Background

Dr. Christian Simon and PhD student Mr. Florian Gerstner are involved in neuroscience research, in particular investigating spinal cord sections and motor neuron functionality in mouse models.

Dr. Simon described his work, “For the spinal cord, if you break it into small sections, you cut off all the dendritic trees and this doesn’t really work, we are using a new technique where you divide the spinal cord into very thick sections, where you can visually target the surface motor neurons and still have the circuitry intact.”

Mr. Gerstner told us more, “We are doing a lot of electrophysiology on these thick samples, primarily patch clamp recordings, but we are also doing fluorescence and DIC microscopy to target certain cells and check their viability. We are using GFP and Atto dyes towards red.”

Challenge

For thicker samples a high sensitivity camera is vital due to the scattering that occurs for both DIC and fluorescence, as well as high sensitivity across a range of wavelengths, considering the use of traditional visible GFP and another dye further towards infrared that better suits thick samples.

Dr. Simon had a previous camera that lacked sensitivity, “With our old camera the sensitivity wasn’t sufficient, especially in these thicker tissues and in DIC. We are targeting cells beneath the surface, so we need good image quality, we have to image the layer beneath to get more intact cells.”

Mr. Gerstner outlined more challenges, “We are using two types of objective, a 10x to locate the area we want to see, then for patching we use a 60x in order to target individual cells. Our previous camera imaged at around 30 fps which was low for us, we need more speed in order to get the patch pipette to the desired cell.”

Overall, this application requires high and broad sensitivity, as well as high speed and a pixel size suited to high-resolution imaging at a range of different magnifications.

Prof. Jan Pielage

Department of Biology, RPTU University of Kaiserslautern-Landau, Kaiserslautern, Germany

Background

Professor Jan Pielage’s research focuses on identifying molecular mechanisms controlling synapse formation, function, and stability. Prof. Pielage told us more, “We use Drosophila models to identify novel genes that are required to maintain the stability or function of the neuromuscular junction (NMJ), the connection between nerve and muscle. We have identified a number of genes, and these are conserved and can lead to disabilities in mouse or human models.”

“We want to look at neurodegeneration across a whole Drosophila NMJ and see how the signal pattern changes, to learn more about potential compensatory mechanisms and disease progression.”

Challenge

Prof. Pielage described some of the challenges this application involves, “We want to monitor potential defects in synaptic transmission at the resolution of single active zones in the NMJ, for this, we require extreme sensitivity as the signal is very low.”

“We also need high-speed imaging to identify these events in real-time, this is in the range of 10-100 Hz, but we also want to image voltage-sensitive dyes, and for this, we would need to go up to 1000 Hz. So, we need to really push the potential of the camera.”

“We also want to image the entire NMJ, it is critical to understand which release sites are active. Previously we relied on electrophysiology and could detect release events, but didn’t know where they occur. For this, we need optical methods.”

This application needs a combination of high speed and high sensitivity across a large field of view, in order to observe all the events across an entire Drosophila NMJ at sub-cellular resolution.

Lukas Jarzembowski, Prof. Barbara A. Niemeyer

Centre for Integrative Physiology & Molecular Medicine (CIPMM), Saarland University, Homburg, Germany

Background

The lab of Prof. Barbara A. Niemeyer at CIPMM is interested in the molecular mechanisms underlying the regulation of calcium (Ca2+) signals in physiology and disease. The lab uses Zeiss microscopy systems, where Zen software is critical for the multi-user environment.

We spoke with a PhD student from the lab, Lukas Jarzembowski, “My research focuses mostly on the mechanisms of how calcium enters the cell, specifically how this is dysregulated in neurons and in the pre synapse for neural transmission. I am studying this at a single synapse level with primary hippocampal neurons using imaging and optical physiology tools.”

“One calcium entry route is through ion channels which interact with the endoplasmic reticulum (ER), I am using GCaMP fluorescent calcium sensors targeted to different cellular compartments to understand the role of this pathway in neural transmission.”

Challenge

Mr Jarzembowski further described the imaging challenges of this application, “We are using a low light dose with our live primary neural cells, so the signal from GCaMP is quite low. Also, the pre-synapse is already quite tiny, and in the ER the pre-synapse is even tinier, so it is a very small volume containing very little GCaMP. Responses from individual synapses are also unpredictable, so I need to be able to record very faint signals from as many synapses as possible.”

“Ideally, I want to image at around 40 fps across the full FOV, but in the future, I may want to perform voltage imaging, which requires a much faster acquisition speed. As for resolution, I am looking for calcium signals in sub-cellular compartments, we have a Prime 95B on our Zeiss system which works perfectly fine, but the pixels are a little too large for our magnification and we want higher resolution, so we need a smaller pixel size.”

“We also have a Photometrics Evolve EMCCD and while it was sensitive, the FOV was way too small and it doesn’t make sense for me to do any experiments there.”

To this end, this application requires a camera that can maximize the field of view of the Zeiss imaging system, while also having high sensitivity in order to capture this very faint signal across the field at a sufficiently high speed, with sub-cellular resolution. Futureproofing this system for high-speed voltage imaging is also desirable, requiring a camera that can operate at 1000 fps or 1 kHz.

