Four-Channel, Cerna®-Based Confocal Systems


  • Complete Upright Confocal Imaging Microscopes
  • Up to 4 Channels of Excitation/Emission
  • Supports Widefield Imaging with
    Cerna® Accessories

Each Four-Channel Confocal System includes a computer, DAQ, and ThorImage®LS Data Acquisition Software. The optical table is sold separately.

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Sam Tesfai
Sam Tesfai
General Manager,
Thorlabs Imaging Systems

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Stitched Image of Cell Layers in Mouse Retina
Sample Courtesy of Robert Fariss, Biological Imaging Core, NIH, Bethesda, Maryland
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Thorlabs' Confocal Microscopy System

  • Complete Upright Confocal Imaging Systems
  • Up to 4 Channels of Excitation in a Monolithic, Fiber-Coupled Laser Source
  • Up to 4 Channels of Detection with Multialkali or High-Sensitivity GaAsP PMTs
  • Galvo-Galvo or Galvo-Resonant Scanners
  • Full-Frame 4096 x 4096 Pixel Images
    • 2048 x 2048 Pixels (Bi-Directional)
    • 4096 x 4096 Pixels (Uni-Directional)
  • Motorized Pinhole Wheel with 16 Sizes of Round Pinholes
  • ThorImage®LS Data Acquisition Software with Lifetime Support
  • Upright Microscope Body Based on Cerna® System to Support Future Expansion

Thorlabs' Upright Confocal Microscopy Systems are complete, fully equipped microscopes available with galvo-galvo or galvo-resonant scan heads to support a variety of imaging applications. By eliminating signals that originate from outside the focal plane, confocal microscopy provides the ability to acquire high-resolution, optically sectioned images from within a thick sample or to reduce background fluorescence from thin cultures. Each system is tailored to meet individual imaging requirements; contact us using the link in the green box to the upper right for additional information and to receive a quote.

For applications that require rapid imaging of live systems, our galvo-resonant scan head is capable of capturing up to 400 fps. Alternatively, microscopes with galvo-galvo scanners are ideal for experiments involving photo-uncaging. Select one of four pre-configured laser sources with up to four excitation wavelengths. A motorized pinhole wheel with 16 round pinholes provides true diffraction-limited imaging while allowing the user to optimize the pinhole size for their objective. Multi-spectral imaging is enabled by a two- or four-channel detection module with an included filter set that, together with the laser source, has been optimized for the excitation and emission wavelengths of popular fluorophores. Choose from standard multialkali photomultiplier tubes (PMTs) for samples with a large dynamic range or high-sensitivity GaAsP PMTs to image weakly fluorescent samples.

Each microscope includes a PC with DAQ card and the ThorImageLS data acquisition software. ThorImageLS was developed in conjunction with our laser scanning microscopy systems to provide a seamless, logical, intuitive program for acquiring and analyzing images. This open-source software package enables synchronization of external hardware and events, multi-dimensional data acquisition and display, region-of-interest scanning, and multi-user operation. All images are saved in the standard TIFF image format so that they can be viewed using software packages such as ImageJ/Fiji. See the ThorImageLS tab for additonal information on ThorImageLS features. Upon the purchase of a confocal system, Thorlabs provides lifetime support for the ThorImageLS package.

To explore the full range of possible system configurations, including laser source and emission filter options, see the Confocal System tab. For system specifications, please see the Specs tab.

Quick Links
Confocal Scanner
Laser Excitation
Image Detection

Thorlabs' Confocal Microscopy System

Thorlabs’ Four-Channel Confocal Laser Scanning Microscopy Systems consist of a Cerna®-based microscope with a galvo-galvo or galvo-resonant scanner, monolithic laser source, multi-channel PMT detection module control electronics, pinhole wheel, and all fibers and cables needed to interconnect the system. All hardware components are directly controlled by ThorImage®LS software, including automated Z-step control for optical sectioning (via a piezo or stepper motor) and automatic calculation of Airy disk units based on the combination of the objective magnification and pinhole size; the intuitive interface allows novice and experienced users alike to obtain high-resolution microscope images quickly and easily.

Thorlabs' applications engineers install each confocal system and are available to address technical problems that may occur; for additional information, please see our Confocal Microscopy Contact Form.

Scanner

At the heart of our systems is a confocal scan head that can be configured with galvo-galvo or galvo-resonant scanners. Galvo-galvo scanners allow specific regions of interest to be targeted for experiments requiring photo-uncaging, while galvo-resonant scanners support high imaging speeds of up to 400 frames per second (at 512 x 32 pixel resolution). Both types of scanners can create full frame images with high spatial resolution of up to 4096 x 4096 pixels. At either extreme, or anywhere in-between, the control and acquisition system creates high-quality, jitter-free images.

The confocal scan path is integrated with a microscope body based on our Cerna Modular Microscopy System, allowing the system to be customized with widefield imaging accessories. A D1N dovetail on top of the scan path accepts single or double camera ports for scientific cameras, trinoculars, and epi-illuminators. A 95 mm dovetail along the length of the body accepts sample holders including a Z-axis piezo stage, condenser modules, or transmitted illumination modules for brightfield, Dodt contrast, or DIC imaging. In the most basic configuration, a mirror on a manual slider at the front of the scan path allows the user to switch between confocal and widefield imaging modalities. This optic can be upgraded to a dichroic or beamsplitter and the manual slider upgraded to a motorized one.

Our complete systems come standard with a primary dichroic that reflects four laser lines (405 nm, 488 nm, 532 nm, and 642 nm). Other primary dichroics for use with other wavelengths can be provided upon request.

MPH16 without Fiber Connector
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Our A-coated motorized filter wheel shown disconnected from the confocal scan head with the SMA fiber connector removed for free-space output.

Motorized Pinhole Wheel
A motorized pinhole wheel allows the pinhole size to be adjusted for a variety of imaging configurations and objective numerical apertures in order to simultaneously maximize the in-focus light that reaches the PMT detectors and minimize the transmission of signal from outside the focal plane. A rotating glass plate has 16 sizes of lithographic round pinholes deposited to exceedingly tight tolerances, ensuring that optimal alignment is maintained as each pinhole is rotated into the light path.

For thicker samples, the size of the pinhole should be optimized relative to the NA of the objective in order to maximize the signal to noise ratio. With this in mind, our engineers selected each pinhole size to complement a common objective NA. Conversely, for thinner samples that produce less light outside of the focal plane, a larger pinhole size can help improve throughput. Pinhole diameters up to 2 mm provide flexibility so that the system can be easily adapted to different experiments.

