The authors would like to acknowledge Lina DeLuca, Fakhar Singhera, Hannah Williams, Lynn Deng, Osinachi Nwosu and Sarah Wachtman for their assistance in testing the M.A.P.L.E. platform.

1. Semiautomated &#34;plate to plate&#34; sample transfer preparation
<ol>
	<li>Generate a CSV file as shown in Figure 1 containing source and destination plates using a spreadsheet editing application. The CSV file that is generated must have the following header columns in the order listed: Source_barcode; Destination_barcode; Source_well; Dest_well; Transfer_volume. &#160;&#160;</li>
	<li>Under the header columns, make sure to include one row in the CSV file for each desired pipetting operation (i.e., sample transfer) with the following information:
	<ol>
		<li>Source_barcode: Alphanumeric barcode of the source microplate, e.g., S1007372; leave blank if no barcode is associated.</li>
		<li>Destination_barcode: Alphanumeric barcode of the destination microplate, e.g., D0573282; leave blank if no barcode is associated.</li>
		<li>Source_well: Alphanumeric row and column identifier for well to be pipetted out of from source plate, e.g., H10 for row H (8th row), column 10 (ANSI/SLAS standard well designations, e.g., A1, C10&#8230;).</li>
		<li>Dest_well: Alphanumeric row and column identifier for well to be pipetted out of from destination plate, e.g., A3 for row A (1st row), column 3 (ANSI/SLAS standard well designations, e.g., A1, C10&#8230;).</li>
		<li>Transfer_volume: Volume to be transferred from source_well in source_barcode to dest_well in destination_barcode (numeric and unitless: typically in &#181;L).</li>
	</ol>
	</li>
	<li>Open the Microplate Assistive Pipetting Light Emitter Plate to plate GUI application, shown in Figure 2, by opening the Light Guide program (Maple-LightGuide.exe).</li>
	<li>Click the Select cherrypick file button in the upper left corner of the GUI.</li>
	<li>Use the file browser window, shown in Figure 3, to navigate to the CSV file generated in steps 1.1 and 1.2 above and click the Open button. The application will parse the first row of the CSV file and illuminate the corresponding wells in the source and destination plates.</li>
	<li>Use the Previous well and Next well buttons, in the upper right corner of the GUI, shown in Figure 4, to traverse the CSV file as desired. The GUI will highlight in grey any rows which have been previously illuminated and highlight in brown the currently active row.</li>
	<li>Perform pipetting operations as needed to transfer samples between source well of source plate to destination well of destination plate. An example of a M.A.P.L.E. unassisted hand pipetting operation can be seen in Figure 5, with a comparison of the current user pipetting view seen in Figure 4 and Figure 6. In addition to the user GUI, plate barcodes can be verified via the LCD displays attached to the illumination panels.</li>
	<li>Continue until the end of the CSV file is reached via the Next well button. To load a new CSV file, the Select cherrypick file may be clicked at any time. To exit the program the red X at the top right corner of the GUI can be clicked.</li>
</ol>
2. Multi-well illuminations for parallel transfers and serial dilutions
<ol>
	<li>Open the Microplate Assistive Pipetting Light Emitter &#39;Serial dilution&#39;&#160;application by opening the serial dilution program (Maple-SerialDilution.exe).</li>
	<li>Use the GUI, shown in Figure 7 and Figure 8, to specify the desired titration mode (column or row), plate density and start row(s) or column(s). The GUI also allows users to specify a column or row mask to control which LEDs in a given row or column are illuminated. This allows a subset of LEDs in a row or column to be illuminated instead of illuminating the entire row or column.</li>