Background

The lab of Prof. Borner is involved with studying the dynamics of RNA molecules using a range of single-molecule imaging techniques, including single-molecule Forster resonance energy transfer (smFRET) and total internal reflection fluorescence (TIRF) microscopy.

Anxiong Yang from Prof Borner’s group has built a custom two-color smFRET imaging system in a TIRF microscope that uses stroboscopic alternating laser excitation (sALEX) to monitor thousands of RNA molecules in parallel, with time resolutions down to 1 ms.

Mr. Yang further explained his imaging work, “We immobilise molecules onto a coverslip with two dyes, Cy3/5, fluorescence-labelled molecules. Then we use a microfluidic system to adjust the buffer conditions (salt concentration, pH, metabolite concentration) within the sample chamber, and image these surfaces with our TIRF microscope to observe dynamic and optimise or automate the immobilisation process.”

Challenge

Mr. Yang told us about the challenges of these experiments, “We want to capture a big area of our sample to get as much data as we can, because sample preparation is complex and very time-consuming. We want to get more information from our samples.”

“We split the fluorescence emission light in two channels, green and red, and want to image them using each half of the camera sensor. Also, we are using an alternating laser excitation (ALEX) box and we use the TTL output trigger signal of the Prime BSI Express sCMOS to digitally modulate our two laser sources.”

If splitting the sensor to simultaneously image two channels, a camera sensor will be halved in size, meaning a large FOV is paramount to maximize data capture even when reduced in size. Alongside this, this research requires advanced external hardware triggering capabilities in order to control two light sources with alternating excitation.

Dr. Davide Raccuglia

Institute of Neurophysiology, Charité University Berlin, Germany

Background

Dr. Davide Raccuglia and his team use the model organism Drosophila melanogaster (colloquially known as fruit fly) to study how the brain regulates sleep. Dr. Raccuglia told us more about his research, “What I’m particularly interested in is the functional neural architecture of sensory gates for sleep regulation. We investigate how neural networks interact to create a gate that suppresses sensory processing allowing us to fall and stay asleep. Of course, we also want to understand how sensory information can break such a gate in order to awaken and enable us to react.”

“A great advantage to using Drosophila is that we can target specific neural networks, down to the level of a couple of neurons. We express genetically encoded voltage indicators (GEVIs) in these networks to optically derive the membrane potential of these structures. Using voltage indicators of different colors, we can record simultaneously from different neural networks to study how these networks interact when the flies are tired or rested.”

Challenge

Voltage imaging is a challenging technique due to the imaging speeds required, as Dr. Raccuglia mentioned, “For neural signaling that we consider slow, we record at 100 Hz. However, we also look into single neuron activity and want to resolve spikes during bursts, which requires recording speeds between 1-2 kHz. So, we need a camera that performs well at high-frequency rates, and is also very light sensitive.”

When imaging at high speeds the camera exposure time is limited, meaning that only very light-sensitive cameras can collect enough signal for each frame, making voltage imaging a combination of speed and sensitivity.

Dr. Raccuglia also images neural activity across different scales, “We measure, for example, the entirety of presynaptic terminals of a neural network, and this compound signal would be comparable to a network-specific local field potential. However, to determine the contribution of single cells we use GEVIs to perform multi-cellular optical electrophysiology.”

This requires a camera with a large field of view and small pixels, in order to be flexible enough to image across large samples and to focus on the single-cell level.

Background

Dr. Isaac Kauvar is a neuroscientist and engineer, developing tools in order to discover how cortex-spanning neuronal populations support the deployment of internal models during goal-directed behavior.

In order to track and analyze these large-scale activity patterns in the cortex, Dr. Kauvar and graduate student John Kochalka use conventional widefield imaging as well as advanced fluorescent imaging known as ‘cortical observation by synchronous multifocal optical sampling’ (COSMOS) in order to image widespread activity via a transparent window into a mouse brain.

Dr. Kauvar and the team found the need to build a new imaging system for COSMOS due to user demand.

Challenge

In order to measure activity across a dense neuronal sample, both a large field of view and high resolution is needed. Imaging across a tissue while trying to identify individual cells requires a large sensor size in order to image efficiently without excessive stitching/ population tiling, and a small pixel size in order to achieve sub-cellular resolution and pick out which cells are active at what time, across the tissue.

The neuronal activity also occurs on a very short timescale and requires fast detectors, whether using optogenetics or calcium/voltage imaging. This means that a suitable detector also needs to operate at a high speed while still retaining the large field of view.

In order to achieve these high speeds and still suitably detect signal, a highly sensitive detector is needed, especially in order to detect weak signals when imaging at high speeds and having a low exposure time.

Challenge

Dr. Gebbie is using a range of different microscopy and spectroscopy techniques to interrogate surfaces and materials, mainly interferometry, optical spectroscopy and highly-sensitive fluorescence spectroscopy, similar to single-molecule imaging methods. Each of these techniques comes with its own challenges, as Dr. Gebbie explained.