A round pinhole is the ideal shape for maximizing the transmission of light generated in the focal plane of your sample while also optimizing the rejection of signal generated above and below the layer that is being scanned.

The pinhole is conveniently powered and controlled via USB. Additionally, the motorized, encoded control of the pinhole ensures perfect alignment and vibration-free movement. The emitted light from the specimen is focused on the pinhole by an achromatic doublet and then collected by a large-core multimode fiber for transmission to the PMT detector system. To maximize transmission at the desired emission wavelengths, three AR coating options are available for the achromatic doublet.

Confocal Microscope Laser Source
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Four-Channel Laser Source

Excitation

Confocal Laser Source

The solid state multi-laser source minimizes maintenance with an all-fiber design. Each laser line is individually fiber-coupled using a permanent rigid system. The individual fiber-coupled lasers are then combined in an all-fiber coupler with an FC/PC connector. This design ensures the lasers never go out of alignment by keeping the full power of the lasers coupled to the scan head at all times.

Each confocal system also includes a filter set, chosen to complement the excitation wavelengths. Available pre-configured laser source wavelength combinations and the included filter sets are outlined in the table below.

The laser source is controlled by a controller box, which includes a safety interlock system and manual control of laser emission. The laser source can also be controlled remotely by way of an auxiliary input.

Laser Source Options
Laser Source
Identification #a		 # of
Lasersb		 Excitation Wavelengths Included Emission Filters
UV Blue Green Red Emission Filters
(Center Wavelength/Bandwidth)		 Longpass Dichroic Cutoff Wavelength(s)
CLS3 2 - 488 nm 561 nm - 525 nm/45 nm and 600 nm/52 nm 573 nm
CLS5 3 405 nm 488 nm 561 nm - 440 nm/40 nm, 525 nm/45 nm, and 600 nm/52 nm 495 nm and 573 nm
CLS4 3 - 488 nm 561 nm 642 nm 525 nm/45 nm, 600 nm/52 nm, and 647 nm/Longpass 561 nm and 635 nm
CLS6 4 405 nm 488 nm 561 nm 642 nm 440 nm/40 nm, 525 nm/45 nm, 600 nm/52 nm, and 647 nm/Longpass 495 nm, 573 nm, and 647 nm
  • The laser source is not offered separately from the Confocal System, but we have provided a laser source identification # here for convenience when discussing a system configuration with one of our representatives.
  • For sources with fewer than four lasers, slots will be filled from left to right.

Photomultiplier Module
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Expandable PMT modules are designed for multi-channel laser scanning microscopy applications. The photo shows the four-channel module with standard sensitivity PMTs.

Detection

405 nm, 488 nm, 561 nm, and 642 nm Laser Configuration
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The solid lines in the plot show the pass bands of the filters, and the shaded areas show the normalized emission spectra of the fluorophores.
Photomultiplier Module
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The filter sets are mounted in dichroic filter cubes for easy exchange. Here, an emission filter is being removed from a two-channel PMT module with high-sensitivity PMTs.

Detector: Future-proof your experiments with our remotely positioned detector module that can be readily expanded from two to four photomultiplier tubes (PMTs). Sitting in front of each PMT is a quickly exchangeable dichroic mirror and emission filter holder (for included filter sets, see the table above).

The detection module can be configured with our standard sensitivity multialkali PMTs or high sensitivity, ultra-low-noise GaAsP PMTs. The standard sensitivity multi-alkali PMTs provide a low-noise image with high dynamic range that is appropriate for most life-science and industrial applications. For weakly fluorescent or highly photosensitive samples, we also offer the option of high-sensitivity, TEC-cooled GaAsP PMTs. With either choice, the gain of the detector as well as the dynamic range of the digitizer is controlled within the ThorImageLS software.

Filters: Each confocal upgrade includes an appropriate set of emission filters that block laser light from entering the PMTs and provide pass bands at the fluorescence wavelengths of popular fluorophores. The exact configuration is determined by the laser wavelengths in your confocal system. Several common configurations and compatible fluorophores are outlined below. If you have questions concerning the filter set included with a specific laser configuration, please contact ImagingSales@thorlabs.com for more information.

Computer: Each confocal system includes a 64-bit computer with a 24" monitor. The included ThorImageLS software package provides an all-in-one solution for microscope control, automated data collection, and image review. See the ThorImageLS tab for more information.

Sample Filter Configurations

A sample plot of emission filter pass bands is provided to the right as an additional example of how Thorlabs' Confocal System can work with popular fluorophores.

The primary dichroic and emission filter sets in the confocal system are typically optimized for one of two excitation wavelength configurations. The most popular configuration is compatible with 405 nm, 488 nm, 561 nm, and 642 nm excitation lasers. The graph to the right provides an example of this common emission filter configuration, with the emission spectra of four compatible fluorophores superimposed on the filters' transmission profiles.

Thorlabs recognizes that each imaging application has unique requirements.
If you have any feedback, questions, or need a quotation, please contact ImagingSales@thorlabs.com or call (703) 651-1700.

Specifications for Thorlabs' Confocal System are provided in the tables below. For a description of the available modules, see the Confocal System tab. For more information, contact our sales team and applications engineers at ImagingSales@thorlabs.com or (703) 651-1700.

Specifications
Excitation
Laser Source 1 to 4 Channels (See Table Below for Pre-Configured Options)
Primary Dichroic Mirror Quad-Band Dichroic Beamsplitter
(Other Dichroics Available upon Request)
Scanning
Scan Head Galvo-Resonant Scan Head with 8 kHz Resonant Scanner (X) and Galvo Scan Mirror (Y)
Galvo-Resonant Scanning Speed 30 Frames per Second at 512 x 512 Pixels
400 Frames per Second at 512 x 32 Pixels
2 Frames per Second at 4096 x 4096 Pixels
Scan Zoom Up to 2048 x 2048 Bi-Directional Acquisition; Up to 4096 x 4096 Uni-Directional Acquisition
1X - 16X (Continuous)
Digitization / Sampling Density Up to 2048 x 2048 Bi-Directional Acquisition; Up to 4096 x 4096 Uni-Directional Acquisition
Diffraction-Limited Field of View (FOV) FN25 = 442 µm x 442 µm FOV @ 40X; FN23 = 407 µm x 407 µm FOV @ 40X
Emission
Photomultiplier Tubes (PMTs) Standard Multialkali or High-Sensitivity GaAsP
Detection Channels 1 to 4 PMTs
Filters Emission Filter Set and Longpass Dichroic to Complement Multi-Channel Laser Source
(See Table Below for Pre-Configured Options)

The laser wavelengths offered as part of the confocal system are available in one of the pre-configured integrated laser sources listed below. Each source is paired with a set of filters optimized for popular fluorophores that can be mounted in the PMT detection module. If you need help selecting a laser source for you system, you can contact our applications engineers by e-mail at ImagingSales@thorlabs.com or by calling (703) 651-1700.

