This project was funded by the Land Baden-W&#252;rttemberg as well as by the European Union, Europ&#228;ischer Fonds f&#252;r die Regionale Entwicklung. The authors thank Rainer Wittig (ILM Ulm) for providing the U251-MG-L106-Grx1-roGFP2 cell line and Claudia Hintze for skillful technical assistance.

1. Cell Spheroid Growing and Incubation
<ol>
	<li>Experiment 1: Cell spheroids incubated with a chemotherapeutic drug
	<ol>
		<li>Prepare agarose 1.5% in culture medium by adding 0.45 g of agarose to 30 ml culture medium (sufficient for 6 plates).</li>
		<li>Heat the mixture from step 1.1.1 to at least 80 &#176;C while stirring it from time to time.</li>
		<li>Fill 50 &#181;l of the heated mixture from step 1.1.2 into each well of a 96-well plate and let it solidify within 1&#8211;2 hr. Store the plates closed and covered in the refrigerator at 5 &#176;C until use.</li>
		<li>Seed about 150 MCF-7 breast cancer cells in each well of the prepared 96-well plate in Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) with Ham&#8217;s F-12 culture medium supplemented with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin.</li>
		<li>Grow spheroids for 5&#8211;7 days up to a diameter of about 200&#8211;300 &#181;m in an incubator at 5% CO2 and 37 &#176;C.</li>
		<li>Incubate the cell spheroids with the anthracycline antibiotic doxorubicin hydrochloride for 6 hr at a concentration ranging between 2 &#181;M and 8 &#181;M (in culture medium) in an incubator at 5% CO2 and 37 &#176;C.</li>
		<li>Wash the cell spheroids with culture medium or Earle&#39;s Balanced Salt Solution (EBSS) prior to microscopy. </li>
	</ol>
	</li>
	<li>Experiment 2: Oxidation process in spheroids expressing a redox sensor
	<ol>
		<li>Seed about 400 U251-MG-L106 glioblastoma cells permanently transfected with the glutathione sensitive green fluorescent redox sensor Grx1-roGFP2 in agarose gel&#8211;coated wells (procedure described in steps 1.1.1&#8211;1.1.3) of a 96-well plate in DMEM culture medium supplemented with 10% FCS, 1% penicillin/streptomycin and hygromycin B (150 &#181;g/ml).</li>
		<li>Grow spheroids for 5&#8211;7 days up to a diameter of about 200&#8211;300 &#181;m in an incubator at 5% CO2 and 37 &#176;C.</li>
		<li>Add 10 &#181;l of hydrogen peroxide solution (purum p.a., &#8805; 30%) to 990 &#181;l bi-distilled water to create a 100 mM stock solution to be used within 12 hr.</li>
		<li>Dilute the stock solution from step 1.2.3 to 50 &#181;M in EBSS right before the experiment.</li>
		<li>Incubate the cell spheroids via the microfluidic system during measurement with hydrogen peroxide 50 &#181;M in EBSS for oxidation of the redox sensor.</li>
	</ol>
	</li>
	<li>Experiment 3: Initiation and labeling of cellular necrosis within spheroids
	<ol>
		<li>Seed about 400 cells (e.g., U251-MG-L106-Grx1-roGFP2 glioblastoma cells) in agarose gel&#8211;coated wells (procedure described in steps 1.1.1&#8211;1.1.3) of a 96-well plate in DMEM culture medium supplemented with 10% FCS, 1% penicillin/streptomycin and hygromycin B (150 mg/ml).</li>
		<li>Grow spheroids for 5&#8211;7 days up to a diameter of about 200&#8211;300 &#181;m in an incubator at 5% CO2 and 37 &#176;C.</li>
		<li>Incubate the cell spheroids with the mitochondrial electron transport inhibitor rotenone for 3 hr at a concentration of 1 &#181;M in culture medium to induce cellular necrosis. Additionally, co-incubate with a green cytotoxicity dye for 3 hr at a concentration of 1 &#181;l in 1 ml culture medium to label necrotic cells.</li>
		<li>Wash the cell spheroids with culture medium or EBSS prior to microscopy.</li>
	</ol>
	</li>
</ol>
2. Light Sheet Adjustment
NOTE: For achieving best results it has to be ensured that the beam waist of the light sheet is in the focus plane of the objective lens. The position of the beam waist can only be aligned by observing the light sheet in vertical position with respect to the capillary (see steps 2.5 - 2.8).
<ol>
	<li>Use an inverted microscope and mount the SPIM excitation module, as described in Ref. 16, to the baseplate of the positioning table (see Figure 1A).</li>
	<li>Equip the microscope with a 10X/0.3 or 20X/0.5 microscope objective lens and an appropriate long-pass filter (e.g., &#955; &#8805; 515 nm) in the detection path of the microscope.</li>
	<li>Mount an integrating camera to the detection port of the microscope.</li>
	<li>Use a parallel collimated beam of a laser or a laser diode with an excitation wavelength of preferentially 470 nm and apply it to the SPIM module.</li>
	<li>Rotate the cylindrical lens by 90 degrees, which transfers the light sheet into a vertical position for an axial adjustment of the beam waist of the light sheet.</li>
	<li>Place a capillary containing a liquid with a fluorescent dye in the sample holder.</li>
	<li>Attach the sample holder with the capillary to the positioning table of the microscope and align the position of the capillary such that it is centered and the beam waist of the light sheet is in focus.</li>
	<li>Adjust the beam waist by variation of the axial position of the cylindrical lens itself. Turn back the lens after adjustment.</li>
</ol>
3. Cell Spheroid Application and Microscope Feed Synchronization
<ol>
	<li>Place the cell spheroids separately or in groups within rectangular borosilicate glass capillaries16 with an inner cross section of 600 &#181;m x 600 &#181;m and a wall thickness of 120 &#181;m. Two techniques for the application of a cell spheroid have proven to be successful.
	<ol>
