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Products HomeConfocal Microscopy Upgrade
- Convert Inverted Widefield Microscopes into Confocal Imaging Systems
- Thorlabs' Team Ensures Compatibility and Provides Lifetime Support
Four-Channel Confocal Upgrade Installed on a Nikon Microscope (Not Included)
All Confocal Upgrades include a computer, DAQ, and ThorImage®LS Data Acquisition Software.
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Thorlabs' Confocal Microscopy Upgrade
- Up to 4 Excitation/Emission Channels
- Self-Contained Scan Head Connects to a C-Mount Camera Port
- Galvo-Galvo Scanners for User-Defined Scan Geometries
- Galvo-Resonant Scanners for High-Speed Imaging up to 400 fps
- 16-Position Pinhole Wheel
- Round Pinholes for True Diffraction-Limited Imaging
- Motorized Control to Change Pinhole Size with Minimal Disturbance to the Setup
- Standard Multialkali or High-Sensitivity GaAsP PMTs
- Optional Z-Axis Stepper Motor for Z-Stacks
- Up to Video-Rate Image Acquisition: 512 x 512 Pixels at 30 fps
- Capture Resolution
- 2048 x 2048 Pixels (Bi-Directional)
- 4096 x 4096 Pixels (Uni-Directional)
- Includes Computer, DAQ Card, and ThorImage®LS Data Acquisition Software
Thorlabs' Confocal Laser Scanning (CLS) Microscopy Upgrade consists of compact modules designed to bring powerful confocal imaging tools within the reach of any research lab. 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. These CLS upgrades offer turnkey integration with virtually any inverted widefield microscope (not included) using a C-Mount camera port to access the intermediate image plane. The modular components are all developed and built in-house to maximize ease of use, simplify integration with existing imaging setups, and produce high-quality images. The included ThorImageLS software drives the CLS hardware via an intuitive graphical user interface, providing quick data recording and review.
Every Thorlabs confocal upgrade features either a galvo-resonant or galvo-galvo scan head. The galvo-resonant scanner is capable of capturing up to 400 fps and supports rapid imaging of live systems, whereas the galvo-galvo scanner allows users to target selected regions of interest on the sample for applications such as photo-uncaging experiments. Together with a
To explore the full range of possible system configurations, including laser source and emission filter options, see the Confocal Upgrade tab. For system specifications, please see the Specs tab.
Thorlabs also offers complete single-channel and four-channel confocal systems based on our Cerna® microscopy platform, allowing users to combine confocal imaging capability with our Cerna widefield imaging accessories.
Thorlabs' Confocal Microscopy Upgrade
Confocal System Schematic
Thorlabs’ Confocal Laser Scanning (CLS) Microscopy Upgrade consists of compact imaging modules specifically designed for infinity-corrected compound microscopes. They provide the ability to acquire high-resolution optical sections from within a thick sample or to reduce background fluorescence from a thin culture. The CLS upgrades offer turnkey integration to almost any inverted microscope using a C-Mount camera port to acess an intermediate image plane. The included ThorImage®LS software has an intuitive graphical interface that allows data to be quickly recorded and reviewed while providing sophisticated peripheral controls for image acquisition. CLS upgrades are user-installable, although on-site installation is also available.
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Thorlabs' Confocal Microscopy System mounted on an inverted microscope.
All hardware components are directly controlled by the ThorImageLS 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. Our intuitive interface allows novice and experienced users alike to obtain high-resolution microscope images quickly and easily.
All complete CLS upgrades include a multi-channel, fiber-coupled laser source, control electronics, scan head, pinhole wheel, detectors, and all fibers and cables needed to interconnect the system. Additionally, each system includes a Windows® computer with a 24" monitor, data acquisition hardware, control boards, and an installation of the ThorImageLS software.
Thorlabs' applications engineers install each confocal upgrade and are available to address technical problems that may occur. We also include a comprehensive installation and operation manual with basic preventative maintenance instructions to ensure that your system performs optimally for years to come. For further details on our confocal upgrades, please contact us at ImagingSales@thorlabs.com.