Laser Source Options
Laser Source
Identification #a		 # of
Lasersb		 Excitation Wavelengths Included Emission Filters
UV Blue Green Red Emission Filters
(Center Wavelength/Bandwidth)		 Longpass Dichroic Cutoff Wavelength(s)
CLS3 2 - 488 nm 561 nm - 525 nm/45 nm and 600 nm/52 nm 573 nm
CLS5 3 405 nm 488 nm 561 nm - 440 nm/40 nm, 525 nm/45 nm, and 600 nm/52 nm 495 nm and 573 nm
CLS4 3 - 488 nm 561 nm 642 nm 525 nm/45 nm, 600 nm/52 nm, and 647 nm/Longpass 561 nm and 635 nm
CLS6 4 405 nm 488 nm 561 nm 642 nm 440 nm/40 nm, 525 nm/45 nm, 600 nm/52 nm, and 647 nm/Longpass 495 nm, 573 nm, and 647 nm
  • The laser source is not offered separately from the Confocal System, but we have provided a laser source identification # here for convenience when discussing a system configuration with one of our representatives.
  • For sources with fewer than four lasers, slots will be filled from left to right.

Thorlabs recognizes that each imaging application has unique requirements.
If you have any feedback, questions, or need a quotation, please contact ImagingSales@thorlabs.com or call (703) 651-1700.

Sam Tesfai
Sam Tesfai
General Manager,
Thorlabs Imaging Systems

Questions?
Feedback?
Need a Quote?

Contact TSI
Epi-Illuminator Module
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Epi-Illuminator Module Add-On

Post-Installation Upgrades & Add-Ons

Each Four-Channel Confocal Microscope is designed so that imaging capabilities can be added or substituted after installation to accommodate new experimental needs as your research requirements grow. Some upgrades, such as adding Cerna® widefield imaging accessories, can be easily performed by the user. Others require replacing part of the hardware, in which case assistance will be provided by our technical staff; contact us using the link in the green box to the right for additional information.

Widefield Imaging Add-Ons

Confocal Capability Upgrades

Images Obtained with Thorlabs' Confocal Systems

The full source code for ThorImage®LS is available for owners of a Bergamo®, Cerna® Confocal, Veneto®, or confocal microscope. Click here to receive your copy.

ThorImageLS Brochure

ThorImage®LS Software

ThorImageLS is an open-source image acquisition program that controls Thorlabs' microscopes, as well as supplementary external hardware. From prepared-slice multiphoton Z-stacks to simultaneous in vivo photoactivation and imaging, ThorImageLS provides an integrated, modular workspace tailored to the individual needs of the scientist. Its workflow-oriented interface supports single image, Z-stacks, time series, and image streaming acquisition, visualization, and analysis. See the video to the lower right for a real-time view of data acquisition and analysis with ThorImageLS.

ThorImageLS is included with a Thorlabs microscope system purchase and is open source, allowing full customization of software features and performance. ThorImageLS also includes Thorlabs’ customer support and regular software updates to continually meet the imaging demands of the scientific community.

For additional details, see the full web presentation.

Advanced Software Functionality

  • Multi-Column Customizable Workspace
  • Image Acquisition Synced with Hardware Inputs and Timing Events
  • Live Image Correction and ROI Analysis
  • Independent Galvo-Galvo and Galvo-Resonant Scan Areas and Geometries
  • Tiling for High-Resolution Large-Area Imaging
  • Independent Primary and Secondary Z-Axis Control for Fast Deep-Tissue Scans
  • Automated Image Capture with Scripts
    • Compatible with ImageJ Macros
  • Multi-User Settings Saved for Shared Workstations
  • Individual Colors for Detection Channels Enable Simple Visual Analysis

Seamless Integration with Experiments

  • Simultaneous Multi-Point Photoactivation and Imaging with Spatial Light Modulator
  • Fast Z Volume Acquisition with PFM450E or Third-Party Objective Scanners
  • Electrophysiology Signaling
  • Wavelength Switching with Coherent Chameleon Lasers
  • Pockels Cell ROI Masking
  • Power Ramped with Depth to Minimize Damage and Maximize Signal-to-Noise

Features of ThorImage®LS

Supported Imaging Platforms

Veneto® Inverted Microscopes
Bergamo® III Multiphoton Microscopes
Confocal Imaging
Systems

Laser Scanning Microscopy Tutorial

Laser scanning microscopy (LSM) is an indispensable imaging tool in the biological sciences. In this tutorial, we will be discussing confocal fluorescence imaging, multiphoton excitation fluorescence imaging, and second and third harmonic generation imaging techniques. We will limit our discussions to point scanning of biological samples with a focus on the technology behind the imaging tools offered by Thorlabs.

Introduction

The goal of any microscope is to generate high-contrast, high-resolution images. In much the same way that a telescope allows scientists to discern the finest details of the universe, a microscope allows us to observe biological functioning at the nanometer scale. Modern laser scanning microscopes are capable of generating multidimensional data (X, Y, Z, τ, λ), leading to a plethora of high-resolution imaging capabilities that further the understanding of underlying biological processes.

In conventional widefield microscopy (Figure 1, below left), high-quality images can only be obtained when using thin specimens (on the order of one to two cell layers thick). However, many applications require imaging of thick samples, where volume datasets or selection of data from within a specific focal plane is desired. Conventional widefield microscopes are unable to address these needs.

LSM, in particular confocal LSM and multiphoton LSM, allows for the visualization of thin planes from within a thick bulk sample, a technique known as optical sectioning. In confocal LSM, signals generated by the sample outside of the optical focus are physically blocked by an aperture, preventing their detection. Multiphoton LSM, as we will discuss later, does not generate any appreciable signal outside of the focal plane. By combining optical sectioning with incremented changes in focus (Figure 2, below right), laser scanning microscopy techniques can recreate 3D representations of thick specimen.