		<li>Take the empty capillary upright with your thumb and middle finger and seal the upper opening with your forefinger. Bring the lower opening close to the cell spheroid in its surrounding liquid (EBSS or culture medium). Release the forefinger from the upper opening. Liquid with the cell spheroid in it will be soaked in immediately by capillary forces. Adjust the position of the spheroid within the filled capillary by gravitation in respective upright position of the capillary.</li>
		<li>Alternatively, apply the cell spheroid to the capillary via pipetting. Take care that the opening of the tip of the pipette is, on the one hand, big enough for the spheroid size and, on the other hand, less or equal in size to the inner diameter of the capillary.</li>
	</ol>
	</li>
	<li>Place the capillary with the spheroid in it in a special sample holder for microscopy.</li>
	<li>Attach the sample holder with the capillary to the positioning table of the microscope and align the position of the cell spheroid such that it is centered and focused.</li>
	<li>Set the matching between the light sheet and the focal plane of the microscope objective lens in the detection path by adjusting the position of the reflection mirror and the cylindrical lens (see adjustment screw in Figure 1B).</li>
	<li>Adjust the micrometer screw (see Figure 1B) according to the refraction indices and the numerical aperture of the objective lens to compensate the fishtank effect. 
	NOTE: Different refraction indices of the sample and the media surrounding the objective lens lead to a difference between the shift of the objective lens and the shift of the focal plane. Since the light sheet is moved by the objective turret, the mismatch between the shift of the light sheet (corresponding to the shift of the objective lens) and the shift of the focal plane is compensated by a lever arm (see Figure 1B). The micrometer screw is used to adapt the distance between the upper and the lower pin to adjust the shift of the light sheet according to the numerical aperture of the objective lens and the refraction indices.</li>
</ol>
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51993/51993fig1highres.jpg" width="500" /> 
Figure 1. (A) Picture of the single plane illumination module mounted to the baseplate of the positioning table of an inverted microscope, (B) setup of the single plane illumination module and the microscope feed synchronization to compensate the lifting-mismatch (fishtank effect) between focal plane and illumination plane. &#916;zo indicates the shift of the objective lens and &#916;zf the shift of the focal plane and the light sheet. The inlay shows cross sections of spheroid containing capillaries. Left: rectangular capillary with inner diameter of 600 &#181;m (wall 120 &#181;m). Right: Setup used for rotation. An inner round capillary (inner diameter 400 &#181;m, outer diameter 550 &#181;m) is rotated within the outer rectangular capillary. The space between the two capillaries is filled with an immersion fluid.
4. Measurement in Dynamic Liquid Environment
NOTE: The previous protocol is used for static incubation prior to a measurement where the cell spheroid has previously been incubated and then held in place in the capillary by simple gravitation. There is no need for further fixation. However, for measurements in flowing media where a dynamic incubation and, therefore, a dynamic environment is desirable to measure uptake kinetics, one can accomplish this sub-protocol. Steps 4.5&#8211;4.9 are critical to avoid air bubbles reaching the sample. 
<ol>
	<li>Fill the capillary with FCS for 30 min to support later cellular adhesion of a cell spheroid to the inner glass surface.</li>
	<li>Let the remaining FCS coating in the depleted capillary dry out for at least 12 hr.</li>
	<li>Introduce the cell spheroid to the FCS coated capillary as described in step 3.2.</li>
	<li>Leave the capillary in the incubator at 5% CO2 and 37 &#176;C for additional 2&#8211;4 hr to cause cellular adhesion of the spheroid.</li>
	<li>Set up the afflux part of the microfluidic system (see Figure 2). Use a peristaltic pump to fill the afflux of the tubing (inner diameter: 0.89 mm) with culture medium containing the fluorescent dye, drug or agent.</li>
	<li>Connect the afflux part of the microfluidic system to a bubble trap (see Figure 2).</li>
	<li>First clamp the capillary bubble-free to the afflux tubing and then clamp the other side of the capillary to the drain tubing.</li>
	<li>Tune the liquid temperature of the water bath to the desired value (e.g., 37 &#176;C).</li>
	<li>Adjust the peristaltic pump to the desired pump velocity (e.g., flow rate: 9 &#181;l/min; pump velocity in the capillary: 25 mm/min).</li>
	<li>Collect the pumped liquid either in a recipient (open-loop setup), feed it back to its source (closed-loop setup) or feed it directly into the tubing of the peristaltic pump (tight closed-loop setup).</li>
</ol>
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51993/51993fig2highres.jpg" width="500" /> 
Figure 2. Microfluidic setup (open-loop and tight closed-loop); inlay: Illumination of a spheroid within a micro-capillary using light sheet based fluorescence microscopy coupled to an inverted microscope.
5. Data Acquisition and Analysis
<ol>
	<li>Set the laser power and the integration time for the image acquisition.</li>
	<li>Take care that the parameters defined in step 5.1 do not exceed light dose values about 50&#8211;100 J/cm2 for native cells or 10&#8211;20 J/cm2 for cells incubated with a fluorescent dye or transfected with a fluorescent protein encoding plasmid to avoid phototoxicity (for details see Ref. 12).</li>
	<li>Set the increment for the z-stack to &#8710;z = 5&#8211;10 &#181;m which is preferable for 3-dimensional data analysis as the light sheet thickness is about 10 &#181;m.</li>
	<li>Perform measurements of single images or z-stacks by variation of the focal plane within the cell spheroid.</li>
</ol>

