The authors would like to acknowledge The European Research Council (grant no. 336716 to B.S.A.). The authors would also like to thank Irati Lagfragua for her invaluable assistance with recording and editing the video.

1. Preparation of the polydimethylsiloxane (PDMS) layer
<ol>
	<li>Prepare a 10:1 mix of Sylgard 184 monomer and curing agent.</li>
	<li>Place the mixture in a vacuum chamber until air bubbles are gone.</li>
	<li>Pour 1.7 g of PDMS mixture in the center of a 3.5 inch (diameter) Petri dish.</li>
	<li>Place the Petri dish on the vacuum chuck of a spin coater.</li>
	<li>Spin coat the Petri dish for 20 s at 900 rpm, with an acceleration of 75 rpm/s.</li>
	<li>Cure the dish on a hotplate at 50 &#176;C for at least 2 h.</li>
</ol>
2. Sample preparation
<ol>
	<li>Waveguide cleaning
	<ol>
		<li>Prepare 100 mL of a 1% dilution of cleaning detergent concentrate (Table of Materials) in deionized (DI) water.</li>
		<li>Place the chip in a glass Petri dish using a wafer tweezer and cover completely with the detergent solution.</li>
		<li>Place the Petri dish on a hot plate at 70 &#176;C for 10 min.</li>
		<li>While still on the hot plate, rub the surface with a cleanroom tissue swab.</li>
		<li>Remove the chip from the Petri dish. Rinse with at least 100 mL of DI water.</li>
		<li>Rinse with at least 100 mL of isopropanol, taking care that solvent does not dry on the surface to prevent evaporation stains.</li>
		<li>Rinse with at least 100 mL of DI water. Blow the chip dry with a nitrogen gun.</li>
	</ol>
	</li>
	<li>Chamber preparation
	<ol>
		<li>Prepare a layer of 150 &#181;m polydimethylsiloxane (PDMS) in a Petri dish (section 1).</li>
		<li>Use a scalpel to cut a 1.5 cm x 1.5 cm frame from the PDMS layer.</li>
		<li>Lift the frame from the Petri dish with a tweezer. Deposit it flat on a clean and polished chip. The sample is now ready for cell seeding.</li>
	</ol>
	</li>
	<li>Fluorescent labeling
	<ol>
		<li>Prepare the following chemicals: phosphate-buffered saline solution, dye solution(s), dSTORM imaging buffer.</li>
		<li>After cell seeding, remove the chip from the media.</li>
		<li>Use a pipette to remove any excess fluid from outside the PDMS chamber.</li>
		<li>Remove the current fluid from inside the PDMS chamber with a pipette while adding approximately 60 &#181;L of clean PBS at the same time. 
		NOTE: The amount added to the chamber will have to be changed according to the chamber size. Be careful not to remove all media from the cell surface.</li>
		<li>Replace the PBS with 60 &#181;L of clean PBS and let it incubate for 1 min.</li>
		<li>Repeat the previous step, letting it incubate for 5 min this time.</li>
		<li>Remove the PBS and replace it with 60 &#181;L of the dye solution. Leave the sample to incubate for around 15 minutes, shielding it from light. 
		NOTE: This step might have to change significantly, depending on the fluorescent dye used. We use CellMask Deep Red to label the cell membrane for this experiment</li>
		<li>Wash the sample with PBS as in steps 2.3.3-2.3.5.</li>
		<li>Remove the PBS and replace it with 40 &#181;L of the imaging buffer at the same time. 
		NOTE: There are several different imaging buffers for different fluorescent dyes.</li>
		<li>Place a coverslip on top, preventing air bubbles from forming underneath. Gently press the coverslip against the imaging chamber to remove any excess media.</li>
		<li>Use a pipette to remove any excess media outside the coverslip. Clean the area outside the coverslip with a water moist swab to avoid crystals formed by dried immersion media residues.</li>
	</ol>
	</li>
</ol>
3. Imaging procedure
<ol>
	<li>Component setup 
	NOTE: This version of the setup consists of three main components: the microscope, coupling stage, and sample stage. See the Table of Materials.
	<ol>
		<li>Use a microscope with a filter holder, white light source, camera, and objective revolver.</li>
		<li>Use a 3-axis piezo coupling stage with a fiber-coupled laser and a coupling lens.</li>
		<li>Use a one-axis manual sample stage with tip and tilt and a vacuum holder.</li>
		<li>Mount both the coupling and the sample stage on a 2-axis motorized stage for sample translation.</li>
	</ol>
	</li>
	<li>Waveguide coupling
	<ol>
		<li>Place the chip on the vacuum chuck with the coupling facet towards the coupling objective. Make sure the chip is approximately one focal length away from the coupling objective.</li>
		<li>Turn on the vacuum pump.</li>
		<li>Turn on the laser to 1 mW. Roughly adjust the chip height so that the beam hits the edge of it. Turn off the laser.</li>
		<li>Turn on the white light source. Choose a low magnification objective lens (e.g. 10x). Focus the microscope on a waveguide.</li>
		<li>Translate the microscope along the waveguide to see if it is well aligned with the optical path. Move the microscope to the coupling edge.</li>
		<li>Turn on the laser at 1 mW or less. Translate the microscope along the coupling edge to find the laser light. Focus the beam on the chip edge.</li>
		<li>Adjust the coupling stage along the optical path in the direction that reduces the laser beam spot size until it disappears. The beam is now either above or below the chip surface.</li>
		<li>Adjust the coupling stage height until the beam spot reappears and is maximized.</li>
		<li>Repeat the two previous steps until the laser forms a focused spot.</li>
		<li>Move the focused spot to the waveguide of interest.</li>
		<li>Translate the microscope a short distance away from the edge so that the focused beam spot is no longer visible. Turn off the white light.</li>
		<li>Adjust the contrast. If the waveguide is guiding, the scattered light along the waveguide should be clearly visible.</li>
		<li>Adjust the axes of the coupling stage to maximize the scattered light intensity. Turn off the laser.</li>
		<li>Turn on the white light. Adjust the contrast if necessary.</li>
		<li>Navigate to the imaging region.</li>
	</ol>
	</li>
	<li>Diffraction limited imaging
	<ol>
		<li>Focus with the desired imaging objective. Turn the white light off.</li>
		<li>Insert the fluorescence filter and turn the laser power to 1 mW.</li>
		<li>Set the camera exposure time to approximately 100 ms. Adjust the contrast as needed. Ensure that the coupling is still optimized.</li>