 

Figure 1 Widefield Epi-Fluorescence

Wide Field Epi-Fluorescence
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Figure 2 Optical Sections (Visualization of Thin Planes within a Bulk Sample)

Optical Sectioning in Confocal Microscopy

Optical Sectioning in Confocal Microscopy
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Optical Sectioning in Multiphoton Microscopy

Optical Sectioning in Multiphoton Microscopy
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Signal generated by the sample is shown in green. Optical sections are formed by discretely measuring the signal generated within a specific focal plane. In confocal LSM, out-of-focus light is rejected through the use of a pinhole aperture, thereby leading to higher resolution. In multiphoton LSM, signal is only generated in the focal volume. Signal collected at each optical section can be reconstructed to create a 3D image.

 

Contrast Mechanisms in LSM

Biological samples typically do not have very good contrast, which leads to difficulty in observing the boundaries between adjacent structures. A common method for improving contrast in laser scanning microscopes is through the use of fluorescence.

In fluorescence, a light-emitting molecule is used to distinguish the constituent of interest from the background or neighboring structure. This molecule can already exist within the specimen (endogenous or auto-fluorescence), be applied externally and attached to the constituent (chemically or through antibody binding), or transfected (fluorescent proteins) into the cell.

In order for the molecule to emit light (fluoresce) it must first absorb light (a photon) with the appropriate amount of energy to promote the molecule from the ground state to the excited state, as seen in Figure 3A below. Light is emitted when the molecule returns back down to the ground state. The amount of fluorescence is proportional to the intensity (I) of the incident laser, and so confocal LSM is often referred to as a linear imaging technique. Natural losses within this relaxation process require that the emitted photon have lower energy—that is, a longer wavelength—than the absorbed photon.

Multiphoton excitation (Figure 3B, below) of the molecule occurs when two (or more) photons, whose sum energy satisfies the transition energy, arrive simultaneously. Consequently, the two arriving photons will be of lower energy than the emitted fluorescence photon.

There are also multiphoton contrast mechanisms, such as harmonic generation and sum frequency generation, that use non-absorptive processes. Under conditions in which harmonic generation is allowed, the incident photons are simultaneously annihilated and a new photon of the summed energy is created, as illustrated in Figure 3C below.

Further constituent discrimination can be obtained by observing the physical order of the harmonic generation. In the case of second harmonic generation (SHG), signal is only generated in constituents that are highly ordered and lacking inversion symmetry. Third harmonic generation (THG) is observed at boundary interfaces where there is a refractive index change. Two-photon excitation and SHG are nonlinear processes and the signal generated is dependent on the square of the intensity (I2).

The nonlinear nature of signal generation in multiphoton microscopy means that high photon densities are required to observe SHG and THG. In order to accomplish this while maintaining relatively low average power on the sample, mode-locked femtosecond pulsed lasers, particularly Ti:Sapphire lasers, have become the standard.

Another consideration to be made in nonlinear microscopy is the excitation wavelength for a particular fluorophore. One might think that the ideal excitation wavelength is twice that of the one-photon absorption peak. However, for most fluorophores, the excited state selection rules are different for one- and two-photon absorption.

This leads to two-photon absorption spectra that are quite different from their one-photon counterparts. Two-photon absorption spectra are often significantly broader (can be >100 nm) and do not follow smooth semi-Gaussian curves. The broad two-photon absorption spectrum of many fluorophores facilitates excitation of several fluorescent molecules with a single laser, allowing the observation of several constituents of interest simultaneously.

All of the fluorophores being excited do not have to have the same excitation peak, but should overlap each other and have a common excitation range. Multiple fluorophore excitation is typically accomplished by choosing a compromising wavelength that excites all fluorophores with acceptable levels of efficiency.

 

Figure 3 Signal Generation in Laser Scanning Microscopy

LSM Signal Generation
Click to Enlarge

Absorptive Process (A, B):

The absorption of one or more excitation photons (λEX) promotes the molecule from the ground state (S0) to the excited state (S1). Fluorescence (λEM) is emitted when the molecule returns to the ground state.

Non-Absorptive Process (C):

The excitation photons (λEX) simultaneously convert into a single photon (λSHG,THG) of the sum energy and half (for SHG) or one-third (for THG) the wavelength.

Non-Radiative Energy Losses

 

Image Formation

In a point-scanning LSM, the single-plane image is created by a point illumination source imaged to a diffraction-limited spot at the sample, which is then imaged to a point detector. Two-dimensional en face images are created by scanning the diffraction-limited spot across the specimen, point by point, to form a line, then line by line in a raster fashion.

The illuminated volume emits a signal which is imaged to a single-element detector. The most common single-element detector used is a photomultiplier tube (PMT), although in certain cases, avalanche photodiodes (APDs) can be used. CCD cameras are not typically used in point-scanning microscopes, though are the detector of choice in multifocal (i.e. spinning disk confocal) applications.

The signal from the detector is then passed to a computer which constructs a two-dimensional image as an array of intensities for each spot scanned across the sample. Because no true image is formed, LSM is referred to as a digital imaging technique. A clear advantage of single-point scanning and single-point detection is that the displayed image resolution, optical resolution, and scan field can be set to match a particular experimental requirement and are not predefined by the imaging optics of the system.

Figure 4 Confocal Optical Path

Confocal Optical Path

Confocal LSM

In confocal LSM, point illumination, typically from a single mode, optical-fiber-coupled CW laser, is the critical feature that allows optical sectioning. The light emitted from the core of the single mode optical fiber is collimated and used as the illumination beam for scanning. The scan system is then imaged to the back aperture of the objective lens which focuses the scanned beam to a diffraction-limited spot on the sample. The signal generated by the focused illumination beam is collected back through the objective and passed through the scan system.

After the scan system, the signal is separated from the illumination beam by a dichroic mirror and brought to a focus. The confocal pinhole is located at this focus. In this configuration, signals that are generated above or below the focal plane are blocked from passing through the pinhole, creating the optically sectioned image (Figure 2, above). The detector is placed after the confocal pinhole, as illustrated in Figure 4 to the right. It can be inferred that the size of the pinhole has direct consequences on the imaging capabilities (particularly, contrast, resolution and optical section thickness) of the confocal microscope.

