RCM2 | Laser-Scanning Confocal Microscope / Add-On

Modality

Laser-Scanning Confocal Microscopy

Configuration

Widefield & Confocal / Upright or Inverted

Resolution

120 nm (after deconvolution, raw image = 170nm)

Sensitivity

Up to 95% QE

FOV

220 µm x 220 µm (40x/1.4 lens)

Speed

2 fps (at 512 x 512)

Meet RCM2

Brighter images. Larger field of view.

RCM2 is our second generation RCM, with digital scanner technology. It makes bi-directional scanning the standard and allows a speed of 2fps at 512×512 pixels. RCM2 has optics to make it suitable for super-resolution imaging with high NA objectives in the low magnification range, like 40x 1.4. A lower magnification allows for a bigger field of view (FOV), brighter images, and even lower laser power. RCM2 has demonstrated imaging at 10 nano-watt excitation power!

Improvements of sCMOS cameras allow you to sample resolution of low magnification objectives effectively, without increasing the exposure time. In a regular PMT-based confocal this is not possible.

Improve your imaging experience with RCM2

Super-resolution (120nm) imaging with 40x 1.4NA objectives.
Larger FOV without increasing acquisition time.
Bidirectional scanner.
Excellent add-on to any widefield microscope.
Easy to use.
Hardware integration in third-party software.

Discover super-resolution imaging with RCM2

Capture datasets of 170nm resolution raw (120 after deconvolution) over a very large FOV. Study fast live-cell dynamics and perform 4D imaging in optimized conditions. The increased detection efficiency facilitates acquisition in low fluorescence conditions, like single-molecule detection (smFISH).

In combination with silicon objectives, RCM2 allows for deep 3D imaging of organoids, zebrafish embryos, or larger live samples.

The benefits of RCM2

Using RCM2 with a 40x 1.4 objective, you can see more cells at full resolution at once. A larger field of view increases the chances of getting the results you need.

Obtain sharp images with a high signal-to-noise ratio even in samples with a low amount of epitopes or weak stainings. Get more from your samples.

Use even lower laser power to minimize phototoxicity and photobleaching during live-cell imaging.

Getting super-resolution raw images, without averaging or integration, reduces the acquisition time and allows for a more precise analysis of the subcellular structures.

Who should use RCM2 and why?

In combination with silicon objectives, RCM2 allows for deep 3D imaging of organoids, zebrafish embryos, or larger live samples.

What is RCM?

RCM is an easy to use, sensitive, high resolution and affordable confocal imaging system:

An ideal solution for small labs with limited budget, but demanding tasks, particularly when high sensitivity and resolution are desired from the imaging system,
A confocal microscope that works as a camera, no need for an instruction manual
RCM is extremely easy to use: no hardware control or software processing needed, and the images are always RAW.

RCM can be delivered as a total microscope system with a selection of microscopes ( Nikon, Olympus, Leica or Zeiss ), a selection of cameras ( Hamamatsu, PCO, Andor, Photometrics ) and laser solutions (Omicron, Toptica ).

In case you already have a microscope in the lab, RCM is an upgrade to an existing wide-field fluorescence system – RCM can easily be added to the existing wide-field fluorescence microscope system to improve its resolution.

RCM Working Principle

The RCM technique extends standard confocal microscopy with a re-scanning unit, improving lateral resolution by √2 and reducing signal to noise ratio.

Re-scan Confocal Microscopy (RCM) is a new super-resolution technique based on standard confocal microscopy extended with an optical (re-scanning) unit that projects the image directly on a CCD-camera. This new microscope has improved lateral resolution (170 nm at 488 nm excitation), and strongly improved sensitivity, while maintaining the sectioning capability of a standard confocal microscope. It is particularly useful for biological applications where the combination of high-resolution and high-sensitivity is required (but not very high imaging speed).