		<li>Locate a region of interest for imaging. Turn on the piezo stage looping to average out modes. 
		NOTE: 20 &#181;m scan range with a step size of 50 nm is suitable for most waveguide structures.</li>
		<li>Capture at least 300 images.</li>
		<li>Load the captured image stack to Fiji using a virtual stack. From the image menu in Fiji, choose Stacks and z-project.</li>
		<li>Calculate the TIRF image by choosing projection type average intensity.</li>
	</ol>
	</li>
	<li>dSTORM imaging
	<ol>
		<li>Turn on the laser to 1 mW and set the camera exposure time to 30 ms.</li>
		<li>Adjust the contrast and focus.</li>
		<li>Increase the laser power until blinking is observed. 
		NOTE: This might take a while, depending on evanescent field intensity.</li>
		<li>Zoom in on a small region of the sample.</li>
		<li>Adjust the contrast.</li>
		<li>Capture a few images to see if the blinks are well separated.</li>
		<li>Adjust the camera exposure time for optimal blinking. 
		NOTE: Optimizing blinking is a complex task, but a lot of suitable literature is available12.</li>
		<li>Turn on the piezo stage looping.</li>
		<li>Record an image stack of at least 30,000 frames, depending on the blinking density.</li>
	</ol>
	</li>
	<li>dSTORM image reconstruction
	<ol>
		<li>Open Fiji and load the dSTORM stack as virtual images.</li>
		<li>Adjust the contrast, if necessary.</li>
		<li>Use the rectangle tool to select the area to reconstruct.</li>
		<li>Open Run analysis in the Thunderstorm13 plugin in Fiji.</li>
		<li>Set the basic camera settings in Thunderstorm corresponding to the device. The remaining default parameters are usually satisfactory.</li>
		<li>Start the reconstruction. 
		NOTE: For the full field of view, the data might need to be divided in substacks, due to the large file size.</li>
		<li>Filter the localization list provided by the reconstruction software to remove unspecific localizations. Apple an additional drift-correction if necessary.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Juette, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+sub-100+nm+resolution+fluorescence+microscopy+of+thick+samples.">Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples.</a> Nature Methods. 5 (6), 527-529 (2008).</li><li>Huang, B., Wang, W., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+Super-Resolution+Imaging+by+Stochastic+Optical+Reconstruction+Microscopy.">Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy.</a> Science. 319 (5864), 810-813 (2008).</li><li>Lampe, A., Haucke, V., Sigrist, S. J., Heilemann, M., Schmoranzer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-colour+direct+STORM+with+red+emitting+carbocyanines.">Multi-colour direct STORM with red emitting carbocyanines.</a> Biology of the Cell. 104 (4), 229-237 (2012).</li><li>Wazawa, T., Ueda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+internal+reflection+fluorescence+microscopy+in+single+molecule+nanobioscience.">Total internal reflection fluorescence microscopy in single molecule nanobioscience.</a> Advances in Biochemical Engineering/Biotechnology. 95, 77-106 (2005).</li><li>Jalali, B., Fathpour, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silicon+photonics.">Silicon photonics.</a> Journal of Lightwave Technology. 24 (12), 4600-4615 (2006).</li><li>Helle, &. #. 2. 1. 6. ;. I., Coucheron, D. A., Tinguely, J. C., &#216;ie, C. I., Ahluwalia, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscopy+on-a-chip:+super-resolution+imaging+on+the+millimeter+scale.">Nanoscopy on-a-chip: super-resolution imaging on the millimeter scale.</a> Optics Express. 27 (5), 6700-6710 (2019).</li><li>Diekmann, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chip-based+wide+field-of-view+nanoscopy.">Chip-based wide field-of-view nanoscopy.</a> Nature Photonics. 11 (5), 322-328 (2017).</li><li>Tinguely, J. C., Helle, &. #. 2. 1. 6. ;. I., Ahluwalia, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silicon+nitride+waveguide+platform+for+fluorescence+microscopy+of+living+cells.">Silicon nitride waveguide platform for fluorescence microscopy of living cells.</a> Optics Express. 25 (22), 27678-27690 (2017).</li><li>Ahluwalia, B. S., McCourt, P., Huser, T., Helles&#248;, O. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+trapping+and+propulsion+of+red+blood+cells+on+waveguide+surfaces.">Optical trapping and propulsion of red blood cells on waveguide surfaces.</a> Optics Express. 18 (20), 21053-21061 (2010).</li><li>Coucheron, D. A., Wadduwage, D. N., Murugan, G. S., So, P. T. C., Ahluwalia, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chip-Based+Resonance+Raman+Spectroscopy+Using+Tantalum+Pentoxide+Waveguides.">Chip-Based Resonance Raman Spectroscopy Using Tantalum Pentoxide Waveguides.</a> IEEE Photonics Technology Letters. 31 (14), 1127-1130 (2019).</li><li>Structured illumination microscopy using a photonic chip. ArXiV Available from: <a target="_blank" href="https://arxiv.org/abs/1903.05512v1">https://arxiv.org/abs/1903.05512v1</a> (2019)</li><li>Metcalf, D. J., Edwards, R., Kumarswami, N., Knight, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Test+Samples+for+Optimizing+STORM+Super-Resolution+Microscopy.">Test Samples for Optimizing STORM Super-Resolution Microscopy.</a> Journal of Visualized Experiments. (79), (2013).</li><li>Ovesn&#253;, M., K&#345;&#237;&#382;ek, P., Borkovec, J., &#352;vindrych, Z., Hagen, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ThunderSTORM:+a+comprehensive+ImageJ+plug-in+for+PALM+and+STORM+data+analysis+and+super-resolution+imaging.">ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging.</a> Bioinformatics. 30 (16), 2389-2390 (2014).</li><li>Archetti, A., Glushkov, E., Sieben, C., Stroganov, A., Radenovic, A., Manley, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Waveguide-PAINT+offers+an+open+platform+for+large+field-of-view+super-resolution+imaging.">Waveguide-PAINT offers an open platform for large field-of-view super-resolution imaging.</a> Nature Communications. 10, (2019).</li><li>Braet, F., Wisse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+aspects+of+liver+sinusoidal+endothelial+cell+fenestrae:+a+review.">Structural and functional aspects of liver sinusoidal endothelial cell fenestrae: a review.</a> Comparative Hepatology. 1, 1 (2002).</li></ol>

