Page Type: Customer Story

NIR and SWIR ImagingCustomer Stories

Prof. Dr. Oliver Bruns

Department of Functional Imaging in Surgical Oncology, National Center for Tumor Diseases Dresden, Germany

Background

The lab of Dr. Oliver Bruns is dedicated to the development of imaging techniques and novel contrast agents in the short-wave infrared (SWIR) and near-infrared (NIR) range. This range uses wavelengths between 700 and 2000 nm (beyond the visible light range), in order to generate high-resolution in vivo images at depths not possible with conventional imaging.

Imaging at longer NIR and SWIR wavelengths can be very beneficial due to less scattering with thicker samples (such as tissue slices or whole animals), reduced autofluorescence, lower energies (less bleaching and photodamage allowing for longer experiments), and less absorption.

One issue with NIR imaging is a lack of usable probes or contrast agents at longer wavelengths, but the lab of Dr. Bruns continually develops new dyes with sensitivity in the NIR and SWIR, with these dyes and NIR and SWIR sensitive cameras, deep multicolor imaging can be performed in vivo on large samples.

Figure 1: Ex vivo histological validation of tumor labeling after injection of FNIR-872-Pan. Samples are 15 mm sections of tumor mounted with DAPI. The Prime BSI was used to detect fluorescence of IR-800CW (at 20x) and DAPI (at 4x) across the whole tumor, images shown together with H&E stained adjacent section in the top row. The second row shows a magnified view of the central necrotic area, with the red square further magnified and shown in the bottom row (all at 20x). Scale bars displayed for IR-800CW-Pan images. Figure adapted from Bandi et al. 2022.

Challenge

Imaging at longer wavelengths can be challenging due to the fact that silicon is transparent at wavelengths longer than 1100 nm, with typical sCMOS cameras sharply dropping in sensitivity beyond 1000 nm. When using silicon-based sensors for NIR imaging, it is vital to have a broad quantum efficiency curve in order to retain sensitivity from 800 nm and above.

In addition, NIR imaging lends itself well to large, thick samples due to the penetrative ability to image at greater depth than visible imaging. This means that large samples are often used and a camera with a large field of view would be most suitable, in order to avoid excessive stitching and tiling to image in vivo samples.

Small pixel size is also desirable to allow for sub-cellular resolution across these larger images at optimized magnifications, allowing for high image quality at scale and fine details at smaller ROIs.

The [Prime BSI] has great sensitivity in the NIR around 900 nm, and performed well in our experiments.
Figure 2: Ex vivo histological validation of tumor labeling after injection of IRDye800CW-Pan. Samples are 15 mm sections of tumor mounted with DAPI. The Prime BSI was used to detect fluorescence of IR-800CW (at 20x) and DAPI (at 4x) across the whole tumor, images shown together with H&E stained adjacent section in the top row. The second row shows a magnified view of the central necrotic area, with the red square further magnified and shown in the bottom row (all at 20x). Scale bars displayed for IR-800CW-Pan images. Figure adapted from Bandi et al. 2022.

Solution

The Prime BSI features a broad quantum efficiency curve, with a peak of 95% and ranging from 200 nm to over 1000 nm, resulting in high sensitivity in the 700-900 nm NIR-I band. Combining this maximized signal collection with minimized noise thanks to the low-noise CMS mode of the Prime BSI results in a highly sensitive solution for NIR imaging.

The Prime BSI uses a small 6.5 μm pixel across a large 19 mm array, resulting in the combination of a large field of view while retaining high resolution at a range of magnifications.

Dr. Bruns recently used the Prime BSI for NIR imaging of murine tumor sections, using NIR dyes such as IRDye 800CW and FNIR-872 (emitting at around 900 nm) in combination with visible dye DAPI. These dyes were used for large format, high-resolution multicolor imaging across thick mouse organ sections in a recent Nature Methods publication.

