Laboratory of Neural Dynamics and Cognition, The Rockefeller University
Background
Dr. Priya Rajesethupathy and colleagues in the Rockefeller University Laboratory of Neural Dynamics and Cognition are interested in the process of memory. Integrating approaches ranging from genomics, transcriptomics, optogenetics and imaging, they aspire to address memory formation and recall at scales ranging from the synapse through to collections of hundreds to thousands of neurons and finally animal behavior.
The hippocampus region of the brain has long been thought as the place where memories form, but Dr. Rajesethupathy recently showed that a bundle of neurons that connect the hippocampus to the pre-frontal cortex is important for retrieving memories
Figure 1Left Diagram of a mouse brain with red and green activity sensors in two different cell populations whose axon bundles are projecting to the same downstream brain region. Fiber optic cannulas are used to collect signals from the cell bodies, as well as the nerve terminals.
Right Plot of the change in fluorescence over the initial fluorescence acquired with the Prime 95B from the nerve terminals, which previously had been too weak to detect reliably by photometry, is illustrated. Using the Prime 95B, dF/F values reaching 10% or greater can reliably be detected.
Challenge
Dr. Rajesethupathy’s lab often measure neuronal activity of deep brain regions in a live mouse behaving in a virtual 3-dimensional world. To obtain neural activity information from select cell bodies and nerve bundle termini, fiber optics are implanted to deliver excitation light and recover fluorescence changes from neural activity sensors in brain regions of interest.
Collecting signals from cell-bodies can be easier than collecting signals from nerve bundle terminals. The sensor they use is cytoplasmic and because nerve terminal bundles contain less volume, they contain less sensor. This means they tend to have signals that are too weak to detect by traditional PMTs and sCMOS sensors. They also aim to record signals at speeds relevant to neuronal activation, which can be as fast as milliseconds.
The low noise and high quantum efficiency of the Prime 95B allows us to get good signal to noise and fast sampling from even sparsely innervated nerve termini
Solution
The Neural Dynamics and Cognition lab now uses the Teledyne Photometrics Prime 95B sCMOS camera to collect the light through fiber optics from various regions in the brain.
Fibers collect light from characterized regions but give up spatial information in the process. Previous camera solutions allowed good signal to noise and high temporal precision from fibers implanted near groups of cell bodies, but not from regions rich in axonal synapses.
Dr. Rajasethupathy told us “Using the Prime 95B we collect these weak signals from nerve terminals very efficiently with good signal to noise (as shown in Figure 1). This reveals new biology that we are excited to explore.”
Director of the Group of Bio-Photonics and Microfluidics Technology Huazhong University of Science and Technology
Background
Dr. Fei Peng’s lab at the Huazhong University of Science and Technology is interested in the development of new technologies for life science. Some of the fields that they focus on include developmental biology, tissue engineering and regenerative medicine.
One of their research interests is the imaging of cleared samples with light-sheet illumination. Dr. Fei told us, “We want to image the whole mouse brain with high throughput and high resolution.”
Figure 1. 2D view of Thy1-GFP labeled rat brain acquired with the Iris 15 Scientific CMOS camera using light sheet microscopy
Challenge
Cleared samples are typically very large which means that low magnification is often necessary to image the entire sample. However, this comes at the cost of resolution.
The current sCMOS camera used in Dr. Fei’s lab has an 18.8 mm diagonal field of view and 6.5 µm pixels. However, they would prefer a camera with a larger field of view, higher resolution and high quantum efficiency.
Dr. Fei explained, “The main challenge in our research is how to balance the trade-off between field of view and axial resolution.”
The low noise and high quantum efficiency of the Prime 95B allows us to get good signal to noise and fast sampling from even sparsely innervated nerve termini
Solution
With the larger, 25 mm chip size provided by Iris 15, Dr. Fei could use higher magnification and get the same field of view of the sample.
Dr Fei told us, “This extra magnification combined with the smaller 4.25 µm pixel size helps us to achieve much higher resolution. At the same time, the Iris 9 and Iris 15 have a high enough quantum efficiency that we also don’t need to use too much laser power.”
Dr. Fei continued, “The Iris 15 has a very efficient field of view for mouse brain imaging with high magnification without stitching. For some other samples, the field of view of the Iris 9 is also enough. Furthermore, both of the cameras can maintain a high frame rate of up to 30 frames per second (fps), which helps a lot when chasing high throughput.
Super Resolution Bacterial ImagingCustomer Stories
Dr Seamus Holden
Centre for Bacterial Biology. Institute for Cell and Molecular Biosciences Newcastle University
Background
Dr. Holden’s research lies between biophysics and microbiology, using super-resolution microscopy to study basic principles of bacterial spatial organization. In particular, the Holden Lab focuses on how the Gram-positive model bacterium Bacillus subtilis divides, and how the bacterial cytoskeleton guides the construction of a mid-cell cross-wall or septum.
