Flamingos are light sheet microscopes, a microscopy illumination technology that was introduced to modern biology in the early 2000s. The vast diversity of biological samples and experimental requirements resulted in a variety of light sheet setups being developed over the years.
The classic L-SPIM configuration with one illumination arm and one detection arm is well suited for imaging developing embryos over time, with the option of rotating the sample for multi-view imaging.
The T-SPIM orientation with a second illumination arm offers more homogeneous fluorescence excitation across larger and/or more complex samples.
An X-SPIM configuration has an additional detection arm that provides more coverage for larger and delicate specimen without the need for rotation.
A V-SPIM configuration resembles an upright compound microscope and supports more traditional mounting techniques for large pieces of tissue or brain imaging in naturally oriented fish, among others.
To accommodate as many experiments as possible, the Flamingo supports multiple configurations. Due to the modular design we can convert between the configurations by removing and adding a few components.
The Flamingo is designed in a modular way. An illumination arm is a module, as is a foot or a stage. By combining modules in different ways, we create custom light sheet microscopes for a variety of biological imaging applications.
An example is shown here. By adding another illumination arm module, a Flamingo L-SPIM can be converted to a T-SPIM configuration.
Flamingo microscopes feature fast & low-noise camera, such as the pco.panda 4.2. We integrate several detection lenses, e.g. selected models of the Nikon CFI60 and CFI75 water dipping series. Each lens can be combined with tube lenses of different focal length to adapt magnification to the imaging needs. Emission filters for extracting fluorescence signals are mounted in custom-built motorized filter wheels in the detection path.
Field of View and Spatial Sampling
Several detection lenses can be used to adjust the field of view and the spatial sampling. Representative images and numbers shown here assume a camera chip with 2048×2048, 6.5 μm pixels. The field of view can be reduced by cropping the camera chip to increase acquisition speed or reduce data size, and several fields of view can be combined to cover a larger area (tiling).
|Detection Lens||Lateral Resolution||200 mm Tube Lens||400 mm Tube Lens|
|Magnification||Field of View||Pixel Size||Spatial Sampling||Magnification||Field of View||Pixel Size||Spatial Sampling|
|Nikon 10x/0.3||833 nm||10x||1330 µm||650 nm||1.3||20x||665 µm||433 nm||2.6|
|Nikon 16x/0.8||313 nm||16x||831 µm||406 nm||0.8||32x||416 µm||203 nm||1.5|
We facilitate PI L-505 or PI M linear stages for motorized sample translation and enable motorized sample rotation using PI U-651 or U-628 stages. The Flamingo can record z-stacks and supports multi-position recordings and tiled acquisition. In an L-, T- or X-SPIM configuration, the vertically mounted sample can be rotated around its central axis. In a V-SPIM configuration, the surface with the sample resting on top can be rotated and two orthogonal z-stacks can be recorded.
Laser illumination in Flamingo light sheet microscopes is achieved by using fibre-coupled laser modules, such as a Toptica iChrome CLE. Static Gaussian light sheets are formed by cylindrical lenses and feature the options to pivot the sheet around the center of the field of view (mSPIM) and illuminate the sample from two sides in T- and X-SPIM configurations. Our main illumination lens is the water-dipping Nikon 10x/0.3 from the CFI60 series. For additional brightfield illumination, we use an RGB LED.
A light weight microscope construction and off-the-shelf components tend to be incompatible. We designed the Flamingo to require the smallest number of highly functional – sometimes multi-functional – custom components. Connecting off-the-shelf components effectively and efficiently was challenging. In addition, we had to adapt to the more unique challenges scientist would generate in different experiments. To produce custom parts for the Flamingo we employ 3D printing in multiple formats and materials, as well as rapid prototyping and manufacturing.
3D printing was used for Flamingo components in the most demanding cases. While 3D-printed exotic metals exist and are becoming more common they are neither low cost nor readily accessible. We chose to stay with lower cost easily obtained hardware and focused on taking advantage of breakthrough materials compatible with these printers in both stereolithography (SLA) and fused filament modeling (FFM) formats.
Complex detailed components like our chambers that do not require high structural strength are purposefully designed to leverage all the potential of the SLA process using a Formlabs Form 2 printer while taking advantage of the new and growing number of biocompatible resin materials originally developed for medical and dental applications. Our chambers could have easily been one of the most expensive custom components on a Flamingo, but through 3D printing they are one of least expensive and are easily modified if unforeseen needs arise.
For highly complex structural parts that adapt directly to their mating components we use FFM desktop printers modified and tuned to work with carbon composite filaments for their high strength and stiffness while being very light weight. While technically not as strong or stiff as metal, in practice being free to optimize part geometry saves us time, money, weight and overall system volume. Being able to place material exactly where needed, and omit unnecessary material through key reinforcements and asymmetric load driven shapes, these composite components have met or exceeded metal performance while saving the project development time and money over traditional metal components in key locations.
The electronics box contains laser module, control PC, stage controllers, circuit boards, power supplies and an uninterruptible power supply. Although a Flamingo requires an electronics box to run, a single box can be swapped between Flamingo microscopes.
Each Flamingo has an integrated computer controlling the hardware and coordinating imaging experiments. The researcher connects remotely to the Flamingo to manually control the microscope and to set up experiments. A computer running macOS 10.12 or later is required for this step.
Flamingo image data is streamed from the camera to computer memory and, in a parallel thread, saved to external USB data storage. For reliable imaging sessions we recommend SSDs mounted in a USB 3.1 enclosure.
A Flamingo can generate a large amount of image data even during short experiments. Some examples of typical experiments and the generated data size is shown below. Keep in mind that you might want to reserve additional storage space to save processed images.
|Z-stack with 200 planes and 3 channels||5 GB|
|Multi-view recording with 6 angles, 200 planes and 3 channels||30 GB|
|12 hour time-lapse with 100 planes, 2 channels and 5 min interval||240 GB|
|2 day time-lapse with 2 positions, 150 planes, 2 channels and 10 min interval||1.5 TB|
|2 hour continuous recording with 25 ms exposure time||2.5 TB|
A Flamingo in T-SPIM configuration is about 350 mm in diameter and 500 mm tall. The control box is 480 x 380 x 275 mm in size. Microscope and control box each fit in a Pelican 1660 Protector Case. Additional accessories are kept in a Pelican 1485 Air Case. All cases fit in a compact car with the rear seats folded down.
The Flamingo requires a workspace of at least 90 x 70 cm (35 x 28″). We recommend a workspace of at least 130 x 70 cm (50 x 28″) to have additional space for samples and laptop. The L-/T-/X-SPIM needs about 510 mm (20″) of clearance.