CX7 LZR for High Content Confocal Microscopy of 3D Cell Culture Models

As a company that specializes in three-dimensional tissue imaging, we have worked with just about every type of optical imaging device there is and have even built our own (Clara™) when one didn’t exist. In the last two years with the rapid adoption of 3D cell culture models (e.g. organoids, spheroids, microtissues) we have gotten a significant amount of interest from researchers to characterize these models in 3D using our patented Visikol® HISTO-M™ tissue clearing approach and image analysis algorithms. Because of this interest, we decided to purchase our own high content confocal system specifically for these 3D cell culture service projects and evaluated all of the imagers in the marketplace for this application before eventually purchasing the Thermo Fisher CellInsight CX7 LZR. Buying a high content confocal system is a big decision for any lab but boils down to three main factors: 1) technical capabilities, 2) throughput and 3) cost.

Technical Capabilities

In visualizing fixed 3D cell culture models, the largest challenge is characterizing these models in their entirety as light attenuation will typically limit imaging depth to 1-3 cell layers. This limitation manifests itself in imaging assays (e.g. widefield, confocal) that bias results towards only characterizing peripheral cells instead of the entire population of cells within these models. To address this limitation, we have developed our Visikol HISTO-M tissue clearing technique which allows for complete 3D cell culture model characterization.

However, one of the challenges we encountered with high content systems is their excitation source as LED-based systems are highly limited in their ability to image 3D cell culture models in their entirety. The reason for this is that the diffuse nature of this excitation source compared to a coherent source requires that the overall light intensity is many times greater than the laser-based system to achieve the same fluorophore excitation as depth increases. Additionally, the non-coherent nature of an LED excitation source reduces the crispness of images acquired when compared to a laser-based system.

When we compare laser vs. LED side-by-side using the CX7 platform which has both excitation configurations for 3D cell culture models in PBS, what we see is that at the center plane of a 250 – 300 um spheroid both are incapable of imaging the interior of models. The reason for this is that both suffer from optical attenuation when imaging into tissues which limits their overall imaging depth. If we then compare these two systems with cleared tissues that have been cleared with the Visikol HISTO-M technique, what we see is that the images acquired with the laser system are significantly crisper than those acquired on the LED system.

CX7 LED with and without Visikol HISTO-M tissue clearing

 Left) Cleared and Right) Non-Cleared NCI-H2170 + SYTOX Green + CX7 LED                    

Left) Cleared and Right) Non-Cleared NCI-H2170 + SYTOX Green + CX7 LED                    

CX7 LZR with and without Visikol HISTO-M tissue clearing

 Left) Cleared and Right) Non-Cleared HepG2 + DAPI + CX7 LZR

Left) Cleared and Right) Non-Cleared HepG2 + DAPI + CX7 LZR

Therefore, after testing several systems we determined that non-laser-based systems would have significant technical limitations in imaging 3D cell culture models and thus we gravitated towards the Perkin Elmer Opera Phenix, the newly released CellInsight CX7 LZR, the GE IN Cell 6000/6500 and the Yokogawa CV8000.

Outside of the hardware aspects of these devices, there are considerable differences between the software packages available and each package has its own unique advantages and disadvantages depending upon your specific research question and your lab’s image analysis workflow.

Throughput

For any 3D imaging application, it is vitally important to understand the minimum amount of data required to address your research question as more data is not always better and will result in lower throughput. The reason that this is so important is that this data density (i.e. magnification, Z-step size, pixels) will dictate your throughput which is typically the major bottleneck in any high content imaging campaign.  When considering the instruments described above, the Perkin Elmer Opera Phenix has the greatest throughput with up to simultaneous 4-channel imaging and several advanced optical features (e.g. microlens, water dipping objectives). The CV8000 has simultaneous 4-channel imaging capabilities as well and then the other instruments described above all are equipped with single channel at a time imaging.

Cost

The reason we did not start off by describing price is that there is no sense in purchasing an imager that is not capable of addressing your research question or throughput no matter how little it might cost. The difference in price between the CX7 LZR and the Perkin Elmer Opera Phenix or CV8000 is roughly equivalent to the difference between the imaging throughput between these devices. The GE IN Cell 6000/6500 price falls in between these two devices on cost, but has an equivalent throughput to the CX7 LZR.

Instrument Cost
CX7 LZR, CX7 LED, Operetta CLS,
ImageXpress
$
GE IN Cell 6000/6500 $$
Phenix Opera, CV8000 $$$

Ultimately as a company we decided to purchase the CX7 LZR for several practical reasons with the first of which being that today we do not require the throughput capability of the Opera Phenix and its additional features were not requirements for us. Additionally, there is the practical consideration of instrument maintenance and down time where purchasing three CX7 LZRs can provide similar throughput to one Opera Phenix without the potential of having all imaging going down if there is an instrument breakdown. In the HCS market for 3D cell culture models we would consider the the GE IN Cell 6000/6500 system, the CX7 LZR, the CV8000 and the Perkin Elmer Opera Phenix as very capable for 3D cell culture imaging and the specific system that you choose to purchase will be entirely dependent upon your lab’s needs.

CX7 LZRs can provide similar throughput to one Opera Phenix without the potential of having all imaging going down if there is an instrument breakdown. In the HCS market for 3D cell culture models we would consider the the GE IN Cell 6000/6500 system, the CX7 LZR, the CV8000 and the Perkin Elmer Opera Phenix as very capable for 3D cell culture imaging and the specific system that you choose to purchase will be entirely dependent upon your lab’s needs.

Michael Johnson