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No conflicts of interest declared.

The present manuscript describes a light sheet or single plane illumination microscopy (SPIM) device which is optimized for 3-dimensional cell systems, e.g., multi-cellular tumor spheroids (MCTS). Three exemplary applications include (1) uptake of a cytostatic drug and its partial conversion to a degradation product (whose contribution to chemotherapeutic efficacy still remains to be evaluated), (2) measurements of the redox state by use of a genetically encoded glutathione sensor upon addition of an oxidizing a...

In addition to well established methods (confocal or multi-photon laser scanning microscopy1-4, structured illumination microscopy5,6) light sheet or single plane illumination microscopy (SPIM) has proven to be a valuable method of 3D imaging7,8,9. Of particular interest is its application to 3-dimensional cell cultures, e.g., multi-cellular tumor spheroids (MCTS), which are used increasingly for drug discovery research10,11. Furthermore, SPIM is a preferential method when even upon long-term exposure or repetitive measurements low light doses are required to maintain the sample&#8217;s viability, since for measurement of each plane of the sample only this plane is exposed to light. This is in contrast to other microscopy techniques, where for detection of each focal plane the whole sample is illuminated, so that upon recording of numerous planes the light dose sums up and may damage the sample12.
Light sheet microscopy or SPIM is based on illumination of the sample in perpendicular direction to the observation path either by use of a cylindrical lens or by scanning the exciting laser beam (for a review see Ref. 8). This often requires special sample chambers13,14 or matrices, e.g., agarose7,15, implemented in special high-cost microscopes. As an alternative to those systems a comparably simple illumination device for SPIM has been developed and adapted to a conventional inverted microscope16 (see Figure 1). It consists of a laser beam expanded to a diameter of 8&#160;mm and focused by a cylindrical lens (focal length: 50&#160;mm, numerical aperture: 0.08) to a light sheet of 6&#8211;10&#160;&#181;m thickness over a depth of field of about 100&#160;&#181;m. Samples are located in a rectangular capillary of 600&#8211;900&#160;&#181;m inner diameter placed in front of the microscope objective lens for fluorescence detection. These main features are presently completed and optimized by the use of advanced micro-capillary approaches for holding and for rotating the samples, the synchronous adjustment of the illuminating light sheet (in axial direction) and the objective lens used for fluorescence detection (identical optical path lengths of displacement require a correction of the mechanical feed), and the adaptation of a microfluidic system for application of fluorescent dyes, pharmaceutical agents or drugs, thus minimizing the required quantities and expenses.

