FAQs

We have a Luxendo image registration and fusion tool as a separate software application that allows to register and fuse in the simplest case two opposing view stacks (as acquired with our two cameras) but also opposing view stacks taken from different angles (thus at least 4 stacks). And then the fused image stack can be imported into Imaris or Arivis for further 3D display or analysis.  

The time required for a fusion of the 2 stacks (mentioned above) depends also on the contrast and content of the images. A typical time would be 120 s. The software team is working on a GPU-based implementation to speed this up significantly.

Currently the 405 nm laser is not supported in the MuVi-SPIM, because of the chromatic aberrations coming along with UV light and because most of the optical components including the objective lenses are specified for a typical spectral range of 440-750 nm 

The rate-limiting step of the MuVi-SPIM acquisition speed is indeed the camera. Z movement is facilitated by a so-called Piezo crawler which is effectively a Piezo stage with a travel range significantly increased to up to a few mm (different for x, y, z, please see the specs in the flyer). At minimized exposure time the full-frame readout speed is 65 fps (both cameras and thus 2 views simultaneously). Note: With a next firmware version, cropping of e.g. to the central quarter of the chip (1024×1024) will increase speed accordingly by ca. a factor of 2.

The current z travel range is 1.2 mm and is limited by the space available between the objective lenses and some safety buffer to not bump the sample into the lenses.

Cell biology, especially early stage long term embryo development, including human embryo development, due to the extremely low photo-toxicity of the InVi system.

Technically there is no real competitor of InVi-SPIM on the market. Application wise, spinning disk system has similar target areas with InVi-SPIM. As compared to Spinning Disk system, the advantage of light-sheet microscope including higher speed, deeper field of view, and low photo-toxicity and so on.

Because we are using two sets of two galvano scanners. The perfect synchronization takes time. However, we are working on developments to get faster scanning speed.

No, all microscopes (Leica, Zeiss, Nikon and Olympus) can be used, but please check the physical space around the C-mount to see if the RCM unit will fit. Up-right microscope is possible too with side port adapter, please do check the leggings, orientation and physical space as well.

At this moment we can acquire up to 4 images sequentially. We are working on the solution for acquiring 2 colours simultaneously (RCM-FRET tba).

The pinhole is fixed but it is possible (on request) to install any size in production. In the traditional confocal microscope, pinhole size is reduced to increase the lateral resolution. In the RCM, the lateral resolution is achieved by the re-scanning invention. So the pinhole size does not affect anymore the resolution – hence, the pinhole is now only used for optical sectioning. we just need a wide open pinhole (currently 1.5AU for 100x objective @ 510 nm) in RCM to have high sensitivity. Currently, the RCM is optimised for 60x and 100x. In future, the goal is to have 20x and 40x support. In that case the default pinhole size might be changed.

The RCM is equipped with Quad-band dichroic mirror (45°) ZT405/488/561/640rpc and Quad-band emission filter ZET405/488/561/640m. On special request, RCM can be equipped with any other filter combination (single, dual triple or quad).

The RCM has its own laser box, but yes, it does support 3rd party laser combiner. However the RCM has 4 analog channels (to control intensity) and 4 digital channels our (laser on/off), which can be used for 3^rd party lasers (mainly through the digital TTL trigger signals) and a FC/APC fiber connection is needed. 

1. The laser combiner shall be able to be controlled by Digital I/O signal (laser ON/OFF) and Analog Signal (Laser power).  2. The laser combiner shall has a FC/APC fiber output connection. 3. Laser power shall be small, preferably <60mW. Since RCM has high light efficiency, high laser power will bleach the sample easily. 4. Laser wavelength can be selected based on available CHROMA filters.

The RCM unit does not have an internal emission filter-wheel. For most applications, channel separation on the excitation side (switching lasers on and off) is sufficient. An additional external filter wheel can be installed to further improve channel separation.

The RCM can be controlled with:

1. uManager/ImageJ(Fiji), which is free of charge and compatible with most of the microscopes/cameras/accessories available on the market. For detailed lists of hardware supported, please refer to uManager website: https://micro-manager.org/wiki/Device_Support
2. Nikon NIS Elements. If a customer has existing Nikon microscope and NIS Elements software, it can be upgraded to RCM with the minimum hassle. And below are the requirements:
   • Nikon NIS Elements AR or BR, version 4.4-4.6.
   • Nikon NIS Elements Wavelength Switcher Module
   • RCM NIS Plugin (at cost)

Yes, user can select up to 6 wavelengths from 340, 369, 385, 405, 455, 470, 505, 528, 595, 625, 660, 730, 820 nm. Filter sets will be adjusted as well based on wavelength selection.

Yes, the Imaging Machine supports laser auto-focus as well as software autofocus for best image quality in long time-lapse imaging.

ACQUIFER is the combination of ACQUIsition and datatransFER. It is a most efficient IT infrastructure platform to handle and master the rapidly growing data streams of new microscopy methods and modern imaging technologies in biomedical applications and the life sciences. In particular, the HIVE is optimally suited to receive, store and process the data in High Content Screening Projects, in Microscopy Core Facilities and Multi-User Microscopy Laboratories. It can cope with the challenging data rates of modern Light Sheet Fluorescence Microscopes, Confocal and Super Resolution Microscopes, Spinning Disc Confocal or Electron Microscopes to name a few.

