S E C O M

TABLE OFcontents
The SECOM platform

4

Applications - sections

5Applications - whole cells

8Features

9Integrated workflow

12

Automated overlay

13

ODEMIS - integrated software

16Specifications

17Samples provided byT. Templier and R.H.R. Hahnloser,
University of Zurich and ETH Zurich.

The SECOM platform
at a glance+ Streamlines your correlative
workflow+ Seamless switching between
fluorescence and electron		 microscopy+ Best optical performance of		 any integrated system+ Fully automated overlay with		 an accuracy better than 50		 nm, independent of sample		 and user
+ Modular design &		 open-source software

4SECOM

Integrated Correlative Lightand Electron Microscope
The SECOM platform is a fluorescencemicroscope made to be integrated with ascanning electron microscope. It enables you to
do correlative microscopy extremely fast, withthe highest optical quality and overlay accuracy.
Thanks to its integrated design, switching fromfluorescence to electron imaging is seamlessand instantaneous. And with the automatedalignment procedure, you are directly imaging
the right location at a high resolution.
Imaging with the SECOM is just like using afully equipped high-end optical wide-fieldmicroscope, without compromise on either
optical or electron performance.
The system is installed as a retrofit by replacing
the door to the vacuum chamber and can be
fitted to most scanning electron microscopes.

5

APPLICATION

Imaging thin sections
Using the SECOM for thin sections allows you to
use fluorescent markers for pinpointing regionsof interest, locating rare events, screening large
areas and identifying subcellular structures
on a molecular basis. By using multicolor
labelling you can have the enormous toolboxof fluorescent markers available in nanometer
resolution electron images.
Sectioning biological material that is resin
embedded or cryo fixed is the ideal method
to reveal subcellular details. This is why mostelectron microscopists are used to working
with sectioned material. For fluorescencemicroscopy, the axial resolution is greatly
enhanced as the sections can be as thin as 50nm.

Ultrathin sectioning can be applied to manydifferent biological samples, such as cells insuspension or on a substrate, tissues andanimal embryos.

Localization of the lipid
diacylglycerol within cellular
membranes of HeLa cell expressing
GFP-C1 (Peddie et al., 2014).
Image courtesy ofC.J. Peddie and L.M. Collinson, CRUK LRI
Backscatter detector,
100x /1.40 oil immersion lens
using vacuum compatible immersion oil,laser light source, sCMOS camera.

8APPLICATION

Imaging cultured cells
The SECOM is the perfect tool to accurately
inspect the morphology and surface topologyof cultured cells. With the SECOM platform you
have the unique opportunity to simultaneously
image fluorescent markers together with all thedifferent types of contrast available for SEM.
This provides a fast and straightforward method
to study cell morphology in correlation withspecific proteins of interest.

Growing cells on a substrate is routine workin cell biology. With minor alterations in yoursample preparation, you can take advantage ofthe high resolution and extra contrast provided
by electron microscopy.

Human umbilical vein endothelial cells (HUVEC)
contain rod-like storage granules called WeibelPalade bodies which store Von Willebrand factor(VWF). These organells play an important role inblood coagulation.
Actin (Phalloidin Alexa 488) and VWF (Alexa 568).
Samples courtesy of M.J. Mourik, LUMC

Secondary electron detector,
60x /0.95 lens, multicolor LED light engine,
CCD camera.

9FEATURES
Motorized precision stage
The SECOM sample stage isequipped with precisionpiezoelectric stepping
motors and optical linear
encoders, enabling easy
and accurate sample
navigation.

Retrofit

Compatibility
The space above the sample
stage in the SEM is kept free;thus allowing access to all
the standard detectors. The
system is fully compatible
with a broad range of SEM
detector including: ETD,BSD, EDX and others.

High-end optics
Because only the highest
quality optical components
are used, the SECOM
ensures the best optical
performance of any
integrated system. Itis even possible to use
special immersion oils invacuum. Multiband imaging
is part of the standard
configuration.

The SECOM is installed as a
retrofit to an existing SEM
by replacing the vacuum
chamber door. As aresult, it is easy to switch
between the SECOM
platform and the original
door.

12

INTEGRATED WORKFLOW
Fast and precise

Correlative imaging with an integrated system
is very different from traditional correlativeprocedures. With the SECOM platform you
acquire your fluorescence and electron images
in one microscope, allowing you to quickly
switch between both modalities. This drasticallyreduces the time from sample preparation tocorrelative imaging. After loading your sample,
you immediately get your results: in one
acquisition you get your fluorescence and highresolution electron images combined with anaccurate overlay. Moreover, by easily switching
between imaging modes you can quickly assess
the quality of your sample preparation andadapt your imaging parameters accordingly.
Thanks to the automated alignment procedureof the SECOM, you can accurately correlate
subcellular structures, without the riskof introducing a user-bias. The use of an
integrated sample preparation protocol
prevents unpredictable sample shrinkage anddeformation.