The excitation lasers (blue and yellow lines) are directed via a dichroic mirror towards the first scanning unit SM1. As in a standard confocal microscope, the scanning unit scans the laser light in the sample and de-scans the emission light, directing it at the pinhole PH (green and red lines). After the pinhole, a second re-scan unit SM2 directs the light onto a camera chip.[/caption]

During scanning, re-scan mirrors (SM2) move faster than the first scan mirrors (SM1). This magnifies the image on the camera chip compared to the sample, and eventually results in the higher resolution of the image. The resolution of the system is improved with the re-scan step by a factor of √2 (i.e. 1.41 times), compared to Abbe’s resolution limit by changing the angular amplitude of the re-scanner (SM2). Reduction of pinhole is no longer necessary to increase resolution. Closing down the pinhole only limits the amount of light passing through and decreases the signal to noise ratio due weaker signal. Since the re-scan is a purely optical method with no further image processing required, there is cost in time while improving the resolution. By using a sensitive camera as detector, the signal-to-noise ratio of the RCM is 4 times higher than in standard confocal microscopy.

To fully understand the principle of rescanning, resolution improvement and the optical layout of the RCM, please watch the video below that explains the components and the light path of the RCM (animation credits to StudioFlip). Additional technical details and test images can be found in De Luca et al (2013).

Watch the working principle here

The Re-scan Confocal Microscopy (RCM) module can be used to turn any fluorescent microscope into a confocal microscope. For an upgrade, laser(s) and a camera are needed.

Check magnificent Turnkey examples from our customers for your inspiration:

Let us know which equipment you have available, and we will make a custom tailored upgrade solution for you.

Detector	Camera
Resolution	120 nm (after deconvolution, raw image = 170nm)
Quantum Efficiency	Up to 95%
Scan Speed	2 FPS @ 512 x 512
FOV	220×220 µm (40x, super-resolution)
Optimized For	100x, 60x, 40x (high NA)
Scanner	Digital (closed-loop)
Speed	2fps @ 512×512 pixels
Wavelength	VIS
Software	Micromanager, Volocity, NIS Elements, Zen, LAS X, Cellsens
Integration	Hardware – USB Connection
PSF for deconvolution with	Microvolution, SVI Huygens
Bypass mode	Yes

Check out the More Information tab for comparison of RCM2 with Widefield, Confocal and other similar systems!

Confocal imaging

RCM has improved lateral resolution and the same axial resolution compared to conventional confocal microscope.

Ratio-imaging and multi-color applications

RCM can work in multi-colour mode for different colour combinations and ratio-imaging (applications like FRET, FRAP, pH, and Ca2+ imaging).

Live cell imaging

RCM has a very high signal-to-noise ratio and quantum efficiency (80-95%, depending on the camera), which means no need for high laser power.

HO1N1 cells stained with Bacmam 2.0 Mito-GFP (green), ER-tracker (blue) and SiR-DNA (red). One 3-color image was taken every 10 seconds for 1.5 hours, giving a total of 1000 images. Because the RCM is very sensitive laser power can be kept very low (few microwatts), enabling long-term imaging. The native lateral resolution of the RCM is 170 nm. After deconvolution by SVI Huygens the resolution is improved to 120 nm! Movie taken by Jeroen Kole (Confocal.nl), sample courtesy Dandan Ma (ACTA, Amsterdam), equipment provided by Marko Popovic (Nikon Center of Excellence, Amsterdam University Medical centers, VUmc, Amsterdam.)

HO1N1 cells expressing Mitochondria-RFP through the Bacmam expression system. One image was taken every 10 seconds for 61 hours (giving a total of almost 22,000 images!). Laser power was measured to be 1 microwatt at the sample plane. Movie taken by Jeroen Kole (Confocal.nl), sample courtesy Dandan Ma (ACTA, Amsterdam), equipment provided by Marko Popovic (Nikon Center of Excellence, Amsterdam University Medical centers, VUmc, Amsterdam.)

The first video shows the RAW data from RCM, the entire 61 hour time lapse.

The second video shows the benefits of deconvolution. We used SVI Huygens CMLE deconvolution to improve the resolution to 120 nm and have an even better signal-to-noise ratio.