Reference: Bandi, V. G., Luciano, M. P., Saccomano, M., Patel, N. L., Bischof, T. S., Lingg, J., Tsrunchev, P. T., Nix, M. N., Ruehle, B., Sanders, C., Riffle, L., Robinson, C. M., Difilippantonio, S., Kalen, J. D., Resch-Genger, U., Ivanic, J., Bruns, O. T., & Schnermann, M. J. (2022). Targeted multicolor in vivo imaging over 1,000 nm enabled by nonamethine cyanines. Nature methods, 10.1038/s41592-022-01394-6. Advance online publication. https://doi.org/10.1038/s41592-022-01394-6

Calcium Imaging OptogeneticsCustomer Stories

Dr. Benjamin Rost, Prof Dietmar Schmitz, Prof. Peter Hegemann

German Centre for Neurodegenerative Diseases (DZNE), Charité Berlin, Germany

Background

Dr. Benjamin Rost is a senior scientist at the German Center for Neurodegenerative Diseases (DZNE) in Berlin and describes his research as follows, “I am interested in creating and applying new molecular tools to investigate signaling cascades underlying synapse function. Deciphering these pathways will allow us to understand how neurons communicate in the brain under physiological and pathological conditions. I develop novel optogenetic tools, and by combining optogenetic actuators and genetically encoded fluorescent indicators, I can modulate and image neuronal activity with light.”

In a recent collaboration with Prof. Peter Hegemann from Humboldt University Berlin, Dr. Rost combined electrophysiology and live-cell calcium imaging to characterize newly developed calcium-permeable channelrhodopsin (ChR) variants in hippocampal neurons. Light-sensitive ChRs are vital for optogenetics, and developing ChRs with a selectivity for calcium ions (Ca2+) has the potential to be a highly impactful tool for neuroscience. Dr. Rost and colleagues from Charité and Humboldt Universities have recently published their work in Nature Communications, demonstrating a major step forward for understanding Ca2+ interactions with ChR, and empowering neuroscientists with new tools (Fernandez Lahore et al 2022).

Figure 1: Optogenetics, calcium imaging, and electrophysiology of primary hippocampal neurons, acquired using the Prime BSI Express. Top left: neurons transduced with AAVs encoding ChRs fused to YFP. Top right: Cal-630 is perfused into neurons using a patch pipette. Bot left: Cal-630 baseline fluorescence after 630 nm excitation. Bot right: imaging with 630 and 470 nm flashes to see increased Cal-630 fluorescence indicating robust calcium influx due to ChR activity.

Challenge

The combination of optogenetics and electrophysiology brings unique challenges to this neuroscience application, which Dr, Rost outlines, “We need to perform whole-cell patch clamp experiments using brightfield illumination, followed by imaging of faint calcium signals at a reasonable speed, with minimal noise.”

Optogenetics requires high speed, high sensitivity fluorescence microscopy in order to image dynamic activity, and in this case the transport of calcium ions. On the other hand, electrophysiology benefits from a high-resolution camera with a large field of view, as individual neurons need to be selected from dense networks and carefully patch-clamped with a micro-pipette.

Dr. Rost further explained his experimental process, “We used the calcium indicator Cal-630 with neurons previously transfected with ChRs fused to YFP. This means we used 630 nm light for excitation of Cal 630 and 470 nm for excitation of the ChR, so we needed a camera that was synched to the excitation.”

This application needs a camera that is sensitive enough to image with a high quantum efficiency across a broad range. This is all done with low exposure times, meaning a low read noise is also required in order to capture the signal while imaging at the desired speed.

The [Prime BSI] has great sensitivity in the NIR around 900 nm, and performed well in our experiments.

Solution

The Prime BSI Express is an ideal solution for neuroscience applications, featuring high sensitivity thanks to a combination of 95% peak quantum efficiency that extends over a broad spectral area, and 1 e- of read noise for low noise contributions. The Prime BSI Express can also image at 95 fps for capturing dynamic functional activity, and all across a 19 mm field of view.

The Prime BSI Express also features advanced hardware triggering capabilities, allowing for synchronisation with multiple light sources at different exposure times and with staggered timings. As well as flexible hardware, the Prime BSI Express has flexible software options, with full control available in Micro-Manager.

Overall, the Prime BSI Express is a great option for Dr. Rost’s work, having widespread usage across optogenetics, calcium imaging and electrophysiology, a truly proven solution.

Reference: Fernandez Lahore R.G., Pampaloni N.P., Peter E., Heim M.M., Tillert L., Vierock J., Oppermann J., Walther J., Schmitz D., Owald D., Plested A.J.R., Rost B.R., and Hegemann P (2022) Calcium-permeable channelrhodopsins for the photocontrol of calcium signaling. Nat Commun 13, 7844 https://doi.org/10.1038/s41467-022-35373-4

Plant Calcium ImagingCustomer Stories

Prof. Rob Roelfsema

Molecular Plant-Physiology and Biophysics, University of Wurzburg, Germany

Background

Prof. Rob Roelfsema is studying plant signaling and transport, typically using fluorescent ion sensors to see changes in calcium concentrations during certain conditions. Prof. Roelfsema told us more, “We use Arabidopsis, tobacco, and the Venus flytrap as models to show people that plants are actually sensing organisms, and they are more alive than most people realize.”

