Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Platforms for Optogenetic Stimulation and Feedback Control

doi: 10.3389/fbioe.2022.918917

Figure Lengend Snippet: Optogenetic stimulation and target output observation in cellular context. Optogenetic studies involve placing a desired genetic construct under a light-activated tool (e.g. light-inducible transcription factors illustrated here) with an observable target output (e.g. fluorescent proteins illustrated here). Depending on the application requirements, one can choose from a plethora of optogenetic platforms (left: DMD-based platform , LPA , Chi.Bio ) and measurement devices (right) to stimulate and investigate the optogenetic constructs, respectively.

Article Snippet: When integrated with an appropriate measurement device, some of these platforms also provide optogenetic in silico feedback control (Cybergenetics) capability, with which different feedback control strategies can be implemented and investigated.

Techniques: Construct

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Platforms for Optogenetic Stimulation and Feedback Control

doi: 10.3389/fbioe.2022.918917

Figure Lengend Snippet: Optogenetic in silico feedback control framework. Given a target cell (engineered with desired optogenetic constructs and fluorescent reporter) culture, fluorescence measurements from target cells (measured via specialized devices, e.g. microscope, flow-cytometer, etc .) are sent to a computer where they are quantified. The quantified output is then used to run a controller (e.g. PI, MPC, etc .) simulation. The controller computes the required light input intensity for cells in order to achieve a desired output behaviour, such as tracking a desired set-point. This input light intensity is then applied to the target cells via a suitable light stimulation platform, thus closing the feedback loop. This measurement-computation-stimulation loop is performed at fixed intervals driving the output dynamics as desired.

Article Snippet: When integrated with an appropriate measurement device, some of these platforms also provide optogenetic in silico feedback control (Cybergenetics) capability, with which different feedback control strategies can be implemented and investigated.

Techniques: In Silico, Control, Construct, Fluorescence, Microscopy, Flow Cytometry

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Platforms for Optogenetic Stimulation and Feedback Control

doi: 10.3389/fbioe.2022.918917

Figure Lengend Snippet: Diverse optogenetic platform architectures. (A) Microscope-coupled DMD-based platform illustration. The illumination light from LEDs is steered to fall onto a DMD at a particular angle. A DMD has micrometer-sized mirrors that can be individually tilted to ON and OFF positions. ON position allows the incident light to follow a defined path towards the microscope opening port whereas OFF position lets the incident light divert away after reflection. Once a given mask image is sent to the DMD controller, it sets the tilt-position of all DMD micromirrors accordingly with each micromirror representing an individual pixel of the image. These micromirrors oscillate between ON and OFF positions at a very high frequency, and the duty cycle of each micromirror is determined by the corresponding pixel intensity (gray value) in the mask image. When incident light is applied, the oscillating micromirrors allow the projection of the mask image towards the microscope opening port. The projection light then follows a series of optical elements (lens, filters, apertures, etc .) before entering the microscope port which then guides it towards the microscope objective lens. All optical elements are chosen and positioned in such a way that the projection image is focused onto the microscope sample plane allowing spatio-temporal and graded illumination of target cells placed under the microscope field of view. (B) Microtiter multiwell plate LED array illustration. These devices, in general, have a layered structure. The main layer is the electronic board fit with LEDs at regular matrix locations aligned with the target plate wells. This board can have multiple LEDs grouped together to provide multiple wavelengths per well. It also contains LED driver electronics and micro-controllers needed to set LED intensities and turn them ON/OFF as and when desired in a pre-programmed manner. To provide independent illumination to each well, an opaque adapter can be placed on top of the LED board isolating individual LEDs. The multiwell plate containing target cell culture is then placed on top of the adapter such that each well receives light from the LED underneath independent and isolated from other wells. One can also place a light diffuser film between the plate and the adapter to provide more homogeneous illumination within each well. Furthermore, if required, one can also add a heat sink or other cooling system under the LED board to dissipate heat generated by the electronics board which may adversely effect the target cell culture. (C) Milli-culture optogenetic platform illustration. The target cell culture is usually placed in a glass vial which can be externally illuminated with LEDs. Cell culture incubation temperature is maintained with a heat plate below the vial or a heated water bath. Aeration is achieved using devices such as magnetic stirrers. Some platforms also have an integrated system to measure and regulate growth conditions, such as maintaining a desired cell culture density during an experiment. They have dedicated OD sensors as well as media replenishing and culture removal mechanisms to provide turbidostat, chemostat, or morbidostat modalities. These platforms usually have separate micro-controllers/processors to execute these functionalities either in a pre-programmed manner or a live run by receiving updated commands from a computer during the course of an experiment. (D) Photobioreactor illustration. This is a generic photo-bioreactor set-up where the cells are usually cultured in a glass (transparent) off-the-shelf bioreactor. Two or more high-power LED panels are placed around the bioreactor to provide sufficient illumination of the target cell culture as and when required. Some designs also involve LED strips wrapped around the bioreactor for better illumination.

Article Snippet: When integrated with an appropriate measurement device, some of these platforms also provide optogenetic in silico feedback control (Cybergenetics) capability, with which different feedback control strategies can be implemented and investigated.

Techniques: Microscopy, Cell Culture, Isolation, Generated, Incubation

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Platforms for Optogenetic Stimulation and Feedback Control

doi: 10.3389/fbioe.2022.918917

Figure Lengend Snippet: Applications of optogenetic in silico feedback control. (A) Cell-to-cell communication studies. Cellular outputs from neighboring cells are used to determine the light stimulation intensity of a target cell. Using this framework, lateral inhibition between neighboring cells has been shown to produce emerging checkerboard patterns in a 2-D grid of cells . Note: M1 and M2 represent the same microscope; C1 and C2 represent the same control computer. (B) Rapid prototyping of biomolecular controller motifs. Stochastic simulation of biomolecular controller networks is run in the in silico feedback control framework with an in vivo target network. This allows rapid benchmarking and characterization of these controller motifs without requiring their in vivo implementations .

Article Snippet: When integrated with an appropriate measurement device, some of these platforms also provide optogenetic in silico feedback control (Cybergenetics) capability, with which different feedback control strategies can be implemented and investigated.

Techniques: In Silico, Control, Inhibition, Microscopy, In Vivo