	<li>Use the Next and Previous buttons to step through the rows or columns in sequence from the initial start row or column to the last row or column in the plate. Each time the Next or Previous button is clicked, the light panel will illuminate the corresponding LEDs of the microplate.</li>
	<li>Continue until the end of the titration sequence is reached. To exit the program, click the red X at the top right corner of the GUI.</li>
</ol>
3. Laboratory training: assay development and screening format techniques
<ol>
	<li>Place a 96- or 384-well microplate into the portable light guide. The portable light guide contains a battery and all electronics necessary to be used independently of a computer. This allows the portable lightguide to be used in a handheld mode which can be controlled with built-in pushbuttons to toggle between demonstration modes.</li>
	<li>Use the power toggle switch on the portable light guide enclosure to power the system on.</li>
	<li>Determine the mode for the portable light guide to be in. By default, the portable light guide will load into the default HTS demo mode which provides users with a visual representation of a typical assay plate as seen in Figure 9. In this mode, one can use the right pushbutton switch at the top of the portable light guide to toggle through the following sample illumination patterns.
	<ol>
		<li>All wells illuminated with red color to simulate the reagent dispense of an assay, e.g., (suspended cells in media).</li>
		<li>All wells illuminated with a yellow color to simulate dye reagent addition.</li>
		<li>First column and last column of wells illuminated green, remaining middle &#39;sample field&#39;&#160;columns illuminated blue to indicate plate being read on microplate reader. Random wells in the sample field will also have green color of varying intensity to represent hits.</li>
	</ol>
	</li>
	<li>To toggle the light guide between HTS demo mode and Titration demo mode, push the left pushbutton switch. Doing so will switch the portable light guide to the Titration demo mode which provides a visual guide to users to understand how titrations can be performed in compound plates. When the light guide enters the Titration demo mode, the following will occur.
	<ol>
		<li>All wells in column 3 and 13 are illuminated with yellow color.</li>
		<li>Subsequent presses of the rightmost pushbutton switch illuminates columns in sequence, e.g., (4 and 14, 5 and 15, etc.).</li>
		<li>When the pushbutton is pressed after columns 12 and 22 are reached, wells in columns 4-12 and 13-22 are illuminated in decreasing intensity of yellow to represent the titration.</li>
	</ol>
	</li>
	<li>To modify the default behavior of the light guide, plug the portable light guide to a computer via a USB cable and follow the detailed instructions for updating the default firmware via the Arduino IDE which can be found on the project GitHub page8. By updating the firmware, you can modify these modes to display other sequences or sets of LEDs.</li>
</ol>
4. Illumination of artifacts in microplates
<ol>
	<li>Place a 96- or 384-well microplate into the portable light guide.</li>
	<li>Switch the light guide to Illumination mode by pressing the leftmost pushbutton switch two times. 
	NOTE: An example of the practical use of this mode can be seen in Figure 10&#160;and&#160;Figure 11, where compounds have precipitated out of solution and can be observed on the bottom of the microplates. Without backlit illumination, most of the precipitate is invisible to the naked eye, but M.A.P.L.E. backlighting reveals precipitate for user inspection and photographic documentation.</li>
	<li>Use the right pushbutton to toggle between a set of predefined colors as needed for the application. The light panel will turn all LEDs on to the following colors in sequence with each push of the right button: red, blue, green, orange, white, violet, yellow and indigo.</li>
	<li>As an optional step, use a camera or smartphone to photograph the illuminated plate for recordkeeping or documenting the work.</li>
</ol>