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The Scan Head of Thorlabs' Confocal Microscopy Upgrade
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Light Path through the Confocal Scan Head.
Scan Head
The confocal scan head module consists of three principal components: the galvo-resonant or galvo-galvo scanner, a wide field of view scan lens, and a pinhole wheel output. The scanner can be attached to virtually any inverted microscope with access to the intermediate image plane via a C-mount-threaded port (e.g., a camera port) as long as 100% of the output can be directed to this port. As shown in the drawing to the right, light enters the scan head through one of the laser inputs, is reflected onto the galvo-resonant scanner mirrors by the primary filter block, and exits the assembly through the wide field of view scan lens. Light then enters the microscope and excites fluorophores in the sample. The resulting fluorescence travels back through the scan lens and is reflected by the galvo-resonant scanner to the pinhole wheel. The pinhole wheel output is connected to the PMT detection module via an SMA-terminated fiber. Our complete systems come standard with a primary dichroic that reflects four laser lines (405, 488, 532, and 642 nm). Other primary dichroics for use with other wavelengths can be provided upon request.
Scanner
At the heart of our confocal upgrade system is an efficiently designed scan head that incorporates either an 8 kHz resonant scanner and a galvanometer for fast image acquisition or a galvo-galvo scan system to image user-defined scan geometries. The resonant scanner supports high imaging speeds of up to 400 frames per second (at 512 x 32 pixel resolution). Both systems can image 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.
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Cross-Section Schematic of the Motorized Pinhole Wheel
Wide Field of View Scan Lens
To complement the large angular range over which the resonant scanner is used, Thorlabs' engineers developed a scan lens optimized for large fields of view. The lens features excellent chromatic aberration correction from 405 to 1100 nm (antireflective coating effective from 405 to 750 nm), superb field flatness, and very low distortion within the field of view. This broad wavelength range adds to the functionality of the system by enabling the use of laser sources down to 400 nm while color correcting fluorescence emissions from even the deepest of red-emitting fluorophores. Coupled with ultra-sensitive, low-noise detectors and control electronics, we are able to provide systems with superb contrast, resolution, and imaging speed at an affordable cost.
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The motorized pinhole wheel shown disconnected from the confocal scan head with the SMA fiber connector removed for free-space output.
Motorized Pinhole Wheel
The motorized pinhole wheel (also available separately as the MPH16) 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 signal to noise. 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 over 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 and then collected by a large-core multimode fiber for transmission to the PMT detector system. More details on our motorized pinhole are available here.
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Four-Channel Laser Source
Excitation
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. 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. For added flexibility, a second fiber output is provided that is dedicated to an optional 405 nm laser diode.
The combined visible output is contained in a single mode fiber with an FC/PC connector. The optional 405 nm laser output is delivered on its own single mode fiber and is combined after the beam expander in the Scan Head module. By combining the 405 nm light after the beam expander, we are able to couple the UV laser into the light path with a 4 mm beam diameter, which increases stability and maintains the color correction of the system.
Each confocal upgrade 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 entire laser source is controlled by a single USB connection, which allows the user to turn each laser on and off as well as control its intensity.