The lateral resolution of a confocal microscope is determined by the ability of the system to create a diffraction-limited spot at the sample. Forming a diffraction-limited spot depends on the quality of the laser beam as well as that of the scan optics and objective lens.

The beam quality is typically ensured by using a single mode optical fiber to deliver the excitation laser light as a Gaussian point source, which is then collimated and focused into a diffraction-limited beam. In an aberration-free imaging system, obtained by using the highest quality optical elements, the size of this focus spot, assuming uniform illumination, is a function of excitation wavelength (λEX) and numerical aperture (NA) of the objective lens, as seen in Equation 1.

Spot Size

Equation 1 Spot Size

In actuality, the beam isn't focused to a true point, but rather to a bullseye-like shape. The spot size is the distance between the zeros of the Airy disk (diameter across the middle of the first ring around the center of the bullseye) and is termed one Airy Unit (AU). This will become important again later when we discuss pinhole sizes.

The lateral resolution of the imaging system is defined as the minimum distance between two points for them to be observed as two distinct entities. In confocal (and multiphoton) LSM, it is common and experimentally convenient to define the lateral resolution according to the full width at half maximum (FWHM) of the individual points that are observed.

Using the FWHM definition, in confocal LSM, the lateral resolution (Rlateral,confocal) is:

Lateral Resolution, Confocal

Equation 2 Lateral Resolution, Confocal LSM

and the axial resolution (Raxial,confocal) is:

Axial Resolution, Confocal

Equation 3 Axial Resolution, Confocal LSM

where n is the refractive index of the immersion medium.

It is interesting to note that in a confocal microscope, the lateral resolution is solely determined by the excitation wavelength. This is in contrast to widefield microscopy, where lateral resolution is determined only by emission wavelength.

To determine the appropriate size of the confocal pinhole, we multiply the excitation spot size by the total magnification of the microscope:

Pinhole Diameter

Equation 4 Pinhole Diameter

As an example, the appropriate size pinhole for a 60X objective with NA = 1.0 for λEX = 488 nm (Mscan head = 1.07 for the Thorlabs Confocal Scan Head) would be 38.2 μm and is termed a pinhole of 1 AU diameter. If we used the same objective parameters but changed the magnification to 40X, the appropriate pinhole size would be 25.5 μm and would also be termed a pinhole of 1 AU diameter. Therefore, defining a pinhole diameter in terms of AU is a means of normalizing pinhole diameter, even though one would have to change the pinhole selection for the two different objectives.

Theoretically, the total resolution of a confocal microscope is a function of the excitation illumination spot size and the detection pinhole size. This means that the resolution of the optical system can be improved by reducing the size of the pinhole. Practically speaking, as we restrict the pinhole diameter, we improve resolution and confocality, but we also reduce the amount of signal reaching the detector. A pinhole of 1 AU is a good balance between signal strength, resolution, and confocality.

Figure 5 Multiphoton Optical Path

Multiphoton Optical Path

Multiphoton LSM

In multiphoton LSM, a short pulsed free-space laser supplies the collimated illumination beam that passes through the scanning system and is focused by the objective. The very low probability of a multiphoton absorption event occurring, due to the I2 dependence of the signal on incident power, ensures signal is confined to the focal plane of the objective lens. Therefore, very little signal is generated from the regions above and below the focal plane. This effective elimination of out-of-focus signal provides inherent optical sectioning capabilities (Figure 2, above) without the need for a confocal pinhole. As a result of this configuration, the collected signal does not have to go back through the scanning system, allowing the detector to be placed as close to the objective as possible to maximize collection efficiency, as illustrated in Figure 5 to the right. A detector that collects signal before it travels back through the scan system is referred to as a non-descanned detector.

Again using the FWHM defintion, in multiphoton LSM, the lateral resolution (Rlateral,multiphoton) is:

Lateral Resolution, Multiphoton

Equation 5 Lateral Resolution, Multiphoton LSM

and the axial resolution (Raxial,multiphoton) is:

Axial Resolution, Multiphoton

Equation 6 Axial Resolution, Multiphoton LSM

These equations assume an objective NA > 0.7, which is true of virtually all multiphoton objectives.

The longer wavelength used for multiphoton excitation would lead one to believe (from Equation 5) that the resolution in multiphoton LSM, compared to confocal LSM, would be reduced roughly by a factor of two. For an ideal point object (i.e. a sub-resolution size fluorescent bead) the I2 signal dependence reduces the effective focal volume, more than offsetting the 2X increase in the focused illumination spot size.

We should note that the lateral and axial resolutions display a dependence on intensity. As laser power is increased, there is a corresponding increase in the probability of signal being generated within the diffraction-limited focal volume. In practice, the lateral resolution in a multiphoton microscope is limited by how tightly the illumination beam can be focused and is well approximated by Equation 5 at moderate intensities. Axial resolution will continue to degrade as excitation power is increased.

 

Image Display

Although we are not directly rendering an image, it is still important to consider the size of the image field, the number of pixels in which we are displaying our image (capture resolution) on the screen, and the lateral resolution of the imaging system. We use the lateral resolution because we are rendering an en face image. In order to faithfully display the finest features the optical system is capable of resolving, we must appropriately match resolution (capture and lateral) with the scan field. Our capture resolution must, therefore, appropriately sample the optical resolution.

In LSM, we typically rely on Nyquist sampling rules, which state that the pixel size should be the lateral resolution divided by 2.3. This means that if we take our 60X objective from earlier, the lateral resolution is 249 nm (Equation 2) and the pixel size in the displayed image should be 108 nm. Therefore, for a 1024 x 1024 pixel capture resolution, the scan field on the specimen would be ~111 μm x 111 μm. It should be noted that the 40X objective from our previous example would yield the exact same scan field (both objectives have the same NA) in the sample. The only difference between the two images is the angle at which we tilt our scanners to acquire the image.

It may not always be necessary to render images with such high resolution. We can always make the trade-off of image resolution, scan field, and capture resolution to create a balance of signal, sample longevity, and resolution in our images.

 

Considerations in Live Cell Imaging

One of LSM's greatest attributes is its ability to image living cells and tissues. Unfortunately, some of the by-products of fluorescence can be cytotoxic. As such, there is a delicate balancing act between generating high-quality images and keeping cells alive.