“For both interferometry and optical spectroscopy, signal sensitivity is a challenge we have to think about. The more sensitive we can be, the higher framerates we can access. We are aiming for these short acquisition times in order to do high frame rate interferometry.”

“Frame size is also very important for interferometry; we need to be able to image across the full width of the sensor to utilize all our diffraction gratings and the fringe splitting they produce. The number of pixels between two adjacent fringes is very important, as with interferometry we are targeting resolutions down to 3 angstroms (Å), this is approaching the size of a water molecule.”

“With our fluorescence microscopy methods, we need high-performance detectors in order to be confident about the fluorescence shifts that we see. We want to map lithium mobility in ionic liquids, and the detector is vital to see how low a concentration of lithium we can detect. We want to put in the minimal amount of fluorophore and remain highly sensitive to optical shifts.”

Background

Prof. Matthias Weiss and PhD student Ivana Jeremic research challenging problems at the interface of physics and biology, focusing on understanding self-organization processes in living organisms. Prof. Weiss told us about his latest project, “We have built a new light sheet microscopy system for imaging dynamic processes within large samples, with Ivana’s project focusing on the embryogenesis of transgenic nematode Caenorhabditis elegans until gastrulation. Early C. elegans embryos seem to work on autopilot in terms of self-organization, and we have been successful already in monitoring mechanical cues that drive cell positions until gastrulation. With these data, we were able to even predict cell positions and migration paths via a computational model.”

“Now we want to dive a bit deeper and get information on how cells structure themselves internally before undergoing division, so we can learn more about individual steps during embryogenesis that has been missing in our analytical predictions so far.” Prof. Weiss and team are using an inverted SPIM (iSPIM) light sheet imaging system in order to observe cell behavior within C. elegans samples throughout development.

Challenge

While light-sheet microscopy is well-suited to imaging large samples with relatively low illumination levels, the light sheet itself requires fine-tuning for the best results. Prof. Weiss told us more about his imaging challenges, “We want a light sheet that is long and thin enough so that we can get the full volume of the worm embryo, which is about 50 μm in diameter. Bleaching is an issue as excess light can poison the embryo. If we used something like laser scanning confocal, the embryo would never go beyond the four-cell stage and would die, so the light sheet with low laser illumination is ideal as we can even observe hatching.”

“Some of the fluorescent protein constructs within the sample change their expression during embryogenesis so therefore we have to be able to capture signals from low to high intensity without having to change camera modes, so we need a high dynamic range.”

Alongside a sensitive camera with a high dynamic range, this project is also best suited to a detector with a large sensor and a small pixel in order to capture the maximum resolution across the largest field of view.

Background

The lab of Prof. Zhenyu Gao at the Erasmus University Medical Center studies how the brain controls motion, learning, and memory. In order to study these functions, Prof. Gao’s lab uses in vivo methods to detect electric signals in the brains of mice models. This is achieved by either utilizing electrophysiological methods or fluorescent optical methods, or a combination of both.

To visualize activity in the cortex of the mouse brain, voltage imaging can be used. This imaging technique provides an optical readout from fluorescent voltage indicators, which is an incredibly direct method of determining neuronal activities. Voltage imaging experiments are refined in the lab of Prof. Daan Brinks who collaborates tightly with Prof. Gao’s lab.

Challenge

In order to detect voltage signals in neurons, some key criteria need to be fulfilled. As imaging frequency needs to be in the range of 1-2 kHz (1000-2000 fps) in order to be able to precisely describe individual action potentials, a camera is required which is capable of this recording speed. Because of the high frame rate required, signal levels per frame will be very low, which requires a camera that has a very high sensitivity from the quantum efficiency point of view and also a low enough noise level to reliably detect even minute changes in the signal. Only by maximizing signal collection and minimizing noise contributions can a camera detect signals at low enough exposures (less than 1 ms) to operate at 1 kHz or more.

Signal levels of the currently used Archon voltage indicator reports signals from the soma (cell body) of neurons. While signal levels in electrophysiological methods can resolve even very low signal levels with high temporal resolution, voltage indicators work on the basis that their reported signals sometimes are only encoded in 1-10% increases (or decreases) in their baseline signal. These small fluctuations in signal need to be accurately collected and analyzed in order to determine neural function from voltage imaging data.

Challenge

Calcium imaging and live-cell imaging both come with their own challenges, requiring a camera that is sensitive enough to obtain a signal while also maintaining a fast imaging rate. In order to acquire images quickly enough to observe calcium activity a short exposure time is necessary, which in turn reduces the time available to collect signal, resulting in a low signal level. Cameras for this application would need to maximize signal collection and minimize noise levels in order to get a high signal-to-noise ratio while imaging at speed.

For Prof. Pei’s calcium imaging experiments, Fura-2 fluorescence imaging was performed using a Zeiss Axiovert microscope equipped with two filter wheels and an sCMOS camera. With excitation at ~350 nm and emission at ~500 nm, another challenge is using a camera with high sensitivity at a wide range of different wavelengths of light.