“We image the leaves of plants that express an ion sensor, often a calcium sensor, and then we apply stress to the leaf and observe the calcium signaling, which typically moves like a wave through the leaf.”

“With the Venus flytrap we stimulate the trigger hairs twice within 10 seconds and this activates the trap, and with the Arabidopsis, it’s more brutal, we are squeezing or burning the leaves to look at the strong stress signals. It looks brutal but if you think of a caterpillar eating the leaf, it is also brutal, and we want to see what signals the plant sends to defend itself.”

Figure 1: Calcium imaging of plant models using the Retiga E7. On the left is an Arabidopsis leaf after being burned, showing calcium signals propagating from left to right. On the right is a Venus flytrap after being stimulated twice at one of the trigger hairs (seen as bright yellow structures in the trap), and just about to close.

Challenge

Calcium signals are rapid and dynamic, this requires an imaging system that can operate at high speeds while still having the sensitivity to capture the signal.

Prof. Roelfsema described his imaging challenges, “We are imaging calcium waves so we need to capture at about 100 milliseconds or less, but the signal is also in the lower level, and if we reduce the exposure time too much, we drop below the detection level. We also need to consider that plants are very sensitive to light intensity, we are using a UV lamp, and if we are focusing on a single cell the light intensity can be very high.”

Another factor is the field of view, these calcium waves can spread across a whole leaf or section of plant tissue, and the greater the area that can be imaged, the more relevant information can be obtained.

The [Prime BSI] has great sensitivity in the NIR around 900 nm, and performed well in our experiments.

Solution

The Retiga E7 CMOS camera is an ideal entry-level solution for calcium imaging, with the ability to image at 50 fps with low read noise, all with a large field of view.

Prof. Roelfsema explained his experience with the Retiga E7 camera, “We can image the signal moving through the whole leaf tissue like a wave and we couldn’t do that before, our older cameras were much too slow. That’s why we bought this camera, to be able to image these calcium signals in larger tissues at once.”

“There have been cameras that were capable of getting the signal, but they were not affordable, now the Retiga E7 is definitely more in the range of what I could easily afford. Also, setup of the camera in MicroManager was easy.”

High Harmonic Generation Metasurface ImagingCustomer Stories

Dr. Femius Koenderink, Dr. Marko Kamp, Mr. Falco Bijloo

Resonant Nanophotonics Group, Department of Physics, AMOLF, Netherlands

Background

Falco Bijloo is a PhD student in the nanophotonics group of Dr. Femius Koenderink at AMOLF, researching how to generate harmonics and make non-linear metasurfaces.

Mr. Bijloo told us more, “Metasurfaces are basically nano-patterned surfaces, and we build up a substrate with very thin nano-resonators on top, and this is the metasurface consisting of meta-atoms. With these metasurfaces can make lenses that are half a micron thick, or capture certain wavelengths of light and enhance them to generate second and third level harmonics.”

“We have a femtosecond pulse laser at 1200-2500 nm that hits the metasurface sample, we then have a collection objective in transmission and an infrared camera to capture at 1500 nm, we require another visible light or UV camera to capture the harmonics, which can be down to 500 nm at the third harmonic, or further into the UV at greater harmonics.”

“My research is about what kind of photonic and non-photonic processes are happening in these nanostructured metasurfaces, we need to image at a wide range of wavelengths.”

Figure 1: The top schematic shows the third harmonic generation Fourier microscope light path, including an IR
camera, spectrometer, and the Prime BSI Express sCMOS camera on the far right for the harmonics. The bottom
images are the second harmonic generation inspection of 2D materials taken with the Prime BSI Express.

Challenge

Mr. Bijloo described the challenges he faces, “The signal drops down a few orders of magnitude per harmonic, I think the conversion efficiency is 1×10-7 for the third harmonic, and we are interested in even higher harmonics. While we pump an intense amount of energy into the system the harmonics are very low signal and we need a sensitive camera to capture the photons.”

“For some experiments, we want to use an initial wavelength of 650 nm, this means the second harmonic is around 320 nm and that is really into the UV, which we can’t capture with our other cameras. We need a broad OE curve to capture several harmonics in the visible and UV.”

This application requires sensitivity above all, needing a camera with low noise levels and a high quantum efficiency across a broad range of wavelengths, particularly the UV and visible regions.

The [Prime BSI] has great sensitivity in the NIR around 900 nm, and performed well in our experiments.

Solution

The Prime BSI Express CMOS camera is an ideal solution for this application, featuring ultra-high sensitivity thanks to a consistently low read noise (one electron or less in CMS mode) and a near-perfect 95% quantum efficiency peak 500 nm, stretching from 200-1100 nm, covering the UV, visible and near-infrared regions.

Mr Bijloo told us about his experiences with the Prime BSI Express, “The reason we chose this camera is that a large part of the sensitivity is into the UV. We needed a very broad OE curve to capture not only the main wavelength, but several harmonics worth. Our previous camera didn’t capture UV light and that is the most important part of the spectrum for us.”

“I enjoy using the camera, it was easy to set up on our system and it’s a big improvement for us.”

Optogenetic Voltage Clamp ImagingCustomer Stories

Prof. Alexander Gottschalk, Ms Amelie Bergs

Buchmann Institute for Molecular Life Sciences, Goethe University, Frankfurt, Germany

Background

Prof. Alexander Gottschalk and his team at Goethe University study the nervous system of the model organism nematode worm Caenorhabditis elegans, specifically mechanisms of transmission at chemical synapses and function of single neurons within the neuronal networks.

We spoke with PhD student Amelie Bergs, “Our research is focused on C. elegans and optogenetics, particularly developing a new method we call optogenetic voltage clamp or OVC, which combines the advantages of imaging with the control capabilities of electrophysiology and voltage clamping.”

“We use a tandem protein consisting of depolarising and hyperpolarising tools fused together, and we combine this with a genetically encoded voltage indicator, specifically QuasAr2. This shows dim fluorescence depending on the membrane voltage of the cell, which we can keep within a desired level by adapting the wavelengths of our light source, so we clamp the fluorescence and the voltage. OVS is a purely optical approach we are using on specific muscle cells of immobilized C. elegans.“

Figure 1: Optogenetic voltage clamp (OVC) of a C. elegans muscle cell, showing the native open and closed morphologies, and then the forced action potential suppression via OVC control. Acquired with the Kinetix22 across an approx. 30×30 pixel ROI.

Challenge

Voltage signals, even those in the slower firing C. elegans, are rapid and dynamic, requiring the need for high-speed imaging and good sensitivity at low exposure times. Ms Bergs told us more, “We have restrictions in the speed as our GEVI is also rather slow, and we have restrictions from the software side. In the future, we could increase the speed here, but this would be at 1 ms exposure and we would have far fewer photons per frame, so we need high sensitivity to do this.”

“We were previously using an EMCCD, it was difficult to perform clamping due to vibrations from the fan, so we also need a camera that can be water-cooled with no vibrations.”

With custom software scripts for running OVC in Micro-Manager as well, any new device should be compatible with Micro-Manager software for ease of setup and use.

The [Prime BSI] has great sensitivity in the NIR around 900 nm, and performed well in our experiments.

Solution

The Kinetix22 is an ideal solution for this application. Featuring high speed. high sensitivity, water cooling, and great software flexibility. The four main operating modes of Sensitivity, Speed, Sub Electron, and Dynamic Range allow for optimizations to be made, whether performing OVC on a small section or imaging an entire nematode worm.

Ms Bergs outlined her experience with the Kinetix22. “With the water cooling there were no vibrations and it makes quite a difference. we got a lot more measurements done. We use the Dynamic Range mode because we have an intense laser signal.”

“It’s even more sensitive and faster. we can even increase the speed in the future. Some issues we had before weren’t a problem anymore. I think the photon count went up too. Of course, in parallel, you’ve got a higher resolution and the camera is future-proof and flexible for other experiments we can do … I’m very pleased with the Kinetix22, I had no issues using it.”