Images taken with RCM2

References

[1] Drosophila Melanogaster heart tube stained for Actin (green), Myosin heavy chain (red), Alpha-tubulin (blue). Imaged on RCM2 using 40x 1.4 objective.Sample courtesy: Pedro Espinosa Gonzalez, Amsterdam UMC, location VUMC, the Netherlands.
[2] BPAE cells stained for Actin (green), Mitochondria (blue.) Imaged on RCM2 using a 40x 1.4 objective. Sample courtesy: Thermofisher, United States of America.
[3] Neurons in co-culture stained for Actin (red), MAP2 (magenta) and Tau (green). Imaged on RCM2 with a 40x 1.4 objective. Sample courtesy: Vera Wiersma, VU University, Amsterdam, the Netherlands.
[4] HUVECs stained with SPY probes from Spirochrome. Tubulin (yellow), Actin (purple), and DNA (cian). Sample Courtesy of Philippa Phelp. Boon Group, VUMC, Amsterdam.

Deconvolution

Representative nuclear spread from fixed mouse spermatocytes, immunostained for SYCP3 a component of the synaptonemal complex (Alexa 488-labelling) imaged with RCM using without re-scanning (confocal) and with re-scanning (RCM). Left: full spread. Right: individual chromosomes. Sample courtesy of A. Agostinho – Advanced Light Microscopy Facility, Science for Life Laboratory
C. Höög – Department of Cell and Molecular Biology, Karolinska Institutet. Imaging and deconvolution by Erik Manders (Confocal.nl). For deconvolution SVI Huygens software was used.

N1E-115 neuroblastoma cells, made during HOLM 2017 by the students attending one of the workshops. On the left, raw image from RCM, on the right the same image after the Microvolution deconvolution

Mitotic cell – Cos7 cell line with antibody labeling of nucleus (blue), mitochondria (green), and tubulin (red). Samples prepared by Jana Doehner (University of Zurich). Images taken by Stan Hilt (Confocal.nl) during the BioImaging meeting Utrecht. Original RCM image (left), and the result of NIS Elements deconvolution (right).

3D Reconstructions

Hela cell in mitosis stained for DAPI (blue), Actin (green) and Tubulin (red). Sample courtesy of Nicolas Touret, Faculty of Biochemistry, University of Alberta

Data Sheet and Brochures

RCM Brochure

Application Report

Application Notes

Advantages of Re-scan confocal microscopy vs PMT based confocal systems and its benefits for deconvolution

The Re-scan Confocal Microscope (RCM) minimizes motion-blur when imaging fast germination proteins dynamics

Long term mitochondrial live imaging without phototoxicity using RCM system

Lateral resolution of the Re-scan Confocal Microscope 2 measured with the Argolight Argo-SIM slide

Research publications using RCM-VIS

2020

Fabian U. Zwettler, Marie-Christin Spindler, Sebastian Reinhard, Teresa Klein, Andreas Kurz, Markus Sauer, Ricardo Benavente. Tracking down the molecular architecture of the synaptonemal complex by expansion microscopy. Nature Communications. 2020 June 26
Koen Martens, John van Duynhoven and Johannes Hohlbein. Spatiotemporal heterogeneity of κ-carrageenan gels investigated via single-particle-tracking fluorescence microscopy. Langmuir. 2020 Apr 28
R. M. P. Breedijk, J.Wen, V. Krishnaswami, T. Bernas, E. M. M. Manders, P. Setlow, N. O. E.Vischer & S. Brul. A live-cell super-resolution technique demonstrated by imaging germinosomes in wild-type bacterial spores. Nature Scientific reports 2020 March 24.