B.S. Ahluwalia has applied for patent GB1606268.9 for chip-based optical nanoscopy. The other authors declare no competing financial interests.

Chip-based imaging is similar to conventional dSTORM imaging. Image quality can thus be gauged using the same approaches as for traditional dSTORM imaging. The main difference for the user is that the transparent glass slide is exchanged with an opaque Si-wafer. Although they appear very different, the sample handling is practically analogous to a glass slide. The chips are quite sturdy and can easily be handled using wafer tweezers. The imaging procedure and image reconstruction is the same as in a regular dSTORM experiment. Setting up a functional chip-based microscope requires no special components, except for the photonic chips. Further details of the set-up can be found in previous work6,7. The chips used in this work have been fabricated using standard photolithography8.
Sample preparation encompasses the preparation of the sample chamber. When attaching the PDMS frame to the chip, it is crucial to avoid any small folds or rips where air might enter. If the PDMS folds when attaching it, simply remove it carefully with a tweezer and reattach it. When the sample is ready inside the PDMS chamber, the coverglass must be pressed against it, sealing the region. It is important to avoid any air bubbles that might form when attaching the coverglass. If an air bubble is formed, gently remove the coverglass and add PBS to the sample chamber to ensure that the sample is covered. The preparation and attachment of the cover slip can then simply be redone.
Coupling light into the waveguide is simplified using the protocol proposed in this paper. There are, however, a few common challenges that can limit coupling. Firstly, if the chip was not cleaned properly and any leftover PBS removed completely, there might be dirt or crystallized PBS on the waveguide. This can introduce major losses, resulting in very little power in the imaging region. Using a moist swab to clean the region outside the cover glass can improve the power significantly. Secondly, if the coupling facet of the waveguide is damaged (e.g., by improper handling), the coupling loss can increase drastically. Optical inspection of the edge will usually reveal any damages easily. The entire coupling facet of the chip can be polished carefully, much like an optical fiber, and will give a smooth coupling facet, which then increases the coupled power.
After the light has been coupled, the imaging procedure is the same as in any conventional dSTORM setup. If the image has inhomogeneous excitation, as demonstrated in Figure 2A, then most likely the mode averaging did not work well. The two most common reasons for this are: 1) too few images captured in order to create an average stack and 2) too short of an oscillation distance/too big of a step size. Collecting too few images can leave out some excitation patterns and the average will thus be inhomogeneous. This can easily be resolved by increasing the number of images in the average stack. Too short of an oscillation distance can also result in an inhomogeneous image, as not enough mode patterns are excited. This can also easily be resolved by increasing the oscillation distance and/or decreasing the step size. In this work we have used a piezo stage to scan the input laser beam over 20 &#181;m and acquire at least 300 images. Another approach could be to use high-speed galvo-mirrors to scan the light across the input waveguide facet within a single acquisition time, such as 10-30 ms. This option is suitable for live cell TIRF imaging, where sub-cellular organelles are in constant motion.
Chip-based dSTORM offers an unprecedented large area TIRF excitation, which makes it ideally suited for high throughput imaging. The compact character allows for retrofitting to commercial systems, where the chip can be placed upside down for inverted setups or transparent substrates can be developed. The chips are mass fabricated and can be modified to suit many needs. Currently, the main restriction is that it is limited to 2D. The evanescent field is only available approximately 200 nm away from the waveguide surface, so only fluorophores within this region will be excited. Altogether, the field of integrated optics offers many opportunities for chip-based microscopy in the near future, by tackling new imaging questions as well as providing new possibilities to existing ones.