One important consideration is fluorophore saturation. Saturation occurs when increasing the laser power does not provide the expected concurrent increase in the fluorescence signal. This can occur when as few as 10% of the fluorophores are in the excited state.

The reason behind saturation is the amount of time a fluorophore requires to relax back down to the ground state once excited. While the fluorescence pathways are relatively fast (hundreds of ps to a few ns), this represents only one relaxation mechanism. Triplet state conversion and nonradiative decay require significantly longer relaxation times. Furthermore, re-exciting a fluorophore before it has relaxed back down to the ground state can lead to irreversible bleaching of the fluorophore. Cells have their own intrinsic mechanisms for dealing with the cytotoxicity associated with fluorescence, so long as excitation occurs slowly.

One method to reduce photobleaching and the associated cytotoxicity is through fast scanning. While reducing the amount of time the laser spends on a single point in the image will proportionally decrease the amount of detected signal, it also reduces some of the bleaching mechanisms by allowing the fluorophore to completely relax back to the ground state before the laser is scanned back to that point. If the utmost in speed is not a critical issue, one can average several lines or complete frames and build up the signal lost from the shorter integration time.

The longer excitation wavelength and non-descanned detection ability of multiphoton LSM give the ability to image deeper within biological tissues. Longer wavelengths are less susceptible to scattering by the sample because of the inverse fourth power dependence (I-4) of scattering on wavelength. Typical penetration depths for multiphoton LSM are 250 - 500 μm, although imaging as deep as 1 mm has been reported in the literature, compared to ~100 μm for confocal LSM.

Thorlabs recognizes that each imaging application has unique requirements.
If you have any feedback, questions, or need a quotation, please contact ImagingSales@thorlabs.com or call (703) 651-1700.

Thorlabs Dovetail Reference a
TypeShapeOuter DimensionAngle
95 mmLinear95 mm45°
D1NCircularØ2.018"60°
D2NbCircularØ1.50"90°
D2NBbCircularØ1.50"90°
D3NCircularØ45 mm70°
D5NCircularØ1.58"90°
D6NCircularØ1.90"90°
D7NCircularØ2.05"90°
D1TCircularØ1.50"60°
D3TCircularØ1.65"90°
D1YCircularØ107 mm60°
D2YCircularØ2.32"50°
D3YCircularØ1.75"90°
D4YCircularØ56 mm60°
D5YCircularØ46 mm60°
D6YCircularØ41.9 mm45°
D1ZCircularØ54 mm60°
D2ZCircularØ57 mm60°
D3ZCircularØ54 mm45°
  • These dovetail designations are specific to Thorlabs products and are not used by other microscope manufacturers.
  • D2N and D2NB dovetails have the same outer diameter and angle, as defined by the drawings below. The D2N designation does not specify a height. The D2NB designation specifies a dovetail height of 0.40" (10.2 mm).
Mating Circular Dovetails
Click to Enlarge
This photo shows the male D1N dovetail on the trinoculars next to the female D1N dovetail on the epi-illumination arm.
Mating Linear Dovetails
Click to Enlarge
This photo shows the male 95 mm dovetail on the microscope body and the female 95 mm dovetail on the CSA1002 Fixed Arm.

Introduction to Microscope Dovetails

Dovetails are used for mechanical mating and optical port alignment of microscope components. Components are connected by inserting one dovetail into another, then tightening one or more locking setscrews on the female dovetail. Dovetails come in two shapes: linear and circular. Linear dovetails allow the mating components to slide before being locked down, providing flexible positioning options while limiting unneeded degrees of freedom. Circular dovetails align optical ports on different components, maintaining a single optical axis with minimal user intervention.

Thorlabs manufactures many components which use dovetails to mate with our own components or those of other manufacturers. To make it easier to identify dovetail compatibility, we have developed a set of dovetail designations. The naming convention of these designations is used only by Thorlabs and not other microscope manufacturers. The table to the right lists all the dovetails Thorlabs makes, along with their key dimensions.

In the case of Thorlabs’ Cerna® microscopes, different dovetail types are used on different sections of the microscope to ensure that only compatible components can be mated. For example, our WFA2002 Epi-Illuminator Module has a male D1N dovetail that mates with the female D1N dovetail on the microscope body's epi-illumination arm, while the CSS2001 XY Microscopy Stage has a female D1Y dovetail that mates with the male D1Y dovetail on the CSA1051 Mounting Arm.

To learn which dovetail type(s) are on a particular component, consult its mechanical drawing, available by clicking on the red Docs icon (Docs Icon) below. For adapters with a female dovetail, the drawing also indicates the size of the hex key needed for the locking setscrew(s). It is important to note that mechanical compatibility does not ensure optical compatibility. Information on optical compatibility is available from Thorlabs' web presentations.

For customers interested in machining their own dovetails, the table to the right gives the outer diameter and angle (as defined by the drawings below) of each Thorlabs dovetail designation. However, the dovetail's height must be determined by the user, and for circular dovetails, the user must also determine the inner diameter and bore diameter. These quantities can vary for dovetails of the same type. One can use the intended mating part to verify compatibility.

In order to reduce wear and simplify connections, dovetails are often machined with chamfers, recesses, and other mechanical features. Some examples of these variations are shown by the drawings below.

Male Microscope Dovetails
Click to Enlarge
Two examples of how circular male dovetails can be manufactured.
Female Microscope Dovetails
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Two examples of how circular female dovetails can be manufactured.

To schedule an in-person or virtual demo appointment, please email ImagingSales@thorlabs.com.

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Try Our Microscopes In Person or Virtually

Thorlabs' sales engineers and field service staff are based out of nine offices across four continents. We look forward to helping you determine the best imaging system to meet your specific experimental needs. Our customers are attempting to solve biology's most important problems; these endeavors require matching systems that drive industry standards for ease of use, reliability, and raw capability.

Thorlabs' worldwide network allows us to operate demo rooms in a number of locations where you can see our systems in action. We welcome the opportunity to work with you in person or virtually. A demo can be scheduled at any of our showrooms or virtually by contacting ImagingSales@thorlabs.com.

Customer Support Sites
(Click Each Location for More Details)

Demo Rooms and Customer Support Sites
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Selected Confocal Microscopy Publications

2023

 

Egorova, P. A., Marinina, K. S., & Bezprozvanny, I. B. Chronic suppression of STIM1-mediated calcium signaling in Purkinje cells rescues the cerebellar pathology in spinocerebellar ataxia type 2. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 119466 (2023).