Reference:

A.CF. Bergs, J.F. Liewald. S Rodriguez-Rozada, 0 Liu. C Wirt, A Bessel, N. Zeitzschel. H. Durmaz. A Nozownik. J. Vierock. Cl. Bargmann. P. Hegemann. J.S Wiegert. A. Gottschalk (2023) All-optical closed-loop voltage clamp for precise control of muscles and neurons in live animals. Nat Commun 14, 1939 {2023) https://doi.org/10.1038/s41467-023-37622-6

High-Speed Retinal Voltage ImagingCustomer Stories

Dr. Guilherme Testa-Silva

Institute of Molecular and Clinical Ophthalmology Basel (IOB), Switzerland

Background

Dr. Guilherme Testa-Silva is head of Physiological Technologies at 1OB, part of a group committed to developing groundbreaking therapies for restoring sight.

Dr. Testa-Silva told us more, “We draw from areas such as molecular biology, clinical science, and physiological technologies. Our work includes understanding how the eye and the visual system function at a molecular and cellular level, and how alterations in these systems lead to vision loss. The ultimate aim is to develop new therapies that can restore sight, improve the quality of life for those with vision impairments, and contribute to the overall advancement of ophthalmic science.”

“To support our work, we utilize a range of state-of-the-art imaging technologies, including cameras for imaging retina structures; adaptive optics imaging; and two-photon microscopy for observing cellular and tissue properties in the eye. Furthermore, we continue to explore the application of genetically encoded voltage sensors, which allow us to study the electrical activity of excitable cells, giving us greater insights into the mechanisms of sight at a microscopic level.”

Figure 1: The Kinetix sCMOS camera in situ on the retinal voltage imaging system

Challenge

“Low-light levels also pose a challenge. Given that certain procedures must be conducted under minimal light conditions to prevent phototoxicity or because the photoreceptor cells in the retina are sensitive to light, our cameras need to be highly sensitive to capture quality images in these conditions.”

“Lastly, there’s the challenge of imaging depth. To fully understand the complex, multi-layered structures of the eye, we require cameras capable of capturing images at varying depths, which can be quite a technological hurdle.”

“These challenges can significantly affect our imaging work. The inability to capture large fields of view might limit our understanding of the overall retinal structure and cellular interactions. Similarly, cameras with inadequate speed could miss rapid physiological events, reducing the accuracy of our data. Subpar sensitivity in low-light conditions might compromise image quality, while limitations in imaging depth could restrict our knowledge of deeper retinal structures.”

The [Prime BSI] has great sensitivity in the NIR around 900 nm, and performed well in our experiments.

Solution

The Kinetix sCMOS is an ideal solution for this application, answering the need for large field-of-view imaging at high speeds in low-light conditions. Imaging at 500 fps across the entire 10 megapixel, 29 mm sensor, the Kinetix is well- suited to capturing events across large, dynamic samples.

Dr. Testa-Silva described his experience with the Kinetix, “The Kinetix camera has been an invaluable tool in addressing some of our primary imaging challenges. With its larger sensor size, it has greatly enhanced our field of view, allowing us to capture a comprehensive image of the retina in a single frame. Its high pixel resolution has also helped us obtain detailed images of individual cells and intricate structures.”

“Our experience with the Kinetix has been largely positive. The camera’s high speed has been crucial in capturing rapid physiological events. Its high quantum efficiency has proved advantageous in low-light conditions, enabling us to acquire high-quality images even in minimal light.”

Fluorescence and ElectrophysiologyCustomer Stories

Dr. Christian Simon, Mr. Florian Gerstner

Carl-Ludwig-Institute for Physiology, Leipzig University, Germany

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.”

Figure 1: Various DIC and fluorescence images taken using the Moment CMOS. Top left shows a DIC image of electrophysiology, with patch-clamp micro-pipettes approaching a Purkinje cell for stimulation. Top right shows proprioceptive synapses labelled with GFP. Bottom left and right show a neuromuscular junction and some motor neurons respectively, both labelled with tdtomato.

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.

The [Prime BSI] has great sensitivity in the NIR around 900 nm, and performed well in our experiments.

Solution

The Moment CMOS is a high-speed, easy-to-use camera with high sensitivity in both the visible and near-infrared regions. With a small 4.5 µm pixel, the Moment is well suited to both low-magnification localization and high-magnification sub-cellular imaging.

Mr. Gerstner described his experience with the Moment, “It’s easy to use, since with Micro-Manager its plug and play with the single cable, it’s a neat way to do things. It’s been a comfortable camera to use, that produces quality images with a good time resolution. It works perfectly with great resolution at both of our magnifications. We didn’t have any problems.”