| Laser Source Options | ||||||
|---|---|---|---|---|---|---|
| Laser Source #a |
Excitation Wavelengths | Included Emission Filters | ||||
| UV | Blue | Green/ Orange |
Red | Emission Filters (Center Wavelength/Bandwidth) |
Longpass Dichroic Cutoff Wavelength(s) | |
| CMLS-A | - | 488 nm | - | 642 nm | 525 nm/45 nm and 635 nm/Longpass | 562 nm |
| CMLS-Bb | 405 nm | 488 nm | - | 642 nm | 447 nm/60 nm, 512/25 nm, and 635 nm/Longpass | 495 nm and 538 nm |
| CMLS-C | - | 488 nm | 532 nm | 642 nm | 513 nm/17 nm, 582 nm/75 nm, and 635 nm Longpass | 538 nm and 649 nm |
| CMLS-D | - | 488 nm | 561 nm | 642 nm | 525 nm/45 nm, 607 nm/36 nm, and 535 nm/Longpass | 562 nm and 649 nm |
| CMLS-Eb | 405 nm | 488 nm | 532 nm | 642 nm | 447 nm/60 nm, 513 nm/17 nm, 582 nm/75 nm, and 635 nm/Longpass | 495 nm, 538 nm, and 649 nm |
| CMLS-Fb | 405 nm | 488 nm | 561 nm | 642 nm | 447 nm/60 nm, 525 nm/45 nm, 607 nm/36 nm, and 635 nm/Longpass | 495 nm, 562 nm, and 469 nm |
| CMLS-Gb | 405 nm | 488 nm | 588 nm | 642 nm | 447 nm/60 nm, 525 nm/45 nm, 615 nm/24 nm, and 635 nm/Longpass | 495 nm, 605 nm, and 649 nm |
| CMLS-H | - | 488 nm | - | 660 nm | 525 nm/39 nm and 697 nm/58 nm | 562 nm |
| CMLS-I | - | 488 nm | - | - | 525 nm/45 nm | N/A |
| CMLS-J | - | 488 nm | 532 nm | - | 512 nm/25 nm and 582 nm/75 nm | 538 nm |
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Expandable PMT modules are designed for multi-channel laser scanning microscopy applications. The photo shows the 4-channel module with standard sensitivity PMTs.
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Each Confocal System comes with either a Dell (32 bit or 64 bit) or Superlogics (64 bit) computer tower with ThorImageLS installed and a 24" monitor for data acquisition and review.
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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 2-channel PMT module with high-sensitivity PMTs.
Detection
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 upgrade includes a computer with a 24" monitor. Standard configurations use a 64-bit Dell tower with the ThorImageLS software package installed. For systems requiring higher computational power, we also offer a Superlogics tower. The included ThorImageLS software package provides an all-in-one solution for microscope control, automated data collection, and image review.
Sample Filter Configurations
In addition to the filter set list above, the two sample plots of emission filter pass bands are provided below 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 lower left provides an example of this common emission filter configuration, with the emission spectra of four compatible fluorophores superimposed on the filters' transmission profiles.
The second most common 4-laser configuration exchanges the 561 nm laser for a 532 nm laser, useful for samples that are marked with TRITC. To accommodate this wavelength, the two bandpass filters centered at 525 nm and 593 nm would be replaced with a narrower bandpass filter (25 nm pass bandwidth) centered at 512 nm and a wider bandpass filter (75 nm pass bandwidth) centered at 582 nm.
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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 spectrum for the fluorophores.
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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 spectrum for the fluorophores.
Thorlabs' Confocal Microscopy System
The photo above shows a confocal system coupled to an Olympus inverted microscope (not included) on one of our Nexus™ Optical Tables (not included).
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 Upgrade are provided in the tables below. For a description of the available modules, see the Confocal Upgrade 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 | 8 kHz Resonant Scanner (X) and Galvo Scanner (Y) | Galvo-Galvo Scanner |
| Scanning Speeds | 30 Frames per Second at 512 x 512 Pixels (Bidirectional Scan) 400 Frames per Second at 512 x 32 Pixels (Bidirectional Scan) 2 Frames per Second at 4096 x 4096 Pixels (Unidirectional Scan) |
3.2 Frames per Second at 512 x 512 Pixels (Bidirectional Scan) 1.2 Frames per Second at 1024 x 1024 Pixels (Bidirectional Scan) 0.4 Frames per Second at 2048 x 2048 Pixels (Bidirectional Scan) Dwell Time: 0.4 µs |
| Scan Zoom | 1X - 16X (Continuous) | 1X |
| 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 upgrade are available in one of 10 pre-configured integrated laser sources. 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 your 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 #a | Excitation Wavelengths | Included Emission Filters | ||||
