History: optical imaging of cells using photonic crystal enhanced microscopy (PCEM)
Basic principles of PCEM
The principles of PC label-free biosensor surfaces and the PCEM instrument have been described in recent publications and are briefly summarized here (Fig. 10.1) [16,17,2124]. PCEM utilizes the sensing function of PC surfaces and is built upon a conventional benchtop inverted light microscope (e.g., Olympus BX51WI or Carl Zeiss Axio Observer Z1) by integrating it with a line-profiled and polarized light illumination source (e.g., laser or LED), a CCD camera, and a spectrometer as the detection instrument. PCEM provides a novel optical platform that enables label-free imaging of a broad range of surface-bound analytes (e.g., nanoparticles, proteins, antibodies, biomolecules). This capacity of PCEM extends to the analysis of surface-attached cells to enable sensing and imaging of whole, live cells at subcellular resolution at the cell-PC surface interface to temporally and spatially resolve adhesion-mediated cellular activities in situ in real time for life science research and engineering applications (Figs. 10.1-10.2).
The key component in PCEM is the PC biosensor, which is comprised of a periodic- modulated dielectric nanostructure as the optical transducer surface (Fig. 10.1(a) and (b))
Figure 10.1 Schematic of photonic crystal enhanced microscopy. (a) A schematic of a nanoparticle attached to a photonic crystals (PC) surface. Inset: photo of a PC fabricated on a glass microscope slide. (b) SEM image of the PC surface. Inset: zoomed in to show the nanograting surface structures. (c) A schematic of the PCEM setup. (d) Normalized spectrum image (surface plot) obtained from PCEM imaging. Inset: PCEM-acquired 3-D spectrum data. (e) Example spectrum from PCEM with a peak wavelength value (PWV) shift and a peak intensity value (PIV) change with or without a nanoparticle attached on the PC surface (BG, background; NP, nanoparticle). (f) A schematic of nanoreplica molding to fabricate PCs: (i) deposition of a thin layer of liquid UV epoxy polymer between a Si wafer template and a glass substrate, (ii) hardening of the epoxy layer via exposure to UV light, (iii) removal of the Si wafer template, (iv) sputter deposition of a thin layer of TiO2 film on top of the nanograting structure. Reprinted in part with permission from Zhuo Y, et al. Single nanoparticle detection using photonic crystal enhanced microscopy. Analyst 2014;139(5):1007-15, © 2013 RSC Publishing.
Figure 10.2 PCEM for in vitro studies of adhesion-mediated cellular behaviors. (a-b) Bright- field and the corresponding PWV images of Panc-1 cells adhered on the PC surface. PWV images clearly detail cellular protrusions at cell edges and regions of higher or lower mass distribution, noted by the differing magnitudes of the wavelength shift across the attached cell area. (c) Representative PWV spectra of the background (PC portion, nothing attached on top) versus the region where a cell is attached. (d) Time series PWS images of dental epithelial stem cells (mHAT9a) to demonstrate cellular attachment, adhesion, and movement.
Reprinted in part with permission from Chen W, et al. Photonic crystal enhanced microscopy for imaging of live cell adhesion. Analyst 2013;138(20):5886-94, © 2014 RSC Publishing.
[16,17]. The structural features of PC surfaces provide photonic band gaps, where light propagation is prohibited for specific wavelengths [25-27]. When cells attach to a PC surface, the local refractive index of the PC changes. By detecting these changes in the local refractive index of PC surfaces, it is possible to quantify the average response from the entire sensing area (biosensing) or spatially resolve localized responses that can be differentiated from neighboring locations (bioimaging) to achieve highly sensitive label-free sensing and imaging of surface-attached analytes (Fig. 10.1(a)-(e)).
Photonic Crystal (PC) biosensors have recently been demonstrated as a highly versatile technology for a variety of label-free assays including high-throughput screening of small molecule - protein interactions, characterization of protein-protein interactions, and measurement of cell attachment modulation by drugs [16,17,21-24]. A PC is a sub-wavelength grating structure consisting of a periodic arrangement of a low refractive index material coated with a high refractive index layer (Fig. 10.1(a)) . When the PC is illuminated with a broadband light source, high order diffraction modes couple light into and out of the high index layer, destructively interfering with the zeroth-order transmitted light . At a particular resonant wavelength and incident angle, complete interference occurs and no light is transmitted, resulting in 100% reflection efficiency. The resonant wavelength is modulated by the addition of biomaterial upon the PC surface, resulting in a shift to a higher wavelength. The electromagnetic standing wave that is generated at the PC surface during resonant light coupling inhibits lateral propagation, thus enabling neighboring regions on the PC surface to display a distinct resonant wavelength that is determined only by the density of biomaterial attached at that precise location. By measuring the resonant peak wavelength value (PWV) on a pixel-by-pixel basis over a PC surface, an image of cell attachment density may be recorded. PWV images of the PC may be gathered by illuminating the structure with collimated white light through the transparent substrate, while the front surface of the PC is immersed in aqueous media.