The Lab is currently working to understand these biological processes using novel methods based on microfabrication, microfluidics and single molecule and super-resolution microscopy.
Figure 1 Comparison of live cell imaging of FtsZ ring organization during bacterial cell division using a Prime BSI and an EMCCD camera. Images of FtsZ-mNeonGreen fluorescence in live B. subtilis, immobilized vertically in nanofabricated chambers, excited at 488 nm by HILO illumination, were acquired sequentially with a 100× objective using the same effective pixel size on each camera. Each image minimum value cropped to camera baseline of 100. Scale bar, 1 µm.
Challenge
Bacterial cell division takes place below the diffraction limit of microscopy, which is why the Holden Lab uses super-resolution and single molecule imaging to visualize aspects of the bacterial division process, such as the treadmilling dynamics of the bacterial cytoskeleton protein FtsZ during cell division.
The Lab’s work depends on imaging either the dynamics of cytoskeletal protein filaments using only a few fluorescent protein labels on each filament, or individual cell wall synthesis enzymes in live bacteria samples at low illumination power. This combination of low fluorophore density and low illumination intensity means that they require a scientific camera to be sensitive and have a low noise profile.
The low noise and high quantum efficiency of the Prime 95B allows us to get good signal to noise and fast sampling from even sparsely innervated nerve termini
Solution
The Holden Lab is now using the Teledyne Photometrics Prime BSI back-illuminated sCMOS to investigate how B. subtilis divide.
To make this decision, Dr Holden compared the Prime BSI to an EMCCD camera and was impressed with the equivalent level of sensitivity. He told us, “SNR levels are pretty indistinguishable for a tough sample of mNeonGreen-FtsZ in live cells at low illumination power.”
Dr Holden went on to say that, compared to an EMCCD, “[the Prime BSI], is faster and offers a larger field of view with 2048×2048 pixels. The thing that really impressed me is how uniform the sensor is – far fewer hot pixels, noisy pixels and stripes than the last generation of sCMOS cameras. I hardly see a use for EMCCDs anymore.”
The Attwell lab is interested in understanding the interactions that occur between neurons, glial cells and the vasculature of the brain through the use of electrophysiology and imaging techniques.
For years it was believed that brain blood flow, which provides the energy used for neural computation, was controlled solely by constriction and dilation of arterioles in the brain. However, the Attwell lab have since shown that contractile cells called pericytes, located at 30-micron intervals along brain capillaries, also play a major role (Hall et al., 2014, Nature 508, 55; Mishra et al. 2016, Nature Neuroscience 19, 1619).
Capillaries are extremely small (around 4-5 microns in diameter) and therefore red and white blood cells need to change shape in order to pass through them. It is these changes in shape that the Attwell group are interested in imaging.
Figure 1 Pictures taken 1 ms apart with the Prime BSI, showing the passage along a capillary (in an anaesthetised mouse brain) of red blood cells (RBCs; hazy black objects in the middle of the capillary, with orange arrows pointing at them – the bottom RBC moves out of the field of view during the time between the pictures). The Attwell group are interested in how the RBCs interact with, and have their movement affected by, the processes of contractile cells called pericytes which sit on the capillary wall (a pericyte is outlined in red).
Challenge
The problem with imaging such changes of red and white blood cell shape is that the cells themselves move very quickly (~1mm/sec). They are also only 5 microns across, so they pass any given point in only a few milliseconds. They also change shape very quickly. Consequently, to capture images of the changes of shape, a rapid and high sensitivity imaging camera is needed.
The low noise and high quantum efficiency of the Prime 95B allows us to get good signal to noise and fast sampling from even sparsely innervated nerve termini
Solution
The Attwell lab required a high sensitivity and rapid acquisition camera, and thus are now using the Teledyne Photometrics Prime BSI with their imaging system.
Prof. Attwell told us that, “Based on prior interactions, we selected Teledyne Photometrics as a reliable source of information on this and ended up purchasing a Prime BSI for our imaging demands.”
Prof Attwell went on to say that, “The Prime BSI is ideal for our purposes. By aligning a capillary with the camera image x-axis and choosing a smaller field of view in the y-axis, we can acquire images at ~1kHz, allowing us to image changes of the shape of the cells as they pass any given point in the capillary network. The camera has allowed us to acquire images that would otherwise be impossible to obtain, providing a basis for future analysis of how the properties of capillaries, and of red and white blood cells, determine brain blood flow”.