| UV | Blue | Green/ Orange |
Red | Emission Filters (Center Wavelength/Bandwidth) |
Longpass Dichroic Cutoff Wavelength(s) | |
| CMLS-A | - | 488 nm | - | 642 nm | 525 nm/45 nm and 635 nm/Longpass | 562 nm |
| CMLS-Bb | 405 nm | 488 nm | - | 642 nm | 447 nm/60 nm, 512 nm/25 nm, and 635 nm/Longpass | 495 nm and 538 nm |
| CMLS-C | - | 488 nm | 532 nm | 642 nm | 513 nm/17 nm, 582 nm/75 nm, and 635 nm Longpass | 538 nm and 649 nm |
| CMLS-D | - | 488 nm | 561 nm | 642 nm | 525 nm/45 nm, 607 nm/36 nm, and 535 nm/Longpass | 562 nm and 649 nm |
| CMLS-Eb | 405 nm | 488 nm | 532 nm | 642 nm | 447 nm/60 nm, 513 nm/17 nm, 582 nm/75 nm, and 635 nm/Longpass | 495 nm, 538 nm, and 649 nm |
| CMLS-Fb | 405 nm | 488 nm | 561 nm | 642 nm | 447 nm/60 nm, 525 nm/45 nm, 607 nm/36 nm, and 635 nm/Longpass | 495 nm, 562 nm, and 469 nm |
| CMLS-Gb | 405 nm | 488 nm | 588 nm | 642 nm | 447 nm/60 nm, 525 nm/45 nm, 615 nm/24 nm, and 635 nm/Longpass | 495 nm, 605 nm, and 649 nm |
| CMLS-H | - | 488 nm | - | 660 nm | 525 nm/39 nm and 697 nm/58 nm | 562 nm |
| CMLS-I | - | 488 nm | - | - | 525 nm/45 nm | N/A |
| CMLS-J | - | 488 nm | 532 nm | - | 512 nm/25 nm and 582 nm/75 nm | 538 nm |
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.
Images Obtained with Thorlabs' Confocal Systems
We are pleased to announce that ThorImageLS' full source code is available. E-mail us for your copy.
ThorImageLS: Intuitive Workflow-Oriented Software Suite
ThorImage®LS was developed side-by-side with our multiphoton and confocal microscopy platforms to ensure seamless, logical, and intuitive integration between software and hardware. Our workflow-oriented interface only displays the parameters you need for each scan series (such as Z series for volumetric scans, time series for imaging of dynamics, or bleaching series for photoactivation/uncaging experiments). Each software mode offers a gentle learning curve, guides the researcher step by step through data acquisition, and will have you capturing images with just a few clicks.
A complete solution for our microscopy platforms, ThorImageLS provides control of not only the microscope but also a wide range of accessories. In-line panels control the position of our motorized XY and Z stages and the laser power at the sample. This high degree of automation minimizes distractions, allowing you to keep your focus on your research.
Experimental Techniques
- Ramp Power with Sample Depth to Minimize Damage While Maximizing Deep Signal-to-Noise
- Customize Acquisition Parameters for High-Speed Z-Stacks or Image Streams
- Select Region of Interest to Photoactivate/Bleach with an Easy-to-Use Interface
Equipment Control
- Control Power of Independent Excitation Lasers
- Insert/Remove Dichroic Mirrors for Different Scan Types
- Integrate with Electrophysiology Suites Using Master or Slave TTL Signals
- Tune Output Wavelength of Coherent Chameleon™ Ti:Sapphire Lasers
Data Analysis
- Assign a Color to Each Detection Channel
- Calculator Instantly Determines Image Dimensions and Resolution
- Generate 3D Z-Stack Reconstructions
Capture Setup Tab
The ThorImageLS Capture Setup Tab offers a dedicated control panel for each module in your imaging rig. A selection of these panels is shown below.
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Layout of Capture Setup Tab
Selected Capture Setup Tab Panels and Features
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Galvo-Galvo Scanner Control
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Laser Power Control
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Galvo-Resonant or Galvo-Galvo Scanner Control
Laser Power Control
Light Path Control
Pinhole Control (Confocal Systems Only)
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Light Path Control
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Pinhole Control (Confocal Systems Only)
Capture Tab
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Streaming Mode with Triggered Exposure
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Bleaching Mode
The ThorImageLS Capture Tab is a distraction-free area that keeps your focus on the collected data by only showing the parameters you need for the desired workflow. For example, in Streaming Mode (shown at right), it displays the option to acquire data immediately after clicking the Start button or to wait for an external stimulus. Contrast this to Bleaching Mode (also at right), which allows the user to set up different acquisition parameters for before and after the bleaching. In every workflow, the number of frames, scan duration, and required storage space are calculated and presented before each scan so that you know exactly what to expect.