Since the sample is not moved
from ambient to vacuum
conditions, it is guaranteedthat your sample is in the same
conformation for both light andelectron microscopy.
High Numerical Aperture
Because of the excellent optical
performance of the SECOM,
there is no need to switch toanother optical microscope.

1 μm

SECOM workflow
Thanks to the integration of thefluorescence microscope within
the vacuum of the electron
microscope, switching between
fluorescence and electron
imaging can be done over andover again.

13

AUTOMATED OVERLAYHigh accuracy, independent of userand sample
Alignment of fluorescence and electron images
is a crucial step in correlative microscopy.
The alignment procedure of the SECOM isfully automated and achieves an accuracy of
50 nm or better, independent of the sample.
This accuracy is achieved using a patentedalignment procedure. With the SECOM platform
you therefore always look at exactly the same
location with both the fluorescence and theelectron microscope.
The key to this alignment procedure is thephysical principle of cathodoluminescence. Lightis generated at the position where the electron
beam hits the glass. This light can be detectedby the camera of the fluorescence microscope
and acts as a temporary fiducial marker.
By positioning the electron beam such that notone, 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 fortranslation, scaling and rotation; unbiased andindependent of the specimen.
Because the procedure uses thecathodoluminescence of the cover glass
substrate, the procedure is sample independent
and works without any additional fiducial
markers or other landmarks in your sample
preparation.

Grid of temporary fiducial markers
A grid of spots used for the alignmentprocedure. The spots are generated using theelectron beam, and detected using the cameraof the fluorescence microscope.

Cathodoluminescence
When electrons hit aluminescent material, photons
are produced that can bedetected using the optical
microscope.

16ODEMIS

Integrated software
Combining different types of contrast at very
different length scales makes correlative
microscopy a challenging task. The software
package ODEMIS greatly simplifies yourcorrelative imaging workflow. You no longer
have to worry about overlay accuracy and stage
coordinates, giving you time to focus on yourimaging.

users, a scripting interface in Python gives you
full control of the hardware and the imaging
algorithms.
To simplify correlative microscopy, ODEMIS
features a fully automated alignment procedure
resulting in accurate, unbiased overlays on every
specimen. This overlay is directly visualized in
the acquisition software, allowing you to quickly
adapt your imaging conditions. Other powerfultools that improve your imaging workflow
include auto-focus and a history trail thatrecords your previous stage coordinates.

ODEMIS is a user-friendly program that gives
you control over both the fluorescence andelectron microscope. Navigating the sample
is easy with either the fluorescence or SEM
channel and you can use multiple color channels ODEMIS is open-source and makes use of theopen file formats OME-TIFF and HDF5.
with individual settings per channel. For expert

17SPECIFICATIONS
Excitation• Multiband setup in Pinkel configuration
optimized for DAPI, FITC, TRITC, & Cy5 andother like fluorophores.
• Four channel solid-state (LED) excitation
source with digital On/Off and intensity
control.
• Default excitation wavelengths at 387/11,
485/20, 560/25 and 650/13nm.
Others available on request.

Objective Stage• Stage uses piezoelectric stepping motors thatremain full blocking force when not actuated,resulting in small thermal load and thus lowdrift.
• Minimum incremental motion in XY less than
500 nm.
• Repeatability of Z-axis (focusing) 50 nm.
• Use of optical linear encoder for closed loopdriving.

Sample Stage• Total stroke of 18x18mm in XY.
• Equipped with precision piezoelectric
stepping motors and optical linear encoders.
• Minimum incremental motion of 300 nm,
repeatability of 500 nm.

Camera• Scientific CMOS camera allows imaging with
low noise and large field of view.
• 2048x2048 pixels with a pixel size of 6.5 µm
yields• 330x330 µm field of view at 40x magnification

Objective lens

• Plan Apochromat objective lens, magnification
40x, numerical aperture 0.95.
Other objective lenses, including immersion
lenses (up to NA 1.4), available on request.
• User exchangeableSamples provided byP. Ronchi and Y. Schwab, EMBL, Heidelberg

Integration without compromise

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tel. +31 15 7440158 info@delmic.com www.delmic.com