The microscope and camera connect to their normal control/image acquisition PC, and the control PC connects to HIVE system through Ethernet cable. The network card in the microscope control/image acquisition PC has to be CAT 6 or higher. Image data is transferred to HIVE system through Ethernet cable at the max speed of 10Gb/s.

Regular Imaris license does not support Windows Server 2012 R2, however, with a Floating License Module from Bitplane, Imaris can work in Windows Server 2012. 

HIVE uses two RAID systems:
RAID5 on HIVE-CORE for speed
With one redundant “parity stripe”, one disk can fail without data loss.
RAID6 on HIVE-DATA for security
With two redundant “stripes”, two disks can fail without data loss.

All components of the HIVE are secured by the uninterrupted power supply (UPS) located in the NET.
Battery backup is important because power system instability can be fatal for data storage. RAID systems are sensitive to voltage fluctuations which could result in severe loss of data.
With the built-in battery pack, the operating system can initiate a safe shut down process instead of just a blackout.

Here are the commonly used network connection cable types:

• Cat5 supports speeds up to 100Mb/s (100 MHz)
• Cat5e supports speeds up to a Gigabit Ethernet (1,000Mb/s) (100 MHz)
• Cat6 supports speeds up to 10 Gigabit Ethernet and can be connected at a distance of less than 50 meters (1,000Mb/s) (250 MHz)
• Cat6A supports speeds up to 10 Gigabit Ethernet with distance up to 100 meters (10,000 Mb/s)(500 MHz)
• Cat7 & Cat7A support speeds up to 10 Gigabit Ethernet with distance up to 100 meters (10,000 Mb/s)(1000 Mhz)

HIVE supports up to Cat7A, but the hardware in any attached acquisition computers must be matched to the hardware in the HIVE to achieve maximum data transfer rates.

Cat6A or higher is strongly recommended!!

The differences between Copper and Optical Fiber cables are listed below in the table:

	Copper	Optical Fiber
Advantage	Power over Ethernet Less expensive electronics Flexibility Local cable assembly is easy Use existing infrastructure	Transmission and power efficiency No Electromagnetic Interference High Bandwidth Cost – Fibre is less expensive than copper Lightweight compared with copper Transmission distance much greater than copper Ease of installation – higher tension limits when pulling cable (10kg for copper, 40-80kg for optical) Security – cannot be easily tapped (impossible to tap with an EM antenna)
Disadvantage	Fragility Inefficient data transmission Local cable assembly can easily be done incorrectly	Cost of electronics Cable assembly requires more skill

 

In general, there is no need to switch between copper and optical fiber network, just use whatever is available in the current network infrastructure.

Sample preparation is similar to preparation used in fluorescent microscopy. Samples are mounted on 14x14mm glasses coated with ITO (indium tin oxide) to avoid charging later on. After the samples are fixed an immunohistochemically staining can be performed, followed by an additional contrasting step for EM analysis. Samples can be dehydrated via various options and are then ready to be imaged. In the following whitepaper, we discuss several methods suitable for an integrated imaging workflow, and present results obtained using a variety of techniques and samples, including cultured cells and tissue sections, chemical fixation or cryo-fixation, genetic labels and immunolabeled samples. While particularly suited for iCLEM, these protocols may also improve correlation in non-integrated approaches or a combination of both. This is not intended as an extensive list of sample preparation methods, which are many and varied. Furthermore, different types of specimens typically require specifically optimized protocols. This short review provides insights in the possibilities offered by integrated imaging workflows and represents a useful starting point for exploring these techniques. http://request.delmic.com/download-secom-white-paper

Alignment of fluorescence and electron images is a crucial step in correlative microscopy. The alignment procedure of the SECOM is fully automated and achieves an accuracy of 50 nm or better, independent of the sample. This accuracy is achieved using a patented alignment procedure. With the SECOM platform With the SECOM platform you therefore always look at exactly the same location with both the fluorescence and the electron microscope. The key to this alignment procedure is the physical principle of cathodoluminescence. Light is generated at the position where the electron beam hits the glass. This light can be detected by the camera of the fluorescence microscope and acts as a temporary fiducial marker. By positioning the electron beam such that not one, but many spots are created, a grid of such temporary fiducial markers is generated. Using this procedure, the electron and fluorescence images are exactly aligned, correcting for translation, scaling and rotation; unbiased and independent of the specimen. Because the procedure uses the cathodoluminescence of the cover glass substrate, the procedure is sample independent and works without any additional fiducial markers or other landmarks in your sample preparation. 

Normally thin samples are used in SECOM, a typical thickness is around 50-300nm. We need to balance between signal strength and the sample thickness. We can get more fluorescent signals from the thicker sample, but thinner image gives better contrast in EM images.

It depends on which SEM is being used and what kinds of detectors are inside the chamber. but generally speaking, it is a straight-forward procedure and takes less than an hour to do the replacement. Also there will be trainings provided to users during SECOM installation.

Yes, other functionalities are not affected. Nothing is taken away from the functional SEM itself, SECOM has standard sample holder, if a special mounting method or sample holder is required, we can also discuss and customise the sample holder. In principle, every functionality and operation of SEM remain unchanged.

It is typically used in the lower KV range, which gives high contrast images, many people use 1KV or even lower.