A schematic diagram of the PCEM instrument is shown in Fig. 10.1. The system is built upon the body of a standard microscope (Carl Zeiss Axio Observer Z1), but in addition to ordinary brightfield imaging, a second illumination path is provided from a fiber-coupled broadband LED (Thorlabs M617F1, 600 < X < 650 nm) [16, 28]. The fiber output is collimated and filtered by a polarizing beamsplitter cube to illuminate the PC with light that is polarized with its electric field vector oriented perpendicular to the grating lines. The polarized beam is focused by a cylindrical lens (f = 200 mm) to form a linear beam at the back focal plane of the objective lens (10x, Zeiss). After passing through the objective lens, the orientation of the line-shaped beam is rotated to illuminate the PC from below at normal incidence. The reflected light is projected, via a side port of the inverted microscope and a zoom lens, onto a narrow slit aperture at the input of an imaging spectrometer. The width of the adjustable slit can be adjusted according to the need of specific applications (e.g., 30 pm). The reflected light is collected from a linear region of the PC surface, where the width of the imaged line, 1.2 pm, is determined by the width of the entrance slit of the imaging spectrometer and the magnification power of the objective lens. The system incorporates a grating-based spectrometer (Acton Research) with a 512 x 512 pixel CCD camera (Photometrics Cascade 512). The line of reflected light, containing the resonant biosensor signal, is diffracted by the grating within the spectrometer (300 lines per mm-1) to produce a spatially resolved spectrum for each point along the line. Therefore, each pixel across the line is converted to a resonant reflection spectrum, containing a narrow bandwidth (AX « 4 nm) reflectance peak from the PC. The PWV of each peak is determined by fitting the spectrum to a 2nd-order polynomial function, and then mathematically determining the maximum wavelength of the function. By fitting all 512 spectra, in a process that takes 20 ms, a line comprised of 512 pixels is generated that represents one line of a PWV image of the PC surface. With an effective magnification of 26x, each pixel in the line represents a «0.6 pm region of the PC surface and 512 such pixels cover a total width of «300 pm. To generate a two-dimensional PWV image of the PC surface, a motorized stage (Applied Scientific Instruments, MS2000) translates the sensor along the axis perpendicular to the imaged line in increments of 0.6 pm per step. Using this technique, a series of lines are assembled into an image at a rate of 0.1s per line and the same area on the PC surface can be scanned repeatedly. Each image is comprised of 512 by n pixels, where n can be selected during each scan session, and each pixel represents a 0.6 x 0.6 pm region of the PC surface (Fig. 10.1(d)). A biosensor experiment involves measuring shifts in PWV. A baseline PWV image is gathered before the introduction of cells, when the PC is uniformly covered by cell media, which is aligned and mathematically subtracted from subsequent PWV images gathered during and after cell attachment (Fig. 10.1(e)).
Using nanoreplica molding, 1-D PC nanostructures can be mass produced at low cost with high reproducibility (Fig. 10.1(f)) [16,17,21-24]. The molding template is made on silicon wafers or quarts substrates via lithography. To being the process of fabricating a PC, a thin layer of ultraviolet-curable polymer (UVCP; nUVcp = 1.5) with a low refractive index (e.g., grating period of Л = 400 nm, grating depth of d = 120 nm, duty cycle of f = 50%) is first cured on a solid support substrate (e.g., microscope slide) then a thin layer of dielectric material with a higher refractive index (e.g., TiO2; Птю2 = 2.4) is deposited on top (Fig. 10.1(a) and (f)). The thickness of the higher refractive index layer is selected (e.g., thickness of 60 nm) to generate a resonant reflection at a specific wavelength (e.g., resonant wavelength of =620 nm) [21-24]. The sensitivity and the spatial resolution of the PC biosensor can be optimized by the choice of the dielectric materials and the geometry of the nanostructure features. For sensing and imaging of biological analytes, the PC surface can be further modified to immobilize application-specific biomolecules (e.g., antibodies, proteins, peptides) to enhance selectivity . This nanoreplica-molding approach allows simple, rapid, and reliable fabrication of PC surfaces to offer benefits of single-use disposable detection for high-throughput screening applications. Additionally, the PC surface may be made planar by filling the nanograting structure with a polymer using a horizontal dipping process to eliminate potentially unwanted effects arising from analytes reacting to the structured surface topography .