Since the initial demonstration of single molecule localization microscopy, many variations have been developed to solve different challenges1,2,3. One challenge that has remained, however, is large field of view dSTORM imaging. Many dSTORM setups use the same objective lens to both excite the sample and to image it. In order to increase the field of view, a low magnification lens is needed. Low magnification and low numerical aperture (NA) objective lenses typically have a large depth of field, resulting in an increased out-of-plane signal that will reduce the localization precision. TIRF objectives are commonly used to increase the image contrast by reducing out-of-plane fluorescence. Through TIRF, the excitation is limited to an optical thickness of approximately 150 nm from the surface by means of an evanescent field4. TIRF objective lenses require a large NA resulting in a small field-of-view (FOV) (e.g., 50 x 50 &#181;m2), which limits the throughput significantly. There are, however, alternative ways to generate an evanescent field.
An optical waveguide is a structure that will confine and guide light if it is coupled into the structure. Most commonly, waveguides are used in fiber-based telecommunication. Great effort has been made in order to develop 2D integrated waveguides as a main component of photonic integrated circuits. The technology has advanced to a point where fabricating low-loss nano-structured optical waveguides can be routinely done5. Today, several foundries around the world can be used to develop photonic integrated circuits. Waveguides guide light through total internal reflectance also exhibiting an evanescent field at the surface. By careful design of the waveguide structure, a high intensity can be achieved in the evanescent field. A sample placed directly on top of the waveguide surface can thus also be illuminated by the evanescent field for imaging applications. The evanescent field will be generated along the entire length and width of the waveguide, and thus it can be made arbitrarily large6.
We present a novel approach to TIRF dSTORM that offers an arbitrarily large field of view. Instead of using a TIRF lens for both excitation and collection, we excite using the evanescent field from optical waveguides. This decouples the excitation and collection light pathway, allowing for total freedom along the collection light path without compromising the optical sectioning for a given wavelength provided by the waveguide chip illumination. Low magnification lenses can thus be used to image very large regions in TIRF mode, although a smaller NA will reduce the lateral resolution. Furthermore, multicolor imaging is also greatly simplified using waveguides7, as several wavelengths can be guided and detected without readjusting the system. This is advantageous for dSTORM, as low wavelengths can be used to enhance fluorophore blinking and for multicolor imaging. It is worth noting that the penetration depth of the evanescent field will change as a function of wavelength, although it does not affect how the imaging procedure is performed. The chip is compatible with live cell imaging8 and is ideal for applications such as the integration of microfluidics. Each chip can contain tens of waveguides, which can allow the user to image under different conditions or apply optical trapping9 and Raman spectroscopy10.
The chip-based system works equally well for both diffraction-limited and super-resolution imaging. A similar approach was introduced in 2005 using a prism to generate evanescent field excitation4. The photonic chip also excites through the evanescent field, but with modern waveguide fabrication techniques, one can generate exotic light patterns with waveguides. The present chip-based nanoscopy implementation is limited to 2D imaging only, as the excitation field is locked inside the waveguide surface. Future development will aim for 3D applications. Additionally, other super-resolution techniques such as structured illumination microscopy are being developed using the same chip-based microscope11.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-axis sample stage</td>
			<td>Standa</td>
			<td>7T173-20</td>
			<td></td>
		</tr>
		<tr>
			<td>2-axis sample translation stage</td>
			<td>Mad City Labs</td>
			<td></td>
			<td>Custom order</td>
		</tr>
		<tr>
			<td>3-axis NanoMax stage</td>
			<td>Thorlabs</td>
			<td>MAX311D</td>
			<td></td>
		</tr>
		<tr>
			<td>BXFM microscope body</td>
			<td>Olympus</td>
			<td>OLY-LSM-037018</td>
			<td></td>
		</tr>
		<tr>
			<td>CellMask Deep Red, Life technologies</td>
			<td>ThermoFisher</td>
			<td>C10046</td>
			<td></td>
		</tr>
		<tr>
			<td>Cleanroom grade swabs</td>
			<td>MRC Technology</td>
			<td>MFS-758</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber-coupled laser</td>
			<td>Cobolt</td>
			<td>Flamenco</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Holder</td>
			<td>Homemade</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hellmanex III, Hellma Gmbh</td>
			<td>Sigma-Aldrich</td>
			<td>Z805939</td>
			<td>Cleaning detergent concentrate</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma-Aldrich</td>
			<td>563935-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>KL 1600 LED</td>
			<td>Olympus</td>
			<td>OLY-LSM-E0433314</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus Coupling lens</td>
			<td>Olympus</td>
			<td>LMPLFLN 50x/0.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Orca Flash 4.0 V2</td>
			<td>Hamamatsu</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS tablets</td>
			<td>Sigma-Aldrich</td>
			<td>P4417-50TAB</td>
			<td>Mix according to descriptions</td>
		</tr>
		<tr>
			<td>SYLGARD 184 Silicone Elastomer 1.1 kg kit</td>
			<td>Dow</td>
			<td>1673921</td>
			<td></td>
		</tr>
		<tr>
			<td>Tip-tilt stage</td>
			<td>Thorlabs</td>
			<td>APR001</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum holder</td>
			<td>Thorlabs</td>
			<td>HWV001</td>
			<td></td>
		</tr>
		<tr>
			<td>Wafer Tweezers Type 2W</td>
			<td>Agar scientific</td>
			<td>AGT5051</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput total internal reflection fluorescence and direct stochastic optical reconstruction microscopy using a photonic chip

