and plotted in the graph. The light intensity obtained from the part of the image that did not have cells was used to normalize the absorbance. The curves shown in the graphs are the spectra obtained with the reference hyperspectral microscope. Curves measured for two neighboring areas of 2 × 3 pixels are shown for each of Fig. 5(a) and 5(b). Note that the curves show the same characteristic absorption spectra as the curves in Fig. 2 (measured with the particle suspensions in buffer). Imaging results with the QD light source fit within the range of deviation with the curves from the commercial hyperspectral microscope. In Fig. 5(a), the absorbance maximum of the QD light source measurements is found at 700 nm. In Fig. 5(b), the absorbance peak of nanospheres at 520 nm is found with the measurement with the QD light source. Characteristic peaks of both nanorods and nanospheres are clearly visible with the QD imaging method.

 

Fig. 5 Hyperspectral images taken with the QD swept light source for (a) cells stained with nanorods (b) cells stained with nanospheres. The eight images shown on top are taken with the eight colors of the QD light source. Absorbance calculated from the images is plotted along with the curve from the reference hyperspectral microscope measured for the same region (circled in each panel). Scale bars = 20 µm

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6. Conclusion

We have demonstrated a method of using QD-based solid state swept light source. The emission wavelength (520 nm – 800 nm) of the light source can be easily controlled through digital switching of UVLED excitation. We employed the light source in a standard microscope and successfully performed hyperspectral imaging of macrophage cells labeled with gold nanorods and nanospheres. The results suggest that the use of QDs allows for hyperspectral imaging of biosamples with a simple microscope configuration. In addition, proper choice of different gold nanoparticles, such as nanorods and nanospheres, enable absorption-based analysis of multiple biomarkers. Combination of QDs and gold nanoparticles provides a potential on-chip hyperspectral imaging tool for multi-biomarkers recognition at cellular level. In addition to microscopy, our QD light source can be used for several types of absorption spectroscopy techniques, such as pulse oximetry [11] or tissue spectroscopy [12], where the absorption spectra are correlated with physical characteristics. A possible application area beyond biomedical applications is the use as an illumination source alternative to conventional RGB-LEDs. Conventional RGB color spaces, such as sRGB or Adobe RGB, cover only a portion of visible colors [13], while our light source can simulate any spectrum expressed as linear combinations of multiple QD emission peaks. The QD light source can be a strong illumination tool for medical and industrial imaging.