<ol><li>Baillargeon, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Design+of+Microplate-Compatible+Illumination+Panels+for+a+Semiautomated+Benchtop+Pipetting+System%5BTitle%5D)+AND+%22SLAS+TECHNOLOGY%3A+Translating+Life+Sciences+Innovation%22%5BJournal%5D)">Design of Microplate-Compatible Illumination Panels for a Semiautomated Benchtop Pipetting System.</a> SLAS TECHNOLOGY: Translating Life Sciences Innovation. , (2019).</li>
<li>Baillargeon, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Scripps+Molecular+Screening+Center+and+Translational+Research+Institute%5BTitle%5D)+AND+%22SLAS+DISCOVERY%3A+Advancing+Life+Sciences+R%26D%22%5BJournal%5D)">The Scripps Molecular Screening Center and Translational Research Institute.</a> SLAS DISCOVERY: Advancing Life Sciences R&D. 24 (3), 386-397 (2019).</li>
<li>BioSistemika. Pipetting Aid PlatR. , Available from: 
 <a target="_blank" href="https://biosistemika.com/products/pipetting-platr/">https://biosistemika.com/products/pipetting-platr/</a> (2019).</li>
<li>Gilson Trackman Pipetting Tracker. Daigger Scientific. , Available from: 
 <a target="_blank" href="https://www.daigger.com/gilson-trackma-pipetting-tracker-i-gsnf70301">https://www.daigger.com/gilson-trackma-pipetting-tracker-i-gsnf70301</a> (2019).</li>
<li>TRACKMAN Connected US. Gilson. , Available from: 
 <a target="_blank" href="https://www.gilson.com/default/systemm-trackman-connected-us.html">https://www.gilson.com/default/systemm-trackman-connected-us.html</a> (2019).</li>
<li>LI-2100LightOne™ Pro. Embi Tec. , Available from: 
 <a target="_blank" href="http://embitec.com/li2100-lightone-pro-384-and-96-well.html">http://embitec.com/li2100-lightone-pro-384-and-96-well.html</a> (2019).</li>
<li>96 well plate pipette light guide. qit vision. , Available from: 
 <a target="_blank" href="https://www.qitvision.com/projects/#Plate">https://www.qitvision.com/projects/#Plate</a> (2019).</li>
<li>Microplate Assistive Pipetting Light Emitter GitHub repository. , Available from: 
 <a target="_blank" href="https://github.com/pierrebaillargeon/Microplate-Assistive-Pipetting-Light-Emitter">https://github.com/pierrebaillargeon/Microplate-Assistive-Pipetting-Light-Emitter</a> (2019).</li>
<li>Maplebear Electronics Tindie store. , Available from: 
 <a target="_blank" href="http://maplebearelectronics.com">http://maplebearelectronics.com</a> (2019).</li>
<li>Hawker, C. D., Schlank, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+Standards+for+Laboratory+Automation%5BTitle%5D)+AND+%22Clinical+Chemistry%22%5BJournal%5D)">Development of Standards for Laboratory Automation.</a> Clinical Chemistry. 46, 746-750 (2000).</li>
<li>General Laboratory Techniques. An Introduction to the Micropipettor. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22JoVE+Science+Education+Database%22">JoVE Science Education Database</a>. , JoVE. Cambridge, MA. (2019).</li>
<li>General Laboratory Techniques. Introduction to the Spectrophotometer. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22JoVE+Science+Education+Database%22">JoVE Science Education Database</a>. , JoVE. Cambridge, MA. (2019).</li>
<li>General Laboratory Techniques. Introduction to the Microplate Reader. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22JoVE+Science+Education+Database%22">JoVE Science Education Database</a>. , JoVE. Cambridge, MA. (2019).</li>
</ol>