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced contrast due to optical sectioning. The conventional approach is to use high numerical aperture microscope TIRF objectives for both excitation and collection, severely limiting the field of view and throughput. We present a novel approach to generating TIRF excitation for imaging with optical waveguides, called chip-based nanoscopy. The aim of this protocol is to demonstrate how chip-based imaging is performed in an already built setup. The main advantage of chip-based nanoscopy is that the excitation and collection pathways are decoupled. Imaging can then be done with a low magnification lens, resulting in large field of view TIRF images, at the price of a small reduction in resolution. Liver sinusoidal endothelial cells (LSECs) were imaged using direct stochastic optical reconstruction microscopy (dSTORM), showing a resolution comparable to traditional super-resolution microscopes. In addition, we demonstrate the high-throughput capabilities by imaging a 500 &#181;m x 500 &#181;m region with a low magnification lens, providing a resolution of 76 nm. Through its compact character, chip-based imaging can be retrofitted into most common microscopes and can be combined with other on-chip optical techniques, such as on-chip sensing, spectroscopy, optical trapping, etc. The technique is thus ideally suited for high throughput 2D super-resolution imaging, but also offers great opportunities for multi-modal analysis.

TIRF microscopy is a popular technique as it removes out-of-plane fluorescence, increases contrast and thus improves image quality, and is less phototoxic compared to other fluorescence based microscopy techniques. Compared to the traditional objective-based approach, chip-based microscopy offers TIRF excitation without the limited throughput that is usually accompanied with a TIRF lens. An overview of the presented setup can be found in Figure 1A. We present diffraction-limited as well as dSTORM images of liver sinusoidal endothelial cells (LSEC) extracted from mice. A large field of view image of LSECs with labelled microtubulin is also presented, demonstrating the capabilities of high throughput imaging. A conventional dSTORM setup using an oil immersion TIRF lens (either 60x or 100x magnification) typically images an area of 50 &#181;m x 50 &#181;m, which is 100 times smaller than the chip-based image in Figure 2, imaged with a 25x, 0.8 NA objective.
In this method, we use multi-moded Si3N4 waveguides for excitation. The utilized chips consist of a strip-etched guiding layer of 150 nm Si3N4 deposited over a 2 &#181;m oxidized layer of a silicon chip. A schematic of the chip can be found in Figure 1B. Waveguide widths can vary between 200 and 1000 &#181;m. Fabrication details can be found elsewhere8. Through interference between the propagating modes the excitation light will not have a homogeneous intensity distribution, but rather a spatially varying pattern. Figure 2A presents an image with clearly visible mode patterns. This interference pattern will change with the position of the laser beam at the edge of the waveguide. In order to achieve homogeneous excitation in the final images, we use a piezo stage to oscillate along the coupled facet. Over the course of the imaging procedure, enough variation of the interference patterns exists so that they can be averaged, removing intensity fluctuations in the image. The image stack will consist of several images such as in Figure 2A, although with different patterns, but when averaged, the stack will yield an image with homogeneous excitation such as Figure 2B. An alternative approach is to use adiabatic tapering to achieve wide, single moded waveguides8,14, which removes the necessity of mode averaging. However, several millimeters of tapering length are necessary to maintain the single-mode condition to achieve a 100 &#181;m waveguide width. Multi-moded waveguides circumvent this tapering necessity and leave no limitations on the structure width. Beyond the illumination pattern, the highly effective refractive index of the modes allow for unprecedented possibilities towards structured illumination microscopy11 and fluctuation microscopy methods7.
The first step in imaging is to collect a diffraction limited image. The experiment results in a stack of around 300 images and the final image is made by taking the average of the stack. In Figure 2, we present diffraction limited and dSTORM imaging of LSECs labelled with CellMask Deep Red using a 60x, 1.2 NA water immersion objective. Figure 2A shows inhomogeneous illumination caused by insufficient mode averaging. Successful mode averaging is displayed in Figure 2B. Figure 2C is a dSTORM image of the same region, with the marked region shown in Figure 2D. Liver sinusoidal endothelial cells have nano-sized pores in the plasma membrane15, which can be seen here. A Fourier Ring Correlation analysis provided a resolution of 46 nm.
Figure 3 presents a dSTORM image of a 500 &#181;m x 500 &#181;m region, demonstrating the high throughput capabilities of the technique. A zoomed image of Figure 3A, corresponding to a typical dSTORM field-of-view, is presented together with the diffraction limited image in Figure 3B. A Fourier ring correlation to estimate the resolution was performed, yielding a value of 76 nm.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60378/60378fig1.jpg" /> 
Figure 1: Imaging system and waveguide. (A) Photograph of the imaging system. The sample is placed on a vacuum chuck on the sample stage, with the coupling facet of the waveguide towards the coupling objective. A fiber coupled laser and a coupling objective is placed on top of a 3D piezo stage. A lens turret with imaging lenses captures the image from above and relays it to a camera. (B) Schematic of the waveguide with coupling and imaging lenses. The coupling lens couples light into the waveguide. The samples (orange beads) are kept inside a sealed PDMS chamber. The evanescent field along the waveguide will excite the sample and the imaging objective will capture the emitted fluorescence. <a href="https://www.jove.com/files/ftp_upload/60378/60378fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60378/60378fig2.jpg" /> 
Figure 2: Diffraction-limited and dSTORM images. (A) Image of liver sinusoidal endothelial cells with insufficient mode averaging, resulting in a clearly visible excitation pattern. (B) The same region as in (A), but with sufficient mode averaging, resulting in homogeneous excitation. (C) Diffraction limited image of the inset in (B); (D) dSTORM image of the same region. (E) Inset of (D), clearly showing the fenestrations in the plasma membrane of the cell. <a href="https://www.jove.com/files/ftp_upload/60378/60378fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60378/60378fig3.jpg" /> 
Figure 3: dSTORM image of rat LSECs. (A) Large field of view dSTORM image of Alexa 647 stained tubulin in rat LSECs. Scale bar = 50 &#181;m. (B) Larger marked region from (A) comparing diffraction-limited (bottom left) and dSTORM image (top right). (C) Smaller marked region from (A). Scale bar = 1 &#181;m. The image has a resolution of 76 nm. Adapted with permission from Helle et al. 20196. <a href="https://www.jove.com/files/ftp_upload/60378/60378fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip at JoVE.com

