Real-Time Video-Rate Laser Imaging
Real-Time Video-Rate
Laser Imaging
www.thorlabs.com
Real-Time Video-Rate Laser Imaging
Video-Rate Confocal Camera
Confocal Camera Features
■
■
■
■
Confocal Imaging at Video Rates
Mounts onto Camera Port of Optical
Microscope
Diffraction-Limited Resolution Over
a Large Field of View
Convenient Access to Optics
■
■
■
Compatible with Thorlabs' Lens
Tube and Cage Systems
Fiber or Free-Space Coupled Input
and Output
Numerous Filter and Detector
Configurations
Thorlabs’ VCM405 Video-Rate Confocal Camera Head Shown
on an Olympus BX41 Microscope
Confocal scanning optical microscopy is a high-resolution optical imaging technique
that has recently gained wide-spread popularity in the industrial and scientific
communities. Unlike wide-field fluorescence microscopy, 3D images are reconstructed
on a point-by-point basis, leading to higher resolution and the ability to produce infocus images of relatively thick samples.
Thorlabs offers full-featured, video-rate Confocal Camera Systems with laser diode
sources centered at 405 nm, 635 nm, or both wavelengths. In either case, you receive the
optical head, which is packaged in a compact ready-to-use module that connects directly to the
camera port of any standard microscope
via the appropriate microscope
Thorlabs offers preconfigured Confocal Camera
adapter, the confocal control
Systems with source wavelengths centered at 405 nm,
module, imaging software, and a
635 nm, or both wavelengths; we can also fabricate
custom confocal heads that utilize other diodes to
computer. This powerful
provide alternative source wavelengths. Please contact
combination of proven Thorlabs imaging
us to discuss your specific needs.
technology enables a true confocal solution at
a fraction of the cost of competing systems.
For those preferring open access to the microscope, Thorlabs also provides a Confocal
Translation Stand (see page 30), which includes a mechanism for coarse/fine vertical camera
translation, an objective turret, and a sample platform.
(5.94")
150.8 mm
Mouse Kidney
A 16 µm cryostat section of a mouse
kidney simultaneously excited with
403 nm and 471 nm light. The glomeruli
and convoluted tubules are stained with
Alexa Fluor 488 wheat germ agglutinin
(green) while the nuclei are stained
with DAPI (blue). This pseudo-colored
image was obtained by modifying a
VCM405 Video-Rate Confocal Camera
to provide excitation at 403 nm and
471 nm and then mounting it on a
Nikon TE2000 microscope equipped
with a 40X Olympus Objective
(NA = 0.75).
28
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(2.86")
72.7 mm
(1.14")
29.1 mm
(7.38")
187.3 mm
(11.71")
297.3 mm
(3.11")
79.1 mm
(26.32")
668.6 mm
VCM101H Video-Rate Confocal Camera Schematic
Real-Time Video-Rate Laser Imaging
Confocal Camera
Input
Thorlabs' Confocal Camera offers real-time
confocal imaging in a customizable open
platform. The laser source can be free space or
fiber coupled into the scan head; the fibercoupled option ensures a spatially filtered input
beam that is essentially a perfect Gaussian. In
addition, the two single mode fibers used to
deliver the illuminating light and collect the
backscattered signal replace the pinhole that is
used in traditional confocal systems. The confocal
arrangement of the fiber position rejects out-offocus light thus creating a true confocal image.
Output
Confocal Head: Constituent Parts
1.
2.
3.
4.
5.
6.
7.
To Sample
8.
9.
10.
The camera head contains the counter rotation
scanner (resonant scanner), galvo scanner, turning
mirrors, beamsplitter, fiber collimators, and
kinematic mounts, all of which can be accessed by
removing the cover plate (see photo to the right).
The supplied BSW07 50:50 Ø1" Broadband
Beamsplitter for the 400 - 700 nm range, which
fits into the mount labelled as #5 in the photo,
can be easily swapped out for any user-supplied
Ø1" round or 36 mm x 25 mm x 1 mm
rectangular beamsplitter or dichroic mirror. In
addition, an SM1 Lens Tube has been supplied
adjacent to the entrance port (#7 in the photo) as
well as the exit port (#8 in the photo), thereby
enabling the user to insert the Ø1" longpass,
shortpass, bandpass, or notch filter appropriate for
the user's fluroescence imaging application. The
electronic controller module is included in all the
system options we offer. This module drives the
resonant scanner, galvo scanner, and laser diodes;
it also interfaces the computer with the scan head.