Dr. Simon told us more, “[The Moment] improved our approach since we can do everything within one software. With our previous camera fluorescence was horrible, but now even with thick samples we are all very happy.”

Neuromuscular Junction Vesicle ImagingCustomer Stories

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.”

Figure 1: A video of vesicle fusion events across an entire Drosophila larvae NMJ at muscles six and seven, acquired using the Kinetix22. The video shows two motor neurons innervating the NMJ, with the flashes being post-synaptic glutamate receptor responses triggered by spontaneous vesicle fusion events.

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.

The [Prime BSI] has great sensitivity in the NIR around 900 nm, and performed well in our experiments.

Solution

The Kinetix22 is the ideal solution for this application, and Prof. Pielage explained his experience with the Kinetix22, “This system was designed for the Kinetix22, the improvement in temporal resolution and sensitivity is critical. We were previously just at the detection limit but now it looks nice, we were surprised that we can see our signal. We can also look at the kinetics of these events, this is really a great tool and we compared it to competitors, the Kinetix really was the best for our application, it’s the perfect camera.”

“These experiments weren’t possible beforehand, now we can see miniature release events within the NMJ, something that we cannot achieve using electrophysiology. These optical recordings dramatically improve the dimensions of our research, and if we then correlated directly with electrophysiology and immunochemistry, we can really gain novel insights into these processes.”

“It works well in MicroManager, and the setup was very easy, it works from the get-go.”

Sub-Cellular Neuronal Calcium ImagingCustomer Stories

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.”

Figure 1: A video of primary hippocampal neurons expressing a pre-synaptic jGCaMP8f calcium indicator. Images show a whole neuronal network and the calcium activity after stimulation, where each dot is a single synapse. Video acquired with Kinetix22 in Sensitivity mode at 88 fps, 10 ms exposure time.
Figure 2: A Z-plot of activity over time, based on the samples in Figure 1. The graph shows a quiescent period in the beginning, followed by spikes of activity across the whole neuronal network, increasing in intensity.

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.

The [Prime BSI] has great sensitivity in the NIR around 900 nm, and performed well in our experiments.

Solution

The Kinetix22 is an ideal solution for this application, featuring all the benefits of the Kinetix family of cameras while maximizing the FOV of the Zeiss microscope. Mr. Jarzembowski told us about his experience with the Kinetix22, “We tested the Kinetix22 on a wide range of different calcium sensors, such as GCaMP and glutamate signalling, all the way up to over 500 fps and it worked perfectly fine, all with a field of view that is very large, we can get the whole neuron on it which is what we need.”

“We are using Zen for our imaging; in our department we have lots of different users so the flexibility of Zen is critical. Hardware setup was simple with the Kinetix22 on our Zeiss system, we used a T-Mount adapter and this gives us the full frame of view, very useable and homogenous illumination with different light sources, no problem at all. Overall, the [Kinetix22] is working very well.”

Simultaneous Two-Colour TIRF smFRETCustomer Stories

Prof. Richard Börner, Mr. Anxiong Yang

Laserinstitut, Hochschule Mittweida University, Germany

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.”

Figure 1: RNA molecules immobilised with BSA in HW. imaged with two-colour (Cy3 and Cy5) smFRET on a TlRF microscope using the Prime BSI Express sCMOS with a beam splitter. Each set of double images was captured simultaneously by splitting the channels across the sensor.

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.

The [Prime BSI] has great sensitivity in the NIR around 900 nm, and performed well in our experiments.

Solution

The Prime BSI Express CMOS camera is an ideal solution for this application, with high sensitivity thanks to a combination of low-noise CMS mode and 95% quantum efficiency for signal collection. The Prime BSI Express acquires at high speed even in CMS mode. and has a sufficiently large sensor to work well with a splitter and capture two channels simultaneously with thousands of molecule events across each.

Mr. Yang described his experience with the Prime BSI Express, “Your camera sensor is big enough to capture a really big area from our sample, the background signal and noise levels are very low; we can also control the temperature using the fan, this is important for our experiments with fluorescent light detection.”

“We are using Micro-Manager open-source software to run the whole system. and it works great with your camera and our lasers. I can set everything up in the software. The camera triggers also work great with our lasers and system. We have also 3D printed a camera holder for the Prime BSI Express] to match the height of our detection beam pass. as we made the system with this camera in mind.”