High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip

Chip-based super-resolution optical microscopy is a novel approach to fluorescence microscopy and offers advantages in cost effectiveness and throughput. Here, the protocols for chip preparation and imaging are shown for TIRF microscopy and localization-based super-resolution microscopy.

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced ...

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Biochemistry

6.8K Views.  UiT The Arctic University of Norway. Chip-based super-resolution optical microscopy is a novel approach to fluorescence microscopy and offers advantages in cost effectiveness and throughput. Here, the protocols for chip preparation and imaging are shown for TIRF microscopy and localization-based super-resolution microscopy.

Video: High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. The binding of prokaryotic DnaG-like primases to DNA occurs at a specific trinucleotide recognition sequence. It is a pivotal step in the formation of Okazaki fragments. Conventional biochemical tools that are used to determine the DNA recognition sequence of DNA primase provide only limited information. Using a high-throughput microarray-based binding assay and consecutive biochemical analyses, it has been shown that 1) the specific binding context (flanking sequences of the recognition site) influences the binding strength of the DNA primase to its template DNA, and 2) stronger binding of primase to the DNA yields longer RNA primers, indicating higher processivity of the enzyme. This method combines PBM and primase activity assay and is designated as high-throughput primase profiling (HTPP), and it allows characterization of specific sequence recognition by DNA primase in unprecedented time and scalability.&#160;

Protein binding microarray (PBM) experiments combined with biochemical assays link the binding and catalytic properties of DNA primase, an enzyme that synthesizes RNA primers on template DNA. This method, designated as high-throughput primase profiling (HTPP), can be used to reveal DNA-binding patterns of a variety of enzymes.

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. ...

An Economical and Versatile High-Throughput Protein Purification System Using a Multi-Column Plate Adapter

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is also a key component in the development of new therapeutics. The evolving era of functional proteomics is fueling the demand for high-throughput protein purification and improved techniques to facilitate this. It was hypothesized that a multi column plate adaptor (MCPA) can interface multiple chromatography columns of different resins with multi-well plates for parallel purification. This method offers an economical and versatile method of protein purification that can be used under gravity or vacuum, rivaling the speed of an automated system. The MCPA can be used to recover milligram yields of protein by an affordable and time efficient method for subsequent characterization and analysis. The MCPA has been used for high-throughput affinity purification of SH3 domains. Ion exchange has also been demonstrated via the MCPA to purify protein post Ni-NTA affinity chromatography, indicating how this system can be adapted to other purification types. Due to its setup with multiple columns, individual customization of parameters can be made in the same purification, unachievable by the current plate-based methods.

A multi-column plate adapter allows chromatography columns to be interfaced with multi-well collection plates for parallel affinity or ion exchange purification providing an economical high throughput protein purification method. It can be used under gravity or vacuum yielding milligram quantities of protein via affordable instrumentation.

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is ...

Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. Total-internal-reflection-fluorescence (TIRF) microscopy has a high signal-to-background ratio, but it suffers from photobleaching and photodamage of the fluorescent proteins. Label-free techniques such as interference reflection microscopy (IRM) and interferometric scattering microscopy (iSCAT) circumvent the problem of photobleaching but cannot readily visualize single molecules. This paper presents a protocol for combining IRM with a commercial TIRF microscope for the simultaneous imaging of microtubule-associated proteins (MAPs) and dynamic microtubules in vitro. This protocol allows for high-speed observation of MAPs interacting with dynamic microtubules. This improves on existing two-color TIRF setups by eliminating both the need for microtubule labeling and the need for several additional optical components, such as a second excitation laser. Both channels are imaged on the same camera chip to avoid image registration and frame synchronization problems. This setup is demonstrated by visualizing single kinesin molecules walking on dynamic microtubules.

We present a protocol for implementing interference-reflection microscopy and total-internal-reflection-fluorescence microscopy for the simultaneous imaging of dynamic microtubules and fluorescently labeled microtubule-associated proteins.

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. ...

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and regulated by different suites of binding proteins unique for each polymer. Many studies now demonstrate that the dynamics of both cytoskeletal polymers are intertwined and that this crosstalk is required for most cellular behaviors. A number of proteins involved in actin-microtubule interactions have already been identified (i.e., Tau, MACF, GAS, formins, and more) and are well characterized with regard to either actin or microtubules alone. However, relatively few studies showed assays of actin-microtubule coordination with dynamic versions of both polymers. This may occlude emergent linking mechanisms between actin and microtubules. Here, a total internal reflection fluorescence (TIRF) microscopy-based in vitro reconstitution technique permits the visualization of both actin and microtubule dynamics from the one biochemical reaction. This technique preserves the polymerization dynamics of either actin filament or microtubules individually or in the presence of the other polymer. Commercially available Tau protein is used to demonstrate how actin-microtubule behaviors change in the presence of a classic cytoskeletal crosslinking protein. This method can provide reliable functional and mechanistic insights into how individual regulatory proteins coordinate actin-microtubule dynamics at a resolution of single filaments or higher-order complexes.

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence TIRF Microscopy

This protocol is a guide for visualizing dynamic actin and microtubules using an in vitro total internal fluorescence (TIRF) microscopy assay.

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and ...

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens.
The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells.
The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a &#34;dual-addition&#34; approach can detect functional agonist and antagonist &#34;hit&#34; molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z&#39; factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 &#181;M final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

In this work, a high-throughput, intracellular calcium fluorescence assay for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs) is described. The target, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, is expressed in CHO-K1 cells. This assay identifies agonists and antagonists using the same cells in one "dual-addition" assay.

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...

Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence (TIRF) Microscopy

High-resolution imaging techniques have shown that many ion channels are not static, but subject to highly dynamic processes, including the transient association of pore-forming and auxiliary subunits, lateral diffusion, and clustering with other proteins. However, the relationship between lateral diffusion and function is poorly understood. To approach this problem, we describe how lateral mobility and activity of individual channels in supported lipid membranes can be monitored and correlated using total internal reflection fluorescence (TIRF) microscopy. Membranes are fabricated on an ultrathin hydrogel substrate using the droplet interface bilayer (DIB) technique. Compared to other types of model membranes, these membranes have the advantage of being mechanically robust and suitable for highly sensitive analytical techniques. This protocol measures Ca2+ ion flux through single channels by observing the fluorescence emission of a Ca2+-sensitive dye in close proximity to the membrane. In contrast to classical single-molecule tracking approaches, no fluorescent fusion proteins or labels, which can interfere with lateral movement and function in the membrane, are required. Possible changes in ion flux associated with conformational changes of the protein are only due to protein lateral motion in the membrane. Representative results are shown using the mitochondrial protein translocation channel TOM-CC and the bacterial channel OmpF. In contrast to OmpF, the gating of TOM-CC is very sensitive to molecular confinement and the nature of lateral diffusion. Hence, supported droplet-interface bilayers are a powerful tool to characterize the link between lateral diffusion and the function of ion channels.

Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence TIRF Microscopy

This protocol describes how to use TIRF microscopy to track individual ion channels and determine their activity in supported lipid membranes, thereby defining the interplay between lateral membrane movement and channel function. It describes how to prepare the membranes, record the data, and analyze the results.

High-resolution imaging techniques have shown that many ion channels are not static, but subject to highly dynamic processes, including the transient ...