All experimental data is saved in a lossless TIFF format for perfect fidelity. By choosing a standard image format, the images are viewable in ImageJ, Fiji, and many other image analysis programs, preventing lock-in to a specific program and preserving your data for the long term. ThorImageLS's Review Tab (see below) also provides quick and convenient analysis of finished acquisitions.
ThorImageLS directly supports dynamic scans, live streaming, image sequences triggered by a stimulus, and other modalities. Upon request, an SDK can be provided that permits custom acquisition sequences to be programmed by the user.
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Layout of Capture Tab
Review Tab
The ThorImageLS Review Tab lets you intuitively browse through previously acquired images, making it fast and easy to choose the exact image you want to analyze. Use sliders (shown below) to browse an acquisition sequence in time, visualize image planes along the Z-axis, or pick out one image from an entire stream.
Once you find what you're looking for, selectively enable and disable spectral channels to better visualize certain details of your specimen, or hover the mouse over the image to view the pixel's intensity (also shown below). The review tab also offers one-click 3D visualizations.
When you are ready to share your results, ThorImageLS's built-in movie maker will directly export the acquired Z series, time series, or image stream to AVI video.
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Layout of Review Tab
(Image Courtesy of Dr. Hajime Hirase and Katsuya Ozawa, RIKEN Brain Science Institute, Wako, Japan)
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Channel Color Selection and Image Sliders
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Intensity Readout
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.
Laser Scanning Microscopy TutorialLaser 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 |
Figure 2 Optical Sections (Visualization of Thin Planes within a Bulk Sample)Optical Sectioning in Confocal Microscopy Optical Sectioning in Multiphoton Microscopy 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 MicroscopyAbsorptive 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.
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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
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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.
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:
Equation 2 Lateral Resolution, Confocal LSM
and the axial resolution (Raxial,confocal) is:
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:
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
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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:
Equation 5 Lateral Resolution, Multiphoton LSM
and the axial resolution (Raxial,multiphoton) is:
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' sales engineers and field service staff are based out of eight 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 showrooms in a number of locations where you can see our systems in action. We welcome the opportunity for personal interaction during your visit! A demo can be scheduled at any of our showrooms by contacting ImagingSales@thorlabs.com.
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Customer Support Sites
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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.
Selected Confocal Microscopy Publications
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).
| Posted Comments: | |
Poster:biotecnologiasalud Posted Date:2015-12-31 04:23:29.203 I would like to know the Price of this confocal system to use in a olympus BX50 microscope. Poster:jlow Posted Date:2016-01-04 11:46:38.0 Response from Jeremy at Thorlabs: We will contact you directly about this. You can also contact ImagingSales@thorlabs.com or call (703) 651-1700 to request a quote. Poster: Posted Date:2014-10-28 09:11:10.407 Which fiber for delivering light to PMTs do you include in the confocal? can we use 200um or 400 core MMF? Poster:besembeson Posted Date:2014-11-04 02:56:13.0 Response from Bweh at Thorlabs USA: We use a 900um fiber. A 200um or 400um core fiber will lower the collection efficiency and make alignment more tedious. So you can use it but it is not recommended. Poster:cvkelly Posted Date:2014-07-23 14:45:18.953 May I please get ball-park prices for your laser-scanning confocal systems. I currently have my own microscope bodies and lasers, but I would need the high-sensitivity detectors and scan units.
Thank you,
Chris Poster:jlow Posted Date:2014-08-01 11:10:56.0 Response from Jeremy at Thorlabs: We will contact you directly for the pricing. You can also contact us at imagingsales@thorlabs.com to get the pricing. |
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