A parallelized imaging platform for accurate and efficient long-term assessment of brain organoid development
Award last edited on: 1/31/2024

Sponsored Program
Awarding Agency
Total Award Amount
Award Phase
Solicitation Topic Code
Principal Investigator
Mark Harfouche

Company Information

Ramona Optics (AKA: Optical Wavefront Laboratories LLC)

331 West Main Street
Durham, NC 27701
   (919) 590-5851
Location: Multiple
Congr. District: 04
County: Durham

Phase I

Contract Number: 2024
Start Date: ----    Completed: 12/15/2023
Phase I year
Phase I Amount
Significance: High-content microscopy is rapidly accelerating biological research and drugdevelopment. Unfortunately, existing imaging systems fail to efficiently image three-dimensional diseasemodels, such as organoid cultures, at the throughput required by clinical and commercial researchers.Generating mature brain organoids, in particular, is a complex process that requires months of intensiveand costly maintenance with regular imaging to assess health and structure. One of the primarydrawbacks of standard microscopes is their need to step-and-scan across a well plate, which can takeminutes to image all wells in two dimensions, and an hour or more for 3D capture. This not only leads tocumbersome experimentation with a limited number of specimens, but also prevents current high-contentexperiments from measuring short-term (< 1 hour) 3D morphological dynamics, such as neural activity.Proposal: Ramona Optics is developing a new imaging platform for rapid high-content 3D screening ofbrain organoids to effectively assess of dozens of well plates/day across at least 90 days of differentiation.We will also develop image analysis software to identify, measure and track organoid volume and 3Dmorphology over time. These new capabilities should significantly expedite imaging workflow and yieldnovel insights into organoid development within high-content experimentation. After carefully testing the3D imaging performance of our parallelized microscope and assessing higher-resolution optics in thisPhase I proposal, we aim to enhance performance with fluorescence capabilities in a Phase II effort,focused on live-cell whole-plate recording of neural activity in large numbers of brain organoids. SA1: Parallelized microscope hardware improvement: Ramona Optics will produce newparallelized microscope hardware to simultaneously image 96 full wells (8x12 FOV) at 3 µm and 39 µmhalf-pitch lateral and axial resolution, respectively. We will also develop hardware and firmware capableof axial scanning across 1500 µm at 20 µm step size (75 axial slices) to produce a full well plate focalstack in 7.5 sec., >100X less time than what state-of-the-art scanning high-content microscopes require. SA2: Automated organoid analysis software: We will produce software to map acquired whole-plate axial scan measurements into per-organoid 3D measurements and associated statistics in <45 sec.We will also create a user interface to longitudinally track per-organoid measurements. SA3: Longitudinal testing and verification of 3D brain organoid shape/size: With the SteinLab at UNC, we will evaluate the platform for high-content longitudinal measurement of brain organoidshape/size. We will image 20 plates (96 organoids each) daily for 90 days and aim for 95% correlationwith current standard measurement methods across a variety of organoid morphologies and sizes. Wewill also assess 3D scan specs with higher resolution optics (0.8 and 0.5 µm/pixel) and fluorescence.

Public Health Relevance Statement:
Brain organoids are an increasingly popular platform for neuroscientific research, yet their macroscopic size (1-3 mm) prevents current high-content optical microscopes from offering an effective means to rapidly image large specimen collections for longitudinal assessment. In this project, Ramona Optics and the Stein Lab at the University of North Carolina at Chapel Hill will develop and test a parallelized, 96-camera 3D microscope and associated software to rapidly capture (< 10 sec.) the 3D morphology and volume of in vitro brain organoids within full 96 well plates. This new technology aims to drive efficiency and novel bioassay development by delivering 3D volume, surface area and morphology measurements that correlate with current standards by >95% while maintaining a 100X speed-up for experimental workflow.

Project Terms:

Phase II

Contract Number: 1R43MH133521-01A1
Start Date: 11/30/2025    Completed: 00/00/00
Phase II year
Phase II Amount