Resonant Scanner
Galvo Scanner
Scan Lens (f = 80.4 mm)
Kinematic Mirror Mount for Ø1" Optics
Kinematic Mount for Ø1" Beamsplitter
or 25 mm x 36 mm x 1 mm Dichroic Filter
Turning Mirror Mount for Ø1/2" Optics
Kinematic Mount with SM1 Thread and Fiber
Collimator Adapter
Kinematic Mount with SM1 Thread and Fiber
Collimator Adapter
SM1 Lens Tube for Ø1" Filter
SM1 Lens Tube for Ø1" Filter
The green lines and arrows in the photo indicate the path that the excitation light follows. Fluorescence from the sample (when in
the fluorescence imaging mode) or backscattered light (when in the backscattering configuration) will then following the light path
indicated with red lines and arrows.
Confocal Camera Specifications
Optical and Imaging:
■ Central Wavelength: 405 nm and/or
635 nm
■ Bandwidth: ±5 nm
■ Output Power:* ~2 mW
■ Resolution:† 1 µm
■ Imaging Speed:
23 fps @ 800 (X) x 640 (Y) Pixels
DAQ Electronics:
■ Analog Input:‡ 2 Channels, 14 Bits,
125 MS/s
■ Analog Output:‡ 4 Channels, 16 Bits,
1 MS/s, ±10 V
■ Digital I/O: 8 Ports
BPAE Cells
Computer:
Supply Voltage: 100/240 VAC, 50-60 Hz
■ CPU/Memory: Intel® Processor
■ Memory: 2 GB
■ Hard Drive: 250 GB SATA
■ Optical Drive: 16X DVD ±RW
■ Monitor: 19" LCD (1280 x 1024 Pixels)
■ Operating System: Windows® XP
Professional, SP2
■
Confocal Controller:
Supply Voltage:** 100 V - 240 VAC,
50 - 60 Hz
■ Storage/Operating Temperature: 15 - 40 °C
■ Relative Humidity: <85% Non-Condensing
■ Weight of Control Unit: 2.26 kg (5 lbs)
■ Dimensions of Control Unit (L x W x H):
305 mm x 254 mm x 127 mm (12" x 10" x 5")
■
*Output power was measured at output port of VCM101H with no objective
using a 635 nm laser diode light source
‡ Ms/s = Megasamples per second
† Resolution specified using RMS40X objective and 635 nm diode. Actual
resolution will vary based on objective used.
**Control Unit has universal AC input.
F-actin (filamentous actin) and nuclei distribution
in bovine pulmonary artery endothelial (BPAE)
cells obtained using a modified Thorlabs’ videorate confocal camera mounted on a Nikon
TE2000 microscope. The pseudo-colored green
fluorescence indicates F-actin, which was stained
with BODIPY FL phallacidin while blue
fluorescence labels nuclei, which were stained with
DAPI. The image was recorded using
simultaneous scanning of both 403 nm and
471 nm laser sources and a Nikon PlanApo 60X
oil immersion objective with an NA of 1.40.
An adapter is needed to connect the confocal head directly to the
camera port of standard microscopes (e.g., Olympus, Nikon,
Zeiss, and Leica). Please contact us to inquire about the adapter
options we currently have available as well as the possibility of
having our machine shop provide an adapter for your microscope
if there is not one currently available.
VCM Series of Confocal Camera Systems
ITEM#
VCM405
VCM635
VCM405/635
$
£
$ 33,000.00
$ 33,000.00
$ 33,000.00
£ 20,790.00
£ 20,790.00
£ 20,790.00
€
€ 30.690,00
€ 30.690,00
€ 30.690,00
RMB
DESCRIPTION
¥ 315,150.00
¥ 315,150.00
¥ 315,150.00
Confocal System with 405 nm Source
Confocal System with 635 nm Source
Confocal System with 405 nm and 635 nm Sources
www.thorlabs.com
29
Real-Time Video-Rate Laser Imaging
Confocal Camera System
In addition to Confocal Camera Systems in which the confocal head mounts directly to the camera port via an
adapter, leading to a compact ready-to-use module, Thorlabs also offers a VCM-TS Confocal Translation Stand
for those customers preferring open access to the microscope. The VCM-TS Stand includes a mechanism for
coarse/fine vertical camera translation, an objective turret, and a sample platform on
which a user-supplied sample translation stage, such as the MAX312 Flexure Stage
(see photo below) can be mounted.