2019

Doll S, Freitas FP, Shah R, Aldrovandi M, da Silva MC, Ingold I, Grocin AG, Xavier da Silva TN, Panzilius E, Scheel CH, Mourão A, Buday K, Sato M, Wanninger J, Vignane T, Mohana V, Rehberg M, Flatley A, Schepers A, Kurz A, White D, Sauer M, Sattler M, Tate EW, Schmitz W, Schulze A, O’Donnell V, Proneth B, Popowicz GM, Pratt DA, Angeli JPF, Conrad M. FSP1 is a glutathione-independent ferroptosis suppressor. Nature. 2019 Oct 21. doi: 10.1038/s41586-019-1707-0.
Beliu G, Kurz AJ, Kuhlemann AC, Behringer-Pliess L, Meub M, Wolf N, Seibel J, Shi ZD, Schnermann M, Grimm JB, Lavis LD, Doose S, Sauer M. Bioorthogonal labeling with tetrazine-dyes for super-resolution microscopy. Commun Biol. 2019 Jul 19;2:261. doi: 10.1038/s42003-019-0518-z. eCollection 2019.
Steinbach G, Nagy D, Sipka G, Manders E, Garab G, Zimányi L. Fluorescence-detected linear dichroism imaging in a re-scan confocal microscope equipped with differential polarization attachment. Eur Biophys J. 2019 Jul;48(5):457-463. doi: 10.1007/s00249-019-01365-4.
de Leeuw VC, Hessel EVS, Piersma AH. Look-alikes may not act alike: Gene expression regulation and cell-type-specific responses of three valproic acid analogues in the neural embryonic stem cell test (ESTn). (2019) Toxicol Lett. 303:28-37. doi: 10.1016/j.toxlet.2018.12.005.

2017

De Luca, G. M. R., Desclos, E., Breedijk, R. M. P., Dolz-Edo, L., Smits, G. J., Nahidiazar, L., Manders, E. M. M. (2017). Configurations of the Re-scan Confocal Microscope (RCM) for biomedical applications. Journal of Microscopy, 266(2), 166-177. doi: 10.1111/jmi.12526.
De Luca, G., Breedijk, R., Hoebe, R., Stallinga, S., & Manders, E. (2017). Re-scan confocal microscopy (RCM) improves the resolution of confocal microscopy and increases the sensitivity. Methods and applications in fluorescence, 5(1), [015002]. doi: 10.1088/2050-6120/5/1/015002

Research Publications using RCM-NIR

2020

RCM-NIR paper: Raluca Borlan, Andra-Sorina Tatar, Olga Soritau, Dana Maniu, Gabriel Marc, Adrian Florea, Monica Focsan and Simion Astilean. Design of Fluorophore Loaded Human Serum Albumin Nanoparticles for Specific Targeting of NIH:OVCAR3 Ovarian Cancer Cells. Nanotechnology. 2020 Apr 21

Application Notes

Advantages of Re-scan confocal microscopy vs PMT based confocal systems and its benefits for deconvolution

The Re-scan Confocal Microscope (RCM) minimizes motion-blur when imaging fast germination proteins dynamics

Long term mitochondrial live imaging without phototoxicity using RCM system

Lateral resolution of the Re-scan Confocal Microscope 2 measured with the Argolight Argo-SIM slide

The RCM scanning confocal unit can fit to any inverted or upright microscope via C-mount adapter between the microscope and the camera. The laser light is fed into the RCM unit via a single mode FC/APC fiber connector. The RCM comes in two versions: RCM-VIS and RCM-NIR. Both can operate up to 4 colors, RCM-VIS is design to operate with visible light (350-700 nm), while RCM-NIR is designed to operate in the Near-Infrared spectrum (650-1050 nm). The RCM comes with a bypass module to operate the camera in widefield mode.

Microscopes

RCM can be mounted to any inverted or upright microscope. Upright microscope requires the Upright Microscope Mounting Kit. Motorised focussing unit (internal or external) is also needed in most applications. Currently, RCM has been tested and approved for the following microscopes: Nikon Ti & Ti2, Olympus IX, Zeiss Axio Observer, Leica DMi6000 & DMi8 and MiCube. Contact us if you have any question regarding microscope compatibility.

Please note that:

RCM only mounts correctly to the original microscope manufacturers C-mount
A 100/0 beam splitter is mandatory. 50/50, 70/30 and 80/20 beam splitters do not work with RCM, as they are not laser safe.