Authors have no financial interests or conflict of interest with any of the manufactured components suggested in the construction of the M.A.P.L.E. device. Sources presented are strictly for the convenience of the user and any compatible components from alternative sources can be used as needed.&#160;

By releasing M.A.P.L.E. as an open-source platform, we have introduced a laboratory tool that provides utility, but can also be easily extended to meet the evolving needs of the end user. Benchtop microplate sample preparation is a common task that is performed in a wide variety of laboratory environments and this task can be demonstrably improved with a technology such as M.A.P.L.E.
The M.A.P.L.E. platform has been specifically engineered with adaptability to future applications in mind. Each...

As recently published1, the Lead Identification laboratory at Scripps Research2 has developed and released an open-source illumination panel for microplate preparation referred to as the Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.). Manual preparation of microplates, whether they are made for compound management or bio-assay needs, can be prone to human errors that drastically increase as well density of the microplate increases. In addition, proper recordkeeping and data-logging of microplate content/format is also prone to manual entry errors. In high throughput screening (HTS) automation facilities these issues are mitigated through the use of computer-driven robotic workstations that are integrated with automated database recordkeeping; minimizing manual manipulations and reducing the potential of formatting and data recording errors. However, there remain many instances where automation-based instrumentation is simply not feasible or compatible with microplate formatting needs, requiring manual intervention. Moreover, there is also a need to support small-scale laboratory operations that require compact and cost-effective semi-automated devices to improve their throughput, accuracy and automate data-recordkeeping of microplate preparation.&#160;&#160; &#160;
While other microplate illumination systems exist, they are proprietary commercial solutions3,4,5,6,7 limited to select microplate formats and their proprietary closed-source nature prevents user-driven modifications that would allow the adaptation these devices for specialized operations. &#160;M.A.P.L.E. was designed to be an inexpensive open-source device, with source code and all design files available for free online8. Users with knowledge of surface mount soldering techniques can assemble their own M.A.P.L.E. devices with the code and design files available on GitHub, or they can modify the provided printed circuit boards (PCBs) designs, 3D print enclosure computer-aided design (CAD) models and code to meet their specific needs. A full list of parts needed to fabricate the light guide PCBs can be found in Supplementary Tables 1 and 2 and further details regarding the design and implementation of the light panels can be found in recently published documentation1. Users who wish to purchase pre-assembled light guide PCBs based off the open-source files can find them listed online9.
M.A.P.L.E. provides the user with an easily controllable illumination panel which has a microplate-based footprint and LED-to-LED spacing matched to Society for Biomolecular Screening (SBS) specifications for microplates10. M.A.P.L.E. was developed to support 96- and 384-well density microplates and allow users to illuminate wells in any desired configuration, color and intensity. These light panels can be used to illuminate microplates for pipetting operations11, to simulate laboratory formatting operations or instruments such as a microplate reader12,13 for educational and demonstration purposes. The open source nature of the project allows users to easily modify the panels, firmware or graphical user interface (GUI) software to support any new desired functionality. Guidance and data recordkeeping are computer-driven and can be integrated with spreadsheets or ported to a database system. Because M.A.P.L.E. is designed to work with plaintext comma delimited files, any spreadsheet or database software that is able to import or export CSV formatted files can be easily extended to work with M.A.P.L.E. Further, the project enclosure which has been designed for this system tilts the microplate towards the user during pipetting operations, increasing ergonomics by providing a more natural posture for the user while at the lab bench. Specific operational features to the M.A.P.L.E. system include: (i) Facilitating compound management efforts in preparing customized plates by illuminating single source well and destination well across microplates for manual pipetting guidance; assisted through a computer script that can be saved as an electronic record post completion. (ii) M.A.P.L.E. can illuminate any number of wells across microplate rows or columns; which is ideally suited for rapid serial dilution guidance or placement of select replicate controls. (iii) M.A.P.L.E. can be used in a demonstration mode to facilitate laboratory training needs or highlight formatting requirements with respect to sample and control placements or dedicated well usage (e.g., edge-effect barrier gap). (iv) M.A.P.L.E. can backlight transparent/translucent wells to allow visualization of artifacts such as precipitation/crystallization, bubbles, well heterogeneity, empty wells; which also allows end-user to easily photograph plate images for documentation needs

<table><tbody><tr><td>96 or 384 well microplate</td><td></td><td></td><td>https://en.wikipedia.org/wiki/Microplate</td></tr><tr><td>Microplate Assistive Pipetting Light Emitter</td><td>Open source</td><td></td><td>https://github.com/pierrebaillargeon/Microplate-Assistive-Pipetting-Light-Emitter</td></tr><tr><td>Pipettor</td><td></td><td></td><td>https://www.jove.com/science-education/5033/an-introduction-to-the-micropipettor</td></tr><tr><td>Spectrometer</td><td>Ocean Optics</td><td>USB-650 Red Tide</td><td></td></tr></tbody></table>

applications for open source microplate-compatible illumination panels

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well as large-scale high-throughput screening (HTS) campaigns. Though laboratory automation has greatly increased the utility of microplates there remain instances where automation-based instrumentation is not feasible, cost-effective or compatible with microplate formatting needs. In these cases, microplates must be manually prepared. Problematic to manual microplate manipulations is that a number of difficulties can arise pertaining to the accurate tracking of sample operations, data record keeping and quality control (QC) inspection for well artifacts or formatting errors. As microplate well densities increase (i.e., 96-well, 384-well, 1536-well) the potential for introducing errors also drastically increases.&#160; Moreover, for small bench-top laboratory operations there exists a need to improve the ease and accuracy of sample handling in a cost-effective fashion. Herein, we describe a system that acts as a semi-automated pipetting guide referred to as the Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.). &#160;M.A.P.L.E. has multiple uses for supporting compound hit-picking and microplate preparation for assay development in high-throughput screening or laboratory benchtop operations, as well as QC/quality assurance (QA) diagnostic evaluation of microplate quality or visualizing well formatting errors.