2022

 

Pchitskaya, E., Rakovskaya, A., Chigray, M., & Bezprozvanny, I. Cytoskeleton Protein EB3 Contributes to Dendritic Spines Enlargement and Enhances Their Resilience to Toxic Effects of Beta-Amyloid. International journal of molecular sciences, 23(4), 2274 (2022).

Simonova, N. A., Volgushev, M. A., & Malyshev, A. Y. Enhanced Non-Associative Long-Term Potentiation in Immature Granule Cells in the Dentate Gyrus of Adult Rats. Frontiers in Synaptic Neuroscience, 14 (2022).

Zernov, N., Ghamaryan, V., Makichyan, A., Melenteva, D., Hunanyan, L., & Popugaeva, E. Piperazine Derivative Stabilizes Actin Filaments in Primary Fibroblasts and Binds G-Actin In Silico. Current Issues in Molecular Biology, 44(11), 5191-5208 (2022).

Zernov, N., Bezprozvanny, I., & Popugaeva, E. CaMKIIβ knockdown decreases store-operated calcium entry in hippocampal dendritic spines. IBRO Neuroscience Reports, 12, 90-97 (2022).

2021

 

Kraskovskaya, N. A., Erofeev, A. I., Grishina, E. D., Pushkareva, S. A., Gerasimov, E. I., Vlasova, O. L., & Bezprozvanny, I. B. Development of hippocampus-associated cognitive dysfunction in Huntington’s disease mouse model. Journal of Evolutionary Biochemistry and Physiology, 57, 1449-1460 (2021).

2020

 

Caccavano, A. et al. Inhibitory Parvalbumin Basket Cell Activity is Selectively Reduced during Hippocampal Sharp Wave Ripples in a Mouse Model of Familial Alzheimer's Disease. The Journal of Neuroscience
5116–5136 (2020).

Dark, C., Williams, C., Bellgrove, M. A., Hawi, Z., & Bryson-Richardson, R. J. Functional validation of CHMP7 as an ADHD risk gene. Translational Psychiatry, 10(1), 385 (2020).

2016

 

Lewin, A. E. et al. Optogenetic and pharmacological evidence that somatostatin-GABA neurons are important regulators of parasympathetic outflow to the stomach. The Journal of Physiology n/a-n/a (2016). doi:10.1113/JP272069

2015

 

Dechen, K., Richards, C. D., Lye, J. C., Hwang, J. E. C. & Burke, R. Compartmentalized zinc deficiency and toxicities caused by ZnT and Zip gene over expression result in specific phenotypes in Drosophila. The International Journal of Biochemistry & Cell Biology 60, 23–33 (2015).

Jia, Y. et al. Activation of platelet protease-activated receptor-1 induces epithelial-mesenchymal transition and chemotaxis of colon cancer cell line SW620. Oncol. Rep. 33, 2681–2688 (2015).

Lu, W. et al. Inhibiting the mobilization of Ly6Chigh monocytes after acute myocardial infarction enhances the efficiency of mesenchymal stromal cell transplantation and curbs myocardial remodeling. Am J Transl Res 7, 587–597 (2015).

Lu, W. et al. Photoluminescent Mesoporous Silicon Nanoparticles with siCCR2 Improve the Effects of Mesenchymal Stromal Cell Transplantation after Acute Myocardial Infarction. Theranostics 5, 1068–1082 (2015).

Zuo, S., Hughes, M. & Yang, G.-Z. Novel Balloon Surface Scanning Device for Intraoperative Breast Endomicroscopy. Ann Biomed Eng 1–14 (2015). doi:10.1007/s10439-015-1493-2

2014

 

Brown, C. M., Melcher, J. T. & Stranick, S. J. Scan Linearization for Resonant Optomechanical Systems. in IM1C.3 (OSA, 2014). doi:10.1364/ISA.2014.IM1C.3

Partridge, J. G., Lewin, A. E., Yasko, J. R. & Vicini, S. Contrasting actions of group I metabotropic glutamate receptors in distinct mouse striatal neurones. J Physiol 592, 2721–2733 (2014).

Qin, X., Qiu, C. & Zhao, L. Maslinic acid protects vascular smooth muscle cells from oxidative stress through Akt/Nrf2/HO-1 pathway. Mol Cell Biochem 390, 61–67 (2014).

9.Liu, J. et al. Oleanolic Acid Suppresses Aerobic Glycolysis in Cancer Cells by Switching Pyruvate Kinase Type M Isoforms. PLOS ONE 9, e91606 (2014).

2013

 

Hao, X. et al. Contrast reversal confocal microscopy. Optics Communications 298–299, 272–275 (2013).

Lalchandani, R. R., Goes, M.-S. van der, Partridge, J. G. & Vicini, S. Dopamine D2 Receptors Regulate Collateral Inhibition between Striatal Medium Spiny Neurons. J. Neurosci. 33, 14075–14086 (2013).

Standard Mechanical Interfaces on DIY Cerna® Components

The table below gives the dovetail, optical component threads, and cage system interfaces that are present on each DIY Cerna component. If a DIY Cerna component does not have one of the standard interfaces in the table, it is not listed here. Please note that mechanical compatibility does not ensure optical compatibility. Information on optical compatibility is available from Thorlabs' web presentations.