Objective lenses

In order to obtain the 170 nm resolution, the highest NA (>1.40) lenses available have to be used.

Cameras

Any microscope camera with a C-mount connector, external trigger, and similar specifications as the two first ones in the list below should work with the RCM. Please contact us, if you have a camera that you would like to test for the RCM compatibility.

We offer Hamamatsu Flash 4 V2 (LT) and V3, which is tested to work with RCM. Please let us know if you have a camera to verify the compatibilty

A physical pixel size of 6.5 micron or lower is ideal for 60-100x objective lenses, as this results in sufficiently small sampling. At 100x objective magnification and 6.5 micron pixel the sampling is 43 nm at the sample plane.

Beamsplitter

Hamamatsu W-View Gemini A12801-01
Cairn Multisplit V2, four-way emission splitter

Software

RCM drivers are available for LabView (free of charge), Micro-Manager / ImageJ (Fiji) (free of charge), and Nikon NIS Elements (at cost).

Lasers

We offer Oxxius laser combiner. The RCM laser coupling is compatible with single mode fibers with FC/APC connector and NA 0.11. Using other fiber connectors will degrade image quality.

Wavelengths: For RCM-Visible lasers with a wavelength from 405 upwards are suitable. RCM is designed as a 4 color system. We offer a flexible laser solution that can be equipped with 1 to 4 lasers. Laser lines can be upgraded at later stage as well.

As the RCM is sensitive, we only need low power lasers (max 25 mW). The advantage of the low power lasers is the significantly longer lifetime and lower costs.

Rescan Confocal Microscope Comparisons

RCM vs Wide-field & Confocal

In order to demonstrate the image improvement by RCM, we have made images of the same cell from deer skin fibroblast sample using wide-field, conventional confocal microscope and RCM. The image taken by RCM we have further improved using deconvolution.

Indian Muntjac – Deer Skin Fibroblast cells. Staining: Blue: DAPI, Green: Phalloidin-Alexa488, Red: Mitotracker CMXRos
Top: image from full field of view; bottom: zoom in. Images by Jeroen Kole (Confocal.nl).

Method	Microscope	Objective	Detector	Pinhole
Wide-field	Zeiss Axiovert 200M	63x NA 1.4; oil	PCO sensicam (100.3nm/pixel)
Confocal	Nikon A1	60x NA 1.4; oil	GaAsP PMT	1 AU
RCM	RCM +Nikon TiE Eclipse	100x NA 1.45; oil	Hamamatsu Orca Flash 4.0	1 AU

RCM vs PMT-based confocal system

The resolution and sensitivity of the raw RCM image is better, resulting in a better deconvolution result. The light intensity at the sample plane was measured at: 4.5 microwatt (1.0 AU), 12.5 microwatt (0.3 AU) and 3.0 microwatt (RCM).

Nuclear spread from fixed mouse spermatocytes, immunostained for SYCP3 a component of the synaptonemal complex (Alexa 488-labelling). Upper panel: images of a PMT based confocal with 1.0 AU pinhole, 0.3 AU pinhole and RCM image. Lower panel: same images deconvolved using Huygens Essential RCM module (SVI), using an experimental point spread function. Scalebar: 1 micrometer. Sample courtesy of A. Agostinho – Advanced Light Microscopy Facility, Science for Life Laboratory. Imaging and deconvolution performed by Jeroen Kole (Confocal.nl).

RCM vs similar system

We have compared the performance of RCM with that of similar image scanning system (based on technology described in DOI:10.1103/PhysRevLett.104.198101 ). After SVI deconvolution with a measured point spread function the resolution of the deconvolved RCM data is better as shown by the lineplots of the red line.

Nuclear spread from fixed mouse spermatocytes, immunostained for SYCP3 a component of the synaptonemal complex (Alexa 488-labelling). Upper panel: raw images obtained with a similar system* and RCM image. Lower panel: same images deconvolved using a measured point spread function. Scalebar: 1 micrometer. Sample courtesy of A. Agostinho – Advanced Light Microscopy Facility, Science for Life Laboratory. Imaging and deconvolution performed by Jeroen Kole (Confocal.nl).