The M.A.P.L.E. platform is capable of illuminating wells in 96- and 384-well microplates in a variety of user configurable ways, allowing straightforward and independent control of color and light intensity in each well. By helping to reduce opportunities for error in manual pipetting operations, M.A.P.L.E. helps users prepare microplates with increased confidence that each well contains the desired contents. The transfer of samples between plates and the preparation of serial dilution plates, such as the examples seen i...

Watch this Scientific Journal Video about Applications for Open Source Microplate-Compatible Illumination Panels at JoVE.com

Applications for Open Source Microplate-Compatible Illumination Panels

Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.) is a computer-driven device that systematically illuminates microtiter wells to provide guidance for the manual preparation of microplates.&#160; M.A.P.L.E. improves the accuracy of microplate preparation while automating data recordkeeping. &#160;In addition, it can assist with examining microplate quality or aid in the detection of errors.&#160; &#160;

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well ...

applications-for-open-source-microplate-compatible-illumination-panels

Research

JoVE Journal

Bioengineering

7.4K Views.  Scripps Florida. Traditionele handoverdracht methoden van bulk reagentia in microtiter platen zijn traag en gevoelig voor menselijke fouten. Deze technologie verdubbelt meer dan de efficiëntie van het proces en helpt die fout te elimineren. Deze techniek kan gebruikers helpen bij het begrijpen van bewerkingen die in microplaten worden uitgevoerd, de kans op fouten te verminderen en de efficiëntie van de gebruiker te verhogen tijdens handmatige pipettingbewerkingen.Deze techniek zal een positieve invl...

Video: Applications for Open Source Microplate-Compatible Illumination Panels

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 ...

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.

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 ...

Engineering

Luminescence Lifetime Imaging of O2 with a Frequency-Domain-Based Camera System

We describe a method to image dissolved oxygen (O2), in 2D at high spatial (&#60; 50-100 &#181;m) and temporal (&#60; 10 s) resolution. The method employs O2 sensitive luminescent sensor foils (planar optodes) in combination with a specialized camera system for imaging luminescence lifetime in the frequency-domain. Planar optodes are prepared by dissolving the O2-sensitive indicator dye in a polymer and spreading the mixture on a solid support in a defined thickness via knife coating. After evaporation of the solvent, the planar optode is placed in close contact with the sample of interest - here demonstrated with the roots of the aquatic plant Littorella uniflora. The O2 concentration-dependent change in the luminescence lifetime of the indicator dye within the planar optode is imaged via the backside of the transparent carrier foil and aquarium wall using a special camera. This camera measures the luminescence lifetime (&#181;s) via a shift in phase angle between a modulated excitation signal and emission signal. This method is superior to luminescence intensity imaging methods, as the signal is independent of the dye concentration or intensity of the excitation source, and solely relies on the luminescence decay time, which is an intrinsically referenced parameter. Consequently, an additional reference dye or other means of referencing are not needed. We demonstrate the use of the system for macroscopic O2 imaging of plant rhizospheres, but the camera system can also easily be coupled to a microscope.

Luminescence Lifetime Imaging of O2 with a Frequency-Domain-Based Camera System

We describe the use of a novel, frequency-domain luminescence lifetime camera for mapping 2D O2 distributions with optical sensor foils. The camera system and image analysis procedures are described along with the preparation, calibration and application of sensor foils for visualizing the O2 microenvironment in the rhizosphere of aquatic plants.

We describe a method to image dissolved oxygen (O2), in 2D at high spatial (&#60; 50-100 &#181;m) and temporal (&#60; 10 s) resolution. The method employs ...

Chemistry

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular processes. There is a need for culturing and measuring systems that incorporate programmed illumination and stimulation of optogenetic systems. We present a protocol for building and using a continuous culturing apparatus to illuminate microbial cells with programmed doses of light, and automatically acquire and analyze images of cells in the effluent. The operation of this apparatus as a chemostat allows the growth rate and the cellular environment to be tightly controlled. The effluent of the continuous cell culture is regularly sampled and the cells are imaged by multi-channel microscopy. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses in the fluorescence intensity and cellular morphology of cells sampled from the culture effluent are measured over multiple days without user input. We demonstrate the utility of this culturing apparatus by dynamically inducing protein production in a strain of Saccharomyces cerevisiae engineered with an optogenetic system that activates transcription.