<table><tbody><tr><td>Microtiter plate</td><td>Orange Scientific</td><td>4430100</td><td>For cell spheroid growing</td></tr><tr><td>Agarose&amp;nbsp;</td><td>Carl Roth GmbH</td><td>3810.1</td><td>For cell spheroid growing</td></tr><tr><td>MCF-7 cell line</td><td>CLS Cell Lines Service GmbH</td><td>300273</td><td>Cell line</td></tr><tr><td>U251-MG-L106 cell line</td><td></td><td></td><td>Cell line</td></tr><tr><td>DMEM</td><td>Biochrom AG (Merck Millipore)</td><td>FG0435</td><td>Culture medium</td></tr><tr><td>DMEM/Ham&#039;s F-12</td><td>Biochrom AG (Merck Millipore)</td><td>FG4815</td><td>Culture medium</td></tr><tr><td>FCS</td><td>Biochrom AG (Merck Millipore)</td><td>S0615</td><td>Cell culture supplement</td></tr><tr><td>Penicillin/streptomycin</td><td>Biochrom AG (Merck Millipore)</td><td>A 2213</td><td>Antibiotics</td></tr><tr><td>Hygromycin B</td><td>PAA Laboratories&amp;nbsp;&amp;nbsp;</td><td>P02-015</td><td>Antibiotic</td></tr><tr><td>EBSS</td><td>Sigma-Aldrich Inc.</td><td>E3024</td><td>Cell culture supplement</td></tr><tr><td>Doxorubicin</td><td>Sigma-Aldrich Inc.</td><td>D1515</td><td>Fluorescent dye (CAUTION: acute toxicity)</td></tr><tr><td>Green cytotoxicity dye</td><td>Promega GmbH</td><td>G8742</td><td>CellTox - fluorescent cytotoxicity dye</td></tr><tr><td>Rotenone</td><td>Sigma-Aldrich Inc.</td><td>R8875</td><td>Cellular inhibitor (CAUTION: acute toxicity)</td></tr><tr><td>Hydrogen peroxide (H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich Inc.</td><td>95302</td><td>Reagent for oxidation (CAUTION: acute toxicity)</td></tr><tr><td>Capillary</td><td>VitroCom&amp;nbsp;</td><td>8260-050</td><td>Sample preparation</td></tr><tr><td>Microscope</td><td>Carl Zeiss Jena</td><td>Axiovert 200M</td><td></td></tr><tr><td>AxioCam MRc CCD-camera</td><td>Carl Zeiss MicroImaging GmbH</td><td>426508-9901-000</td><td>CCD-camera</td></tr><tr><td>AxioVision data aquisition software</td><td>Carl Zeiss MicroImaging GmbH</td><td>version 4.8.2.</td><td></td></tr><tr><td>Laser diode</td><td>Pico Quant GmbH</td><td>LDH-P-C-470</td><td>Used with driver PDL800-B</td></tr><tr><td>Peristaltic pump</td><td>Ismatec Labortechnik</td><td>MS-1 Reglo</td><td>Re-calibrated to reduce the minimum pump speed by 1/10&amp;nbsp;</td></tr><tr><td>Silicone tubes</td><td>IDEX Health &amp;amp; Science GmbH</td><td>TYGON R3607</td><td></td></tr></tbody></table>

single plane illumination module and micro-capillary approach for a wide-field microscope