Item # Microscope Dovetails Optical Component Threadsa Cage Systemsb
95 mm D1N D2N D2NB D3N D5N D1T D3T D1Y D5Y Internal External
2CM1 - - - - - - - - - - SM1c (1.035"-40) and SM2d (2.035"-40) SM1c (1.035"-40) 60 mmd
2CM2 - - - - - - - - - - SM1c (1.035"-40) and SM2d (2.035"-40) SM1c (1.035"-40) 30 mmc
BSA2000e - - - - Female - - - - - - - -
CEA1350 Male Female - - - - - - - - - - 60 mmd
CEA1400 Male Female - - - - - - - - - - 60 mmd
CEA1500 Male Female - - - - - - - - - - 60 mmd
CEA1600 Male Female - - - - - - - - - - 60 mmd
CFB1500 Male - - - - - - - - - - - -
CSA1000 Female - - - - - - - - - - - -
CSA1001 Female - - - - - - - - - SM1c (1.035"-40) - 30 mmc
CSA1002 Female - - - - - - - - - SM2d (2.035"-40) - 60 mmd
CSA1003 - Female - - - - - - - - - - 60 mmd
CSA1051 Female - - - - - - - Male - - - -
CSA1200e,f - - - - - - - - - - - - 60 mmd
CSA1400e - - - - - - Female - - - - - 60 mmd
CSA1500e,g - - - - - - - - - - - - -
CSA2000e - - - - Female - - - - - SM2d (2.035"-40) - 60 mmd
CSA2001 - - - - Female - - - - - - SM2d (2.035"-40) -
CSA2100e - - - - - - - - - - SM2d (2.035"-40) - 60 mmd
CSA3000(/M) - Male - - - - - - - - - - -
CSA3010(/M) - Male - - - - - - - - - - 30 mmc and 60 mmd
Item # 95 mm D1N D2N D2NB D3N D5N D1T D3T D1Y D5Y Internal External Cage Systems
CSC1001 - - - - Male - - - - - - - -
CSC1002 - - - - Male - - - - - - - -
CSC2001 - - - - Male - - - - - - - -
CSD1001 - Male & Female - Female - - - - - - - - -
CSD1002 - Male & Female - - - - - - - - - C-Mounth -
CSE2000 - Male & Female - - - - - - - - - - 60 mmd
CSE2100 - Male & Female - - - - - Female - - SM1c (1.035"-40) - 30 mmc and 60 mmd
CSE2200 - Male & Female - - - - - Female - - SM1c (1.035"-40) - 30 mmc and 60 mmd
CSN100e - - - - - - - - - - M32 x 0.75 - 60 mmd
CSN110 - - - - - - Male - - - M32 x 0.75 - 30 mmc and 60 mmd
CSNK10 - - - - - - - - - - M32 x 0.75 - 60 mmd
CSNK100e - - - - - - - - - - M32 x 0.75 - 60 mmd
CSN200 - - - - - - Male - - - M32 x 0.75 - -
CSN210 - - - - - - Male - - - M32 x 0.75 - -
CSN1201f - - - - - - - - - - M32 x 0.75 - -
CSN1202f - - - - - - - - - - M25 x 0.75 - -
CSS2001 - - - - - - - - Female - - - -
LAURE1 - Male Female - - - - - - - - - -
LAURE2 - Male Female - - - - - - - - - -
LCPN1 - - - - Male - - - - - SM30 (M30.5 x 0.5) - 30 mmc and 60 mmd
LCPN2 - Male - - - - - - - - SM30 (M30.5 x 0.5) - 30 mmc and 60 mmd
Item # 95 mm D1N D2N D2NB D3N D5N D1T D3T D1Y D5Y Internal External Cage Systems
LCPN3 - Male - - - - - - - Female SM30 (M30.5 x 0.5) - 60 mmd
LCPN4 - Male - - - - - - - - SM2d (2.035"-40) - 60 mmd
LCPN5 - - - - Male - - - - - SM2d (2.035"-40) - 60 mmd
LCPN6 - - Female - - - - - - - SM1c (1.035"-40) - 30 mmc and 60 mmd
LCPY2 - - - - - - - - - Male SM30 (M30.5 x 0.5) - 30 mmc and 60 mmd
LCPY3 - - - - - - - - - Female - - 30 mmc and 60 mmd
OPX2400(/M) - Male & Female - - - - - - - - SM2d (2.035"-40) - 60 mmd
SM1A70 - - - - - - - - - - SM30 (M30.5 x 0.5) SM1c (1.035"-40) -
SM1A58 - - Male Male - - - - - - SM1c (1.035"-40) SM2d (2.035"-40) 30 mmc
SM2A56 - - - - - - - Male - - - SM2d (2.035"-40) -
SM2A59 - Male - - - - - - - - SM2d (2.035"-40) - -
TC1X - - Male - - - - - - - - - -
WFA0150 Female - - - - - - - - - - - -
WFA1000 - - - - - - - - - - - - 30 mmc
WFA1010 - - - - - - - - - - SM1c (1.035"-40) - 30 mmc
WFA1020 - - - - - - - - - - SM1c (1.035"-40) - 30 mmc
WFA1051 - - - - - - - - - - SM1c (1.035"-40) - 30 mmc
WFA1100 - - - - - - - - - - - - 30 mmc
WFA2001 - Male & Female - - - - - - - - SM1c (1.035"-40) SM1c (1.035"-40) -
WFA2002 - Male & Female - - - - - - - - SM1c (1.035"-40) - 30 mmc
Item # 95 mm D1N D2N D2NB D3N D5N D1T D3T D1Y D5Y Internal External Cage Systems
WFA4100 - Male - - - - - - - - SM1c (1.035"-40) C-Mounth -
WFA4101 - Male - - - - - - - - SM1c (1.035"-40) C-Mounth -
WFA4102 - Male - - - - - - - - SM1c (1.035"-40) C-Mounth -
WFA4111 - Male - - - - - - - - - SM2d (2.035"-40) -
WFA4112 - - - Male - - - - - - - C-Mounth -
XT95RC1(/M) Female - - - - - - - - - - - -
XT95RC2(/M) Female - - - - - - - - - - - -
XT95RC3(/M) Female - - - - - - - - - - - -
XT95RC4(/M) Female - - - - - - - - - - - -
XT95P12(/M) Female - - - - - - - - - - - -
ZFM1020 Female - - - - - - - - - - - -
ZFM1030 Female - - - - - - - - - - - -
ZFM2020 Female - - - - - - - - - - - -
ZFM2030 Female - - - - - - - - - - - -
  • Thorlabs' optical component thread adapters can be used to convert between C-Mount threads, SM1 threads, SM2 threads, and virtually every other optical thread standard.
  • Our cage system size adapters and drop-in adapter can be used to convert between 16 mm, 30 mm, and 60 mm cage systems.
  • Our 30 mm cage plates can convert between SM1 lens tubes and 30 mm cage systems.
  • Our 60 mm cage plates can convert between SM2 lens tubes and 60 mm cage systems.
  • Attach to a ZFM focusing module to add a female 95 mm dovetail.
  • The CSA1200 mounting arm is compatible with the CSN1201 and CSN1202 nosepieces.
  • This blank arm is designed for custom DIY machining for non-standard components, threads, and bores.
  • C-Mount and CS-Mount standards feature the same 1.00"-32 threads, but C-Mounts have a 5 mm longer flange-to-sensor distance.

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