We designed a continuous culturing apparatus for use with optogenetic systems to illuminate cultures of microbes and regularly image cells in the effluent with an inverted microscope. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses to illumination can be measured over several days.

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular ...

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of F&#246;rster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.

A method for imaging changes in membrane potential using genetically encoded voltage indicators is described.

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate ...

Neuroscience

LED-Based In Vitro Screening for Assessing Photoactivable Molecules in Bacterial Photodynamic Inactivation

The rise of multi-drug-resistant infections and the lack of new antibiotic classes have renewed interest in alternative therapies like photodynamic inactivation of bacteria (aPDI). This process involves the administration of a photosensitizer (PS) activated by a suitable visible light source, producing exacerbated levels of reactive oxygen species (ROS) that damage crucial cellular biomolecules, ultimately causing bacterial cell death. It is crucial to create standardized, easy-to-use, and reproducible initial tests to assess and compare the effectiveness of light-induced phototoxicity of potential photosensitizers. This study introduces a simple and efficient in vitro technique for assessing photodynamic activity against planktonic bacterial cells. By employing a 96-well microplate format along with a large LED panel, the system facilitates the systematic evaluation of various compounds. Such a configuration allows for high-throughput screening of potential photosensitizers in a controlled and consistent environment, simplifying the process of identifying promising candidates for further development. This flexible platform serves as an important step in advancing the development of innovative photodynamic therapies for managing antibiotic-resistant infections.

This protocol describes a straightforward and economical method for evaluating the effectiveness of potential photosensitizers in antibacterial photodynamic inactivation (aPDI), using a 96-well plate format combined with an LED panel light source. This approach enables the simultaneous testing of multiple experimental conditions, including different concentrations, compounds, and bacterial strains.

The rise of multi-drug-resistant infections and the lack of new antibiotic classes have renewed interest in alternative therapies like photodynamic ...

Biochemistry

Open-Source Miniature Fluorimeter to Monitor Real-Time Isothermal Nucleic Acid Amplification Reactions in Resource-Limited Settings

Traditional methods to detect and quantify nucleic acids rely on polymerase chain reaction (PCR) and require the use of expensive thermocyclers with integrated fluorescence detection of amplicons. Isothermal nucleic acid amplification technologies eliminate the need for thermal cycling; however, fluorescence-based detection of products is still required for real-time, quantitative results. Several portable isothermal heaters with integrated fluorescence detection are now commercially available; however, the cost of these devices remains a significant barrier to widespread adoption in resource-limited settings. Described here is a protocol for the design and assembly of a modular, low-cost fluorimeter constructed from off-the-shelf components. Enclosed in a compact 3D printed housing, the fluorimeter is designed to be placed atop a commercially available heat block holding a PCR tube. The fluorimeter described here was optimized to detect fluorescein isothiocyanate (FITC) dye, but the system can be modified for use with dyes commonly used as reporters in real-time nucleic acid amplification reactions. Clinical applicability of the system is demonstrated by performing real-time nucleic acid detection with two isothermal amplification technologies: recombinase polymerase amplification (RPA) for detection of positive control DNA provided in a commercial kit and reverse transcription loop-mediated isothermal amplification (RT-LAMP) for detection of clinically meaningful levels of SARS-CoV-2 RNA.

Detailed instructions are provided to build an open-source, modular fluorimeter that is compatible with many low-cost heaters to perform real-time, quantitative isothermal nucleic acid amplification.

Traditional methods to detect and quantify nucleic acids rely on polymerase chain reaction (PCR) and require the use of expensive thermocyclers with ...

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

The larvae of Drosophila melanogaster show obvious light-avoiding behavior during the foraging stage. Drosophila larval phototaxis can be used as a model to study animal avoidance behavior. This protocol introduces a light-spot assay to investigate larval phototactic behavior. The experimental set-up includes two main parts: a visual stimulation system that generates the light spot, and an infrared light-based imaging system that records the process of larval light avoidance. This assay allows tracking of the behavior of larva before entering, during encountering, and after leaving the light spot. Details of larval movement including deceleration, pause, head casting, and turning can be captured and analyzed using this method.