A module for light sheet or single plane illumination microscopy (SPIM) is described which is easily adapted to an inverted wide-field microscope and optimized for 3-dimensional cell cultures, e.g., multi-cellular tumor spheroids (MCTS). The SPIM excitation module shapes and deflects the light such that the sample is illuminated by a light sheet perpendicular to the detection path of the microscope. The system is characterized by use of a rectangular capillary for holding (and in an advanced version also by a micro-capillary approach for rotating) the samples, by synchronous adjustment of the illuminating light sheet and the objective lens used for fluorescence detection as well as by adaptation of a microfluidic system for application of fluorescent dyes, pharmaceutical agents or drugs in small quantities. A protocol for working with this system is given, and some technical details are reported. Representative results include (1) measurements of the uptake of a cytostatic drug (doxorubicin) and its partial conversion to a degradation product, (2) redox measurements by use of a genetically encoded glutathione sensor upon addition of an oxidizing agent, and (3) initiation and labeling of cell necrosis upon inhibition of the mitochondrial respiratory chain. Differences and advantages of the present SPIM module in comparison with existing systems are discussed.

Experiment 1: Cell spheroids incubated with a chemotherapeutic drug
A z-stack scan of a previously incubated MCF-7 cell spheroid (8&#160;&#181;M doxorubicin, 6&#160;hr) is depicted in Figure 3. It gives detailed information about the cellular uptake and distribution of doxorubicin17 and its degradation product18,19. Within the outer cell layer of the spheroid red fluorescent doxorubicin is mainly localized in the nucleus, whereas in inner...

Watch this Scientific Journal Video about Single Plane Illumination Module and Micro-capillary Approach for a Wide-field Microscope at JoVE.com

Single Plane Illumination Module and Micro-capillary Approach for a Wide-field Microscope

A module for single plane illumination microscopy (SPIM) is described which is easily adapted to an inverted wide-field microscope and optimized for 3-dimensional cell cultures. The sample is located within a rectangular capillary, and via a microfluidic system fluorescent dyes, pharmaceutical agents or drugs can be applied in small quantities.

A module for light sheet or single plane illumination microscopy (SPIM) is described which is easily adapted to an inverted wide-field microscope and ...

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Universidad San Sebastian

Research

JoVE Journal

Engineering

9.7K Views.  Aalen University. A module for single plane illumination microscopy (SPIM) is described which is easily adapted to an inverted wide-field microscope and optimized for 3-dimensional cell cultures. The sample is located within a rectangular capillary, and via a microfluidic system fluorescent dyes, pharmaceutical agents or drugs can be applied in small quantities.

Video: Single Plane Illumination Module and Micro-capillary Approach for a Wide-field Microscope

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

Bioengineering

Lensless Fluorescent Microscopy on a Chip

On-chip lensless imaging in general aims to replace bulky lens-based optical microscopes with simpler and more compact designs, especially for high-throughput screening applications. This emerging technology platform has the potential to eliminate the need for bulky and/or costly optical components through the help of novel theories and digital reconstruction algorithms. Along the same lines, here we demonstrate an on-chip fluorescent microscopy modality that can achieve e.g., &lt;4&mu;m spatial resolution over an ultra-wide field-of-view (FOV) of &gt;0.6-8 cm2 without the use of any lenses, mechanical-scanning or thin-film based interference filters. In this technique, fluorescent excitation is achieved through a prism or hemispherical-glass interface illuminated by an incoherent source. After interacting with the entire object volume, this excitation light is rejected by total-internal-reflection (TIR) process that is occurring at the bottom of the sample micro-fluidic chip. The fluorescent emission from the excited objects is then collected by a fiber-optic faceplate or a taper and is delivered to an optoelectronic sensor array such as a charge-coupled-device (CCD). By using a compressive-sampling based decoding algorithm, the acquired lensfree raw fluorescent images of the sample can be rapidly processed to yield e.g., &lt;4&mu;m resolution over an FOV of &gt;0.6-8 cm2. Moreover, vertically stacked micro-channels that are separated by e.g., 50-100 &mu;m can also be successfully imaged using the same lensfree on-chip microscopy platform, which further increases the overall throughput of this modality. This compact on-chip fluorescent imaging platform, with a rapid compressive decoder behind it, could be rather valuable for high-throughput cytometry, rare-cell research and microarray-analysis.

A lensless on-chip fluorescent microscopy platform is demonstrated that can image fluorescent objects over an ultra-wide field-of-view of e.g., &#62;0.6-8 cm2 with &#60;4&#956;m resolution using a compressive sampling based decoding algorithm. Such a compact and wide-field fluorescent on-chip imaging modality could be valuable for high-throughput cytometry, rare-cell research and microarray-analysis.