RCM Large FOV vs RCM High resolution

RCM can be used in different imaging modes: Large Field of View allows maximizing the view, but at expense of resolution. In this mode, conventional confocal resolution is achieved. High Resolution, is the RCM mode providing improvement in resolution, but at the expense of the size of view.

Description

Rescan confocal microscope is an easy to use, sensitive, high resolution and affordable confocal imaging system:

- An ideal solution for small labs with limited budget, but demanding tasks, particularly when high sensitivity and resolution are desired from the imaging system,
- A confocal microscope that works as a camera, no need for an instruction manual
- RCM is extremely easy to use: no hardware control or software processing needed, and the images are always RAW.

We can deliver the Rescan Confocal Microscope as a total microscope system with a selection of microscopes ( Nikon, Olympus, Leica or Zeiss ), a selection of cameras ( Hamamatsu, PCO, Andor, Photometrics ) and laser solutions (Omicron, Toptica ).

In case you already have a microscope in the lab, Rescan Confocal Microscope is an upgrade to an existing wide-field fluorescence system – RCM can easily be added to the existing wide-field fluorescence microscope system to improve its resolution.

The new Rescan Confocal Microscope (1.1)

The new RCM (1.1) is based on RCM (1.0) and has all the same features. On top of these, the new RCM offers:

Bypass mode
Two times the field of view
A scanning speed up to 4 frames per second
Integration in SVI Huygens deconvolution software

RCM 1.1 is available in a VIS and NIR version.

Resolution: the standard resolution of the RCM is 170 nm at 488 nm wavelength. This is also called super-resolution. In contrast to other super-resolution systems, RCM offers the super-resolution of the live (raw) image without any processing. It is possible to improve the RCM resolution even further, to 120 nm using deconvolution.

Frame rate: the frame rate of the RCM is 1 fps at 512 x 512, which makes the acquisition time of a 3 colour image about 3 seconds. The frame rate of RCM 1.1 can be increased to 4 fps at 512 x 512, which makes the acquisition time of a 3 colour image less than 1 second. With the new RCM 1.1 applications that require a bit faster imaging are now possible.

Field of view: Rescan Confocal Microscope 1.0 has a Field of View (FOV) of 80 x 80 μm at 100x magnification, RCM 1.1 has the option to increase this FOV to 160 x 160 μm.

Standard mode – high resolution RCM image. FOV 80 x 80 micron

Increased FOV – standard confocal resolution. FOV 160 x 160 micron

Rescan Confocal Microscope Models

Working Principle

The Rescan Confocal Microscopy technique extends standard confocal microscopy with a re-scanning unit. It improves lateral resolution by √2 and reduces signal to noise ratio.

Rescan Confocal Microscope(RCM) is a new super-resolution technique based on standard confocal microscopy extended with an optical (re-scanning) unit that projects the image directly on a camera chip. This new microscope has improved lateral resolution (170 nm at 488 nm excitation), and strongly improved sensitivity, while maintaining the sectioning capability of a standard confocal microscope. It is particularly useful for biological applications that requires combination of high-resolution and high-sensitivity (but not very high imaging speed).

Diagram

During scanning, re-scan mirrors (SM2) move faster than the first scan mirrors (SM1). This magnifies the image on the camera chip compared to the sample, and eventually results in the higher resolution of the image. The re-scan step improves the resolution of the system by a factor of √2 (i.e. 1.41 times), compared to Abbe’s resolution limit by changing the angular amplitude of the re-scanner (SM2). Reduction of pinhole is no longer necessary to increase resolution. Closing down the pinhole only limits the amount of light passing through and decreases the signal to noise ratio due weaker signal. Since the re-scan is a purely optical method with no further image processing required. There is a cost in time for improving the resolution. By using a camera as a detector, the SNR of the RCM is 4 times higher than the standard confocal microscopy.