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Re-scanning confocal microscope

The innovation and commercialization of expensive super-resolution imaging methods (below 50 nm range) has polarized the researchers in two groups: those who have access to super–resolution imaging system – and those who don’t. Obviously the hunger for even more detaillier information has a high value. Unfortunately, the financial aspect of super-resolution can become an obstacle for researchers to discover new.

However, high resolution does has recently become affordable and available for most labs. The new RCM-microscope from confocal.nl has a significantly improved physical resolution (170 nm at 488 nm wavelength) and strongly enhanced sensitivity as compared to conventional confocal microscopes. Since RCM is available as a affordable, versatile modular device it is ideal for labs with wide range of specific interests.

Hardware

The RCM is a standard confocal microscope extended with a re-scan detection unit. With a simple trick a resolution of 170nm can be achieved for any pinhole diameter. The pinhole is only needed for Z-sectioning. Since RCM has also a strongly improved sensitivity there is no need for high laser power, being more friendly for cells. With strong deconvolution algorithm, like Microvolution, RCM produces extremely high contrast and rich in detail images.

High diversity

At this moment RCM can work in multi-colour mode for different colour combinations and ratio-imaging. But it is our mission to go further by extending the spectral range into the IR for deep tissue imaging and real-time acquisition speed for monitoring highly dynamic processes. Applications like FRET, FRAP, pH, and Ca2+ are possible and many more biological applications are on the horizon.

Modular building blocks

The RCM unit is a plug-and-play box that can easily be connected via standard C-mount adapters to the microscope and camera. The laser light can be fed into the RCM unit via a single mode fibre connector.  All standard connectors, no restrictions here!

 Upgrade your old wide-field system to high resolution confocal

To build up a RCM system confocal from scratch you need

  • A wide-field microscope frame (inverted) with one or more side ports
  • A laser light source (e.g. The LightHub)
  • A sCMOS camera (e.g. ORCA from Photometrics)
  • A PC running LabView or MicroManager
  • For even more enhanced high resolution: Microvolution deconvolution

Principle behind RCM – video tutorial

Shrimp

  • Alexa561 labeled Shrimp wing, 1494x996x398 voxel geometry with 35x35x100 nm voxel resolution, 16-bit/1.1 GB
  • Deconvolution with Microvolution using an experimental PSF

Phalloidin-labeled actin

  • 1200x1200x101 image geometry with 34x34x200 nm physical voxel dimensions, 16-bit with 284 MB
  • Max intensity projection of image stacks before (left and after (right) 25 iterations of deconvolution with Microvolution
  • Scale bar 5 um, in cropped detail 1 um (lower panel)

Multi-channel RCM data

  • HeLa cells labeled with Alexa488, Alexa 561 and DAPI
  • 1212x1212x66 image geometry with 35x35x100 nm physical voxel dimensions, 16-bit with 555 MB
  • Max intensity projection of image stacks before (left) and after (right) 25 iterations of deconvolution with Microvolution
  • Scale bar 5 um

Image courtesy Gert van Capellen, Erasmus Optical Imaging Centre, Rotterdam, The Netherlands