Thorlabs’ VCM405 Confocal Camera
System shown with the VCM-TS
Confocal Translation Stand
Confocal Translation Stand
ITEM#
VCM-TS
$
$
€
£
2,000.00 £ 1,260.00 €
RMB
1.860,00
¥
DESCRIPTION
19,100.00
Confocal Translation Stand
Confocal Imaging: Backscattering Mode
The VCM Series of Confocal Camera Systems
offers highly versatile imaging systems.
The standard reflection mode allows surface
imaging of the highly reflective, opaque materials
found in microelectronics, material science, and
surface studies (see the images below). In addition,
this confocal imaging mode can also provide optical
slicing of semitransparent scattering samples, as
evidenced by the image to below.
Leaf
Psuedo-colored 3D
projection and crosssectional confocal
scattering image of a
green leaf. The data was
obtained using a 60X
objective lens and a
VCM635 confocal
camera that was modified
to provide fiber-coupled
excitation at 660 nm.
80 μm
250 μm
Figure 1
185 μm
Microelectronics
Figure 1. Confocal
scattering image of a
memory chip with XZ
and YZ cross-sectional
images taken with a 60X
objective. The total
image size is 75 x 55 µm.
1.
+20
0
-20
55 μm
30
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75 μm
Figure 2. Reconstructed
3D projection model of
the confocal backscattering
signal from a portion of
the circuit of a microchip,
obtained using a 100X
objective.
2
110 μm
150 μm
Real-Time Video-Rate Laser Imaging
Confocal Imaging: Fluorescence Mode
Peach Worm
Below are pseudo-colored confocal
fluorescence images of a peach worm
obtained with a confocal camera system
using a 60X water immersion objective.
A total of 256 Z-slices (0.3 µm step size)
were used to create the pseudo-3D
projection shown at the bottom of the page.
The total 80 µm Z-scan is represented
below by a selection of 8 images that were
taken in 10 µm increment (see images 1-8);
each Z-slice measures 440 µm x 430 µm.
1
Confocal laser scanning microscopy is most frequently used with fluorescent samples since the
technique spatially separates the desired fluorescence signal from the out-of-focus background
fluorescence, thereby allowing optical sectioning along the Z-plane. Combining this method
with imaging processing software enables cross-sectional imaging, 2D projections, and pseudo3D rendering of the optically sectioned sample as demonstrated by the data presented here,
which was obtained with a VCM405 confocal system that has been reconfigured as shown in
Fig. 3 for fluorescence collection.
2
(optional)
3
4
Figure 3 shows a VCM405
confocal head reconfigured for
fluorescence imaging. By
swapping out the beamsplitter in
the standard VCM405 setup,
replacing it with a dichroic filter,
and adding the appropriate
excitation and emission filters, the
system becomes a powerful
fluorescence imaging tool. In
most situations, a PMT should
also replace the fast photodetector
used for confocal imaging in the
reflection mode.
The Confocal Camera is Easily
Reconfigured for Fluorescence Imaging
Figure 3
Pollen
5
6
7
8
Below are pseudo-colored 2D projections and 3D confocal fluorescent images of pollen taken
with a VCM405 confocal camera using a 60X objective. Pollen grains were mounted on a standard
microscope slide and excited with 405 nm light from the laser diode (Thorlabs Item# DL5146-152)
incorporated into the head of the confocal camera. The emission signal was selected using a dichroic
mirror (DMLP505) with a cutoff wavelength of 505 ± 15 nm, collected through a single mode fiber
(P1-460A-FC-5), and directed to a PMT for detection.
(Image size: 150 µm x 110 µm,
Z-scan depth: 80 µm)
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31
Real-Time Video-Rate Laser Imaging
Confocal Fluorescence Imaging
Rabbit Artery
Human Skin and Sweat Gland
A 250 µm x 210 µm image showing the top view projection of a
rabbit artery slice mounted on a standard microscope slide is shown
below. Here, the pseudo-colored confocal fluorescent image was
taken using a confocal camera in fluorescence mode and a 60X
infinity-corrected objective.
Below is a view of a sample slice of human skin and sweat gland that was
mounted on a standard microscope slide and observed with a confocal camera
using an infinitycorrected 60X
water immersion
objective. Here, a
series of individual
Z-slices were used
to create the
pseudo-colored
projection.
Multichannel Fluorescence Imaging
For most fluorescence applications, multiple dyes are used
to distinguish visually and chemically specific cell
structures or metabolic processes. Whether the protocol
uses a combination of external fluorophores like DAPI,
Alexa Fluor 488, and Rhodamine or multiple fluorscent
proteins, it is useful to have multichannel detection. As
shown in Fig. 4, the VCM series of confocal camera
systems is easily reconfigured to meet these experimental
requirements. The system shown to the left was used to
obtain the two-color images below. Although this example
utilizes two-channel detection, simply adding a third PMT
allows the confocal camera to produce three-color images,
making this system comparable to a large commercial
confocal fluorescent microscopy system but with a smaller
footprint and at a fraction of the cost.
Figure 4
Confocal Camera in the
Multichannel Configuration
Plant Sectioning
The pseudo-colored 3D images below show an optically sliced fluorescent plant sample, excited at 405 nm using a modified VCM405
Confocal Camera. Channel 1 (shown in red) collected fluorescence in the 505 – 555 nm range, while channel 2 (shown in green) was
used to collect light from 569 – 600 nm.
32
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Real-Time Video-Rate Laser Imaging
Nonlinear Imaging Using The VCM Series of Confocal Cameras
Many new techniques have been developed to enhance imaging contrast
and biological and chemical specificity. With the advent of turnkey
ultrafast lasers, nonlinear imaging methods such as multiphoton
fluorescence imaging (e.g., 2-photon or 3-photon microscopy), second
harmonic imaging microscopy (SHIM), and resonant Raman techniques
like CARS are increasingly being adopted by microscopists to expand their
biochemical understanding of natural processes. Often, more than one of
these techniques is used simultaneously, which necessitates a custom
system or a modification to a standard confocal laser scanning microscope.
As is the case with other Thorlabs’ products, we have designed the VCM
series of confocal camera systems to provide you with the tools needed to
create your own photonics solutions.
The reconfigurable confocal camera head is ideally suited for nonlinear
microscopy setups. In many cases, it is cheaper and more convenient to
convert an existing confocal laser scanning microscope system into a
multiphoton system rather than purchasing a commercial version; in
particular, for SHIM and CARS applications, a customized system is the
only solution due to the lack of industry options.
In the confocal camera head, internal tap holes are provided for changing
optical components, and the compact footprint allows it to be adapted to
any available microscope port with minimal mounting hardware.
Emission Filter, Laser Blocking 750-1000 nm
Dichroic Filter, 750 nm Long Pass
Focus Lens
Confocal Camera
Reconfigured for
Multiphoton Imaging
Figure 5
Please contact us to inquire about the adapter options currently available to connect the
confocal head directly to standard camera ports as well as the possibility of having our
machine shop provide an adapter for your microscope if there is not one currently available.
Multiphoton Fluorescence Microscopy
Multiphoton microscopy combines fluorescent dyes with an ultrafast laser
to allow fluorescence imaging using less damaging IR radiation. With the
increased penetration depth and the inherently localized nature of the
ultrafast pulse, multiphoton microscopy provides noninvasive sub-cellular
imaging of living systems. The confocal head can be reconfigured for
multiphoton microscopy or second harmonic imaging microscopy (SHIM)
simply by replacing the single mode fiber before the detector with the
appropriate bandpass and emissions filters, as shown in Fig. 5. For SHIM,
the appropriate filters select light at half the excitation wavelength. The
setup shown in Fig. 5
was used to image a slice
of rat kidney using a
confocal head that
was reconfigured for two-photon fluorescence imaging using a TC780-150
femtosecond laser (Menlo GmbH) for excitation. For more information
on this femtosecond laser, please contact Menlo Systems Inc. directly at
973-300-4490 or by emailing [email protected]
Like mulitiphoton microscopy, SHIM uses an ultrafast laser for signal
generation, but in this case, no fluorophore is used; instead, the signal is
generated from noncentrosymmetric features within the sample, making this
technique advantageous in many material science applications. In addition,
SHIM is increasingly being used in biological applications, including
mammalian studies. For example, the large signal created by myelin and
collagen yields excellent cellular structure determination without the need
for either an intrinsic or externally applied fluorophore.
1
2
3
4
5
6
7
8
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33
Real-Time Video-Rate Laser Imaging
Multiphoton Imaging With and Without a Dispersion-Compensating Mirror Pair
Thorlabs has teamed up
with its strategic partner,
Menlo Systems Inc.,
to provide a pair of
Dispersion-Compensating
Mirrors that correct for
the phase distortions that
occur when ultrashort
pulses travel through an optical system. Since femtosecond pulses are
comprised from many different wavelengths of light, pulse broadening,
as a result of dispersion, will occur when the laser light passes through
a dielectric medium (e.g., glass in the optical system). This pulse
broadening is attributed to the wavelength dependence of the refractive
index of the optical components through which the light travels.
Shorter wavelengths are associated with higher indices of refraction
than longer wavelengths; thus, when a femtosecond pulse travels
through an optical system, the shorter wavelengths will travel slower
than the longer ones.
The pulse dispersion caused by the wavelength-dependent nature of
the refractive index can be corrected using Menlo Systems’ dispersioncompensating mirror pair. These mirrors are specifically designed so
that longer wavelengths experience larger group velocity delay than
shorter wavelengths, thereby negating the pulse broadening caused
by the optical elements within the imaging system.
Features
■
■
■
■
■
■
Advanced Coating Layer Composition Corrects for Dispersive
Elements in the Beam Path
Reflectivity: >99.5% from 700-1000 nm
Extremely Flat Polished Substrates to Maintain Beam Quality
Dispersion per Reflection: -175 fs2 at 800 nm
Coated Surface Dimensions: 10 mm x 50 mm
Thickness: 12 mm
For more information on Menlo Systems’ OCTAVIUS-1G Ti:Sapphire Oscillator used to
obtain the mouse images shown below, please contact Menlo Systems Inc. via telephone
(973-300-4490) or email ([email protected]).
Mouse Kidney
The two-photon images of a mouse kidney shown here
demonstrate the benefits of using the DispersionCompensating Mirror Pairs manufactured by Thorlabs’
strategic partner, Menlo Systems Inc., for increasing image
quality. Figure 1 shows an image of a mouse kidney specimen
that was taken without the use of the DispersionCompensating Mirror Pair, whereas Fig. 2 shows the same
image acquired after adding the mirror pair to the
experimental setup. In the mouse kidney specimen (Molecular
Probes®, Invitrogen Corp.), the glomeruli and convoluted
tubules are labeled with Alexa Fluor 488 (green) and cell
nuclei are labeled with DAPI (blue).
These pseudocolored images were obtained using Thorlabs’ inhouse developmental multiphoton microscope equipped with
Figure 1. Uncompressed Pulse
Figure 2. Compressed Pulse
a 40X Olympus objective (NA = 0.75). Two-photon excitation
was provided by Menlo Systems’ Octavius-1G, a Ti:Sapphire oscillator that provides a repetition rate of 1 GHz and ultra short (<6 fs) pulses.
The group delay dispersion (GDD) attributed to the optical elements in the microscope is ~4200 fs2. GDD was compensated by adding the
Dispersion-Compensating Mirror Pair into the beam path prior to the imaging system entrance. An intensity analysis of the images shown in
Figures 1 and 2 indicates that the pulse compression provided by the mirror pair increases the signal to noise by a factor of ~38 (~16 dB), thereby
providing a higher quality image of the mouse kidney.
$
£
€
RMB
DESCRIPTION
$ 5,000.00
£ 3,150.00
€ 4.650,00
¥ 47,750.00
Dispersion-Compensating Mirror Pair
ITEM #
DCMP175
34
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