High-Throughput Screening for Protein Crystallization

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into an ordered lattice amenable to diffraction remains challenging. The crystallization of biomolecules is largely experimentally defined, and this process can be labor-intensive and prohibitive to researchers at resource-limited institutions. At the National High-Throughput Crystallization (HTX) Center, highly reproducible methods have been implemented to facilitate crystal growth, including an automated high-throughput 1,536-well microbatch-under-oil plate setup designed to sample a wide breadth of crystallization parameters. Plates are monitored using state-of-the-art imaging modalities over the course of 6 weeks to provide insight into crystal growth, as well as to accurately distinguish valuable crystal hits. Furthermore, the implementation of a trained artificial intelligence scoring algorithm for identifying crystal hits, coupled with an open-source, user-friendly interface for viewing experimental images, streamlines the process of analyzing crystal growth images. Here, the key procedures and instrumentation are described for the preparation of the cocktails and crystallization plates, imaging the plates, and identifying hits in a way that ensures reproducibility and increases the likelihood of successful crystallization.

Author Spotlight: High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography  

This protocol details high-throughput crystallization screening, ranging from the 1,536 microassay plate preparation to the end of a 6 week experimental time window. Details are included about the sample setup, the imaging obtained, and how users can perform analyses using an artificial intelligence-enabled graphical user interface to quickly and efficiently identify macromolecular crystallization conditions.

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into ...

CMG-Mediated DNA Unwinding Observed by ssDNA-RPA Tract Growth

Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a dynamic complex of proteins termed the replisome. At the heart of the replisome is the&#160;CDC45-MCM2-7-GINS (CMG) helicase, which separates the two strands of the DNA double helix such that DNA polymerases can copy each strand. During genome duplication, replisomes must overcome a plethora of obstacles and challenges. Each of these threatens genome stability, as failure to replicate DNA completely and accurately can lead to mutations, diseases, or cell death. Therefore, it is of great interest to understand how CMG functions in the replisome during both normal replication and replication stress. Here, we describe a total internal reflection fluorescence (TIRF) microscopy assay using recombinant purified proteins, which allows for real-time visualization of surface-tethered stretched DNA molecules by individual CMG complexes. This assay provides a powerful platform to investigate CMG behavior at the single-molecule level, allowing helicase dynamics to be directly observed with real-time control over reaction conditions.

Author Spotlight: Unraveling the Dynamics of Eukaryotic DNA Replication Through Single-Molecule Visualization

This protocol demonstrates performing a single-molecule assay for live visualization of DNA unwinding by CMG helicase. It describes (1) preparing a DNA substrate, (2) purifying fluorescently labeled Drosophila melanogaster CMG helicase, (3) preparing a microfluidic flow cell for total internal reflection fluorescence (TIRF) microscopy, and (4) the single-molecule DNA unwinding assay.

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a ...

Investigating Vitamin A Receptor Kinetics and Function for Retinoid Homeostasis

Methodology for Studying Interactions of Vitamin A Membrane Receptors and Opsin Protein with their Ligands in Generating the Retinylidene Protein

Distribution of dietary vitamin A/all-trans retinol (ROL) throughout the body is critical for maintaining retinoid function in peripheral tissues and generating the retinylidene protein for visual function. RBP4-ROL is the complex of ROL with retinol-binding protein 4 (RBP4), which is present in the blood. Two membrane receptors, Retinol Binding Protein 4 Receptor 2 (RBPR2) in the liver and STimulated by Retinoic Acid 6 Retinol (STRA6) in the eye, bind circulatory RBP4 and this mechanism is critical for internalizing ROL into cells. Establishing methods to investigate receptor-ligand kinetics is essential in understanding the physiological function of vitamin A receptors for retinoid homeostasis. Using Surface Plasmon Resonance (SPR) assays, we can analyze the binding affinities and kinetic parameters of vitamin A membrane receptors with its physiological ligand RBP4. 
These methodologies can reveal new structural and biochemical information of RBP4-binding motifs in RBPR2 and STRA6, which are critical for understanding pathological states of vitamin A deficiency. In the eye, internalized ROL is metabolized to 11-cis retinal, the visual chromophore that binds to opsin in photoreceptors to form the retinylidene protein, rhodopsin. The absorbance of light causes the cis-to-trans isomerization of 11-cis retinal, inducing conformational changes in rhodopsin and the subsequent activation of the phototransduction cascade. Decreased concentrations of serum and ocular ROL can impact retinylidene protein formation, which in turn can cause rhodopsin mislocalization, apoprotein opsin accumulation, night blindness, and photoreceptor outer segment degeneration, leading to Retinitis Pigmentosa or Leber Congenital Amaurosis. 
Therefore, spectrophotometric methodologies to quantify the G protein-coupled receptor opsin-11-cis retinal complex in the retina are critical for understanding mechanisms of retinal cell degeneration in the above-mentioned pathological states. With these comprehensive methodologies, investigators will be able to better assess dietary vitamin A supply in maintaining systemic and ocular retinoid homeostasis, which is critical for generating and maintaining retinylidene protein concentrations in photoreceptors, which is critical for sustaining visual function in humans.

Author Spotlight: Unraveling Vitamin A Transport Mechanisms &#8212; Linking Liver Receptors to Vision Health Through RBPR2 and RBP4 Interactions

Here, we describe two quantitative methods for studying the protein-ligand interactions of vitamin A membrane receptors and photoreceptor opsin with their respective physiological ligands.

Distribution of dietary vitamin A/all-trans retinol (ROL) throughout the body is critical for maintaining retinoid function in peripheral tissues and ...