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

This protocol introduces a light-spot assay to investigate Drosophila larval phototactic behavior. In this assay, a light spot is generated as light stimulation, and the process of larval light avoidance is recorded by an infrared light-based imaging system.

The larvae of Drosophila melanogaster show obvious light-avoiding behavior during the foraging stage. Drosophila larval phototaxis can be used as a model ...

Biology

Fluorescence Lifetime Macro Imager for Biomedical Applications

This paper presents a new photoluminescence lifetime imager designed to map the molecular oxygen (O2) concentration in different phosphorescent samples ranging from solid-state, O2-sensitive coatings to live animal tissue samples stained with soluble O2-sensitive probes. In particular, the nanoparticle-based near-infrared probe NanO2-IR, which is excitable with a 625 nm light-emitting diode (LED) and emits at 760 nm, was used. The imaging system is based on the Timepix3 camera (Tpx3Cam) and the opto-mechanical adaptor, which also houses an image intensifier. O2 phosphorescence lifetime imaging microscopy (PLIM) is commonly required for various studies, but current platforms have limitations in their accuracy, general flexibility, and usability.
The system presented here is a fast and highly sensitive imager, which is built on an integrated optical sensor and readout chip module, Tpx3Cam. It is shown to produce high-intensity phosphorescence signals and stable lifetime values from surface-stained intestinal tissue samples or intraluminally stained fragments of the large intestine and allows the detailed mapping of tissue O2 levels in about 20 s or less. Initial experiments on the imaging of hypoxia in grafted tumors in unconscious animals are also presented. We also describe how the imager can be re-configured for use with O2-sensitive materials based on Pt-porphyrin dyes using a 390 nm LED for the excitation and a bandpass 650 nm filter for emission. Overall, the PLIM imager was found to produce accurate quantitative measurements of lifetime values for the probes used and respective two-dimensional maps of the O2 concentration. It is also useful for the metabolic imaging of ex vivo tissue models and live animals.

This paper describes the use of a new, fast optical imager for the macroscopic photoluminescence lifetime imaging of long decay emitting samples. The integration, image acquisition, and analysis procedures are described, along with the preparation and characterization of the sensor materials for the imaging and the application of the imager in studying biological samples.

This paper presents a new photoluminescence lifetime imager designed to map the molecular oxygen (O2) concentration in different phosphorescent samples ...

A Light-Stimulation Grid Approach to Study Cellular Optogenetic Protein

Mapping the Cellular Distribution of an Optogenetic Protein Using a Light-Stimulation Grid

Our goal was to accurately track the cellular distribution of an optogenetic protein and evaluate its functionality within a specific cytoplasmic location. To achieve this, we co-transfected cells with nuclear-targeted cAMP sensors and our laboratory-developed optogenetic protein, bacterial photoactivatable adenylyl cyclase-nanoluciferase (bPAC-nLuc). bPAC-nLuc, when stimulated with 445 nm light or luciferase substrates, generates adenosine 3&#39;,5&#39;-cyclic monophosphate (cAMP). We employed a solid-state laser illuminator connected to a point scanning system that allowed us to create a grid/matrix pattern of small illuminated spots (~1 &#181;m2) throughout the cytoplasm of HC-1 cells. By doing so, we were able to effectively track the distribution of nuclear-targeted bPAC-nLuc and generate a comprehensive cAMP response map. This map accurately represented the cellular distribution of bPAC-nLuc, and its response to light stimulation varied according to the amount of protein in the illuminated spot. This innovative approach contributes to the expanding toolkit of techniques available for investigating cellular optogenetic proteins. The ability to map its distribution and response with high precision has far-reaching potential and could advance various fields of research.

This protocol involves transfecting cAMP sensors and bPAC-nLuc, an optogenetic protein, to accurately track its cellular distribution and response to light stimulation. The innovative approach of creating a cAMP response map using a point scanning system holds the potential for advancing research with optogenetic proteins in different fields.

Applications for Open Source Microplate-Compatible Illumination Panels

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