On-chip lensless imaging in general aims to replace bulky lens-based optical microscopes with simpler and more compact designs, especially for ...

Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different size scales. In micrometer and millimeter scales, optical microscopy modalities find increasing use owing to the non-ionizing nature of visible light, and the availability of a rich set of illumination sources (such as lasers and light-emitting-diodes) and detection elements (such as large format CCD and CMOS detector-arrays). Among the recently developed optical tomographic microscopy modalities, one can include optical coherence tomography, optical diffraction tomography, optical projection tomography and light-sheet microscopy. 1-6 These platforms provide sectional imaging of cells, microorganisms and model animals such as C. elegans, zebrafish and mouse embryos.
Existing 3D optical imagers generally have relatively bulky and complex architectures, limiting the availability of these equipments to advanced laboratories, and impeding their integration with lab-on-a-chip platforms and microfluidic chips. To provide an alternative tomographic microscope, we recently developed lensfree optical tomography (LOT) as a high-throughput, compact and cost-effective optical tomography modality. 7 LOT discards the use of lenses and bulky optical components, and instead relies on multi-angle illumination and digital computation to achieve depth-resolved imaging of micro-objects over a large imaging volume. LOT can image biological specimen at a spatial resolution of &lt;1 &mu;m x &lt;1 &mu;m x &lt;3 &mu;m in the x, y and z dimensions, respectively, over a large imaging volume of 15-100 mm3, and can be particularly useful for lab-on-a-chip platforms.

Lensfree optical tomography is a three-dimensional microscopy technique that offers a spatial resolution of &lt;1 &mu;m &times; &lt;1 &mu;m &times; &lt;3 &mu;m in x, y and z dimensions, respectively, over a large imaging-volume of 15-100 mm3, which can be particularly useful for integration with lab-on-a-chip platforms.

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different ...

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.

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 ...

Biochemistry

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Immunology and Infection

Label-Free Imaging of Single Proteins Secreted from Living Cells via iSCAT Microscopy

We demonstrate interferometric scattering (iSCAT) microscopy, a method capable of detecting single unlabeled proteins secreted from individual living cells in real time. In this protocol, we cover the fundamental steps to realize an iSCAT microscope and complement it with additional imaging channels to monitor the viability of a cell under study. Following this, we use the method for real-time detection of single proteins as they are secreted from a living cell which we demonstrate with an immortalized B-cell line (Laz388). Necessary steps concerning the preparation of microscope and sample as well as the analysis of the recorded data are discussed. The video protocol demonstrates that iSCAT microscopy offers a straightforward method to study secretion at the single-molecule level.

We present a protocol for the real-time optical detection of single unlabeled proteins as they are secreted from living cells. This is based on interferometric scattering (iSCAT) microscopy, which can be applied to a variety of different biological systems and configurations.

We demonstrate interferometric scattering (iSCAT) microscopy, a method capable of detecting single unlabeled proteins secreted from individual living ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

A Guide to Build a Highly Inclined Swept Tile Microscope for Extended Field-of-view Single-molecule Imaging

Single-molecule imaging has greatly advanced our understanding of molecular mechanisms in biological studies. However, it has been challenging to obtain large field-of-view, high-contrast images in thick cells and tissues. Here, we introduce highly inclined swept tile (HIST) microscopy that overcomes this problem. A pair of cylindrical lenses was implemented to generate an elongated excitation beam that was scanned over a large imaging area via a fast galvo mirror. A 4f configuration was used to position optical components. A scientific complementary metal-oxide semiconductor camera detected the fluorescence signal and blocked the out-of-focus background with a dynamic confocal slit synchronized with the beam sweeping. We present a step-by-step instruction on building the HIST microscope with all basic components.

A detailed instruction is described on how to build a highly inclined swept tile (HIST) microscope and its usage for single-molecule imaging.

Single-molecule imaging has greatly advanced our understanding of molecular mechanisms in biological studies. However, it has been challenging to obtain ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

Single Plane Illumination Module and Micro-capillary Approach for a Wide-field Microscope

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Rozdziały w tym wideo

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Lensless Fluorescent Microscopy on a Chip

Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

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

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Label-Free Imaging of Single Proteins Secreted from Living Cells via iSCAT Microscopy

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

Conducting Multiple Imaging Modes with One Fluorescence Microscope

A Guide to Build a Highly Inclined Swept Tile Microscope for Extended Field-of-view Single-molecule Imaging

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy