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Confocal Microscope


Brochure of Thorlabs' Confocal Microscopes
Confocal System Components
  • Galvo-Resonant Scan Head
    • Provides Video Rate Image Acquisition:
      512 x 512 Pixels at 30 fps
    • 400 fps at 512 x 32 Pixels
    • Maximum Scan Resolution of 4096 x 4096 Pixels
  • Standard Multialkali or High-Sensitivity GaAsP PMTs
  • Fiber-Coupled Laser Source with Up to Four Excitation Wavelengths (See the Specs Tab for Wavelength Options)
  • 16-Position Motorized Pinhole Wheel
  • ThorImageLSTM Software Suite for Automated Data Collection
  • Confocal Z-Stepper Motor (Available Separately)

Thorlabs' Confocal Microscope

  • Designed to Convert Research-Grade Microscopes to Confocal Imaging Systems
  • Compatible with Upright or Inverted Microscopes
  • Compact, Modular Design
  • 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)

Thorlabs' Confocal Laser Scanning (CLS) Microscopy Systems are compact imaging 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. The CLS systems offer turnkey integration with virtually any upright or inverted microscope (not included) with access to the intermediate image plane (e.g., camera port) via a C-Mount threading. The included ThorImageLS™ software drives the CLS hardware via an intuitive graphical user interface, providing quick data recording and review.

Our basic Confocal Laser Scanning System includes an electronics control unit, an expandable dual PMT module, a 16-position pinhole wheel, a two-channel (488 nm & 642 nm) laser source, and the ThorImageLS™ acquisition software and computer workstation. For more sophisticated imaging requirements, systems are available with up to four laser lines and four high-sensitivity GaAsP PMTs.

Images Obtained with Thorlabs' Confocal System

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' Confocal Microscopy System

Confocal System Schematic

Confocal Microscope Schematic
Click to Enlarge

Thorlabs’ Confocal Laser Scanning (CLS) Microscopy Systems consist 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 systems offer turnkey integration to almost any upright or inverted microscope with access to an intermediate image plane (e.g., a camera port) via a C-Mount threading. The included software has an intuitive graphical interface that allows data to be quickly recorded and reviewed while providing sophisticated peripheral controls for image acquisition. CLS systems are user-installable, although on-site installation is also available.

Confocal Microscope
Click to Enlarge

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 systems 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, and control boards as well as the ThorImageLS software. A comprehensive installation and operation manual is also included with basic preventative maintenance instructions to ensure that your system performs optimally for years to come. Also available are complete systems that combine the Thorlabs Confocal package with third party upright and inverted microscopes. For further details on this convenient option, please contact us at ImagingSales@thorlabs.com.

 

Confocal Scanner Head
The scan head of Thorlabs' Confocal System.

Scanner

At the heart of our systems is an efficiently designed Scan Head that incorporates an 8 kHz resonant scanner and a galvanometer for fast image acquisition. This allows for high imaging speeds of up to 400 frames per second (at 512 x 32 pixel resolution) or images with high spatial resolution of up to 4096 x 4096 pixel resolution (at 2 FPS). At either extreme, or anywhere in-between, the control and acquisition system creates high-quality, jitter-free images (see inset at left).

Our confocal scan head consists of three principal components: a high-speed resonant scanner, a wide field of view scan lens, and a pinhole wheel output. The scanner can be attached to virtually any upright or inverted microscope with access to the intermediate image plane via a C-mount-threaded port (e.g., a camera port). Light enters the scan head through one of the laser inputs, is reflected onto the galvo 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 scanner to the pinhole wheel. The pinhole wheel output is connected to the PMT detection module via an SMA-connectorized fiber.

Located within the Scan Head is our kinematic fluorescence filter cube (DFMT1) for quick and repeatable exchange of the primary dichroic mirror. 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.

Wide Field fo 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 across a 22 mm field of view.

The scan lens assembly has been designed for superior imaging performance, and is color-corrected from 400 – 750 nm. This broad 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 which redefine the boundaries of contrast, resolution, and imaging speed at an affordable cost.

 

Confocal Microscope Laser Source
Click to Enlarge

Four-Channel Laser Source

Excitation

The multilaser source minimizes maintenance with an all-fiber design. The 488 nm and 642 nm diodes are permanently pigtailed and the DPSS lasers used for 532 nm, 561 nm, and 588 nm options are rigidly bonded to an optical fiber that feeds into a unique all-fiber combiner. 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 a UV (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 lightpath with a 4 mm beam diameter, which increases stability and maintains the color correction of the system.

Confocal System Laser Configurations
Position in Laser SourceSlot 1Slot 2Slot 3Slot 4
Laser Wavelength
(Choose up to One
from Each Column)a
405 nm488 nm532 nm
561 nm
588 nmb
642 nm
660 nmb
  • At a minimum, Slot 2 and Slot 4 must contain a laser source.
  • By special request.

We offer five standard wavelength options in our laser source (405, 488, 532, 561 nm, and 642 nm) which can be configured as shown in the table to the left; other wavelengths are available upon request. The two-channel systems are provided with 488 nm and 642 nm fiber lasers in the same package as our four-channel source, allowing later expansion. 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.

 

Airy Disk
Airy Disk
Airy Disk Seen Through A Round Pinhole
Airy Disk
A round pinhole cleanly isolates the in-focus light and blocks out-of-focus light.
Airy Disk Seen Through a Large Square Pinhole
Airy Disk
A large square pinhole superscribed on the Airy Disk allows all of the in-focus light to pass, but some of the out-of-focus light will leak through as well.
Airy Disk Seen Through A Small Square Pinhole
Airy Disk
A small square pinhole inscribed on the peak of the Airy Disk blocks out-of-focus light, but also eliminates some of the usable signal.

Detection

Pinhole: An automated 16-aperture pinhole assembly with apertures ranging from Ø25 μm to Ø2 mm enables fine tuning of the balance between resolution and signal (for further details, see the LSM Tutorial tab). A round confocal pinhole is the ideal shape for allowing the maximum transmission of the true signal and optimal
rejection of out-of-focus light. Unlike square pinholes, which are either inscribed on the airy disk (costing valuable signal) or superscribed (allowing out of focus light through), a round pinhole matches the shape of the point spread function. The diagram to the left illustrates this advantage. The result of using a round pinhole is better resolution and higher signal to noise. Our lithographic pinholes are deposited on a glass substrate to exceedingly tight tolerances. This ensures that no realignment is required as the wheel is rotated through each of the 16 different size pinholes.

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.

Photomultiplier Module
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Expandable PMT modules are designed for multi-channel laser scanning microscopy applications.

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.

The detector 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 system 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.

Filters with 405 nm, 488 nm, 588 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 spectrum for the fluorophores.
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 spectrum for the fluorophores.

 

Thorlabs' Confocal Microscope System

Confocal Microscope System

The photo above shows a confocal system coupled to an upright microscope 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.

 CLS-2SSCLS-2HSCLS-4SSCLS-4HSCLS-UV
Excitation
Laser Wavelengths -
Additional Wavelengths
Available Upon Request
405 nm  xxx
488 nmaxxxxx
532 nm or 561 nmb  xx 
642 nmcxxxxx
Primary Dichroic MirrorQuad Band Dichroic Beamsplitter
Scanning
Scan HeadGalvo-Resonant Scan Head with 8 kHz Resonant Scanner (X) and Galvo Scan Mirror (Y)
Galvo-Resonant Scanning Speed30 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 ZoomUp to 2048 x 2048 Bi-Directional Acquisition; Up to 4096 x 4096 Uni-Directional Acquisition
1X - 16X (Continuous)
Capture ResolutionUp 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
Number of PMTs
Included
Standard Sensitivity2 4 2
High Sensitivity 2 4 
Emission Filters
(CWL/Pass Bandwidth;
See Confocal System Tab
for Sample Plots)
447 nm/60 nm
Bandpass
  xxx
512 nm/25 nmd or
525/50 nme
Bandpass
  xx 
525 nm/50 nm
Bandpass
xx  x
582 nm/75 nmd or
593 nm/46 nme
Bandpass
  xx 
645 nm/Longpassxxxxx
Secondary Dichroic
(CWL)
495 nm  xxx
562 nmxxxxx
605 nmd or 649 nme  xx 
  • Source with 488 nm and 660 nm lasers available by special request.
  • 588 nm laser also available by special request.
  • Filters when using the 532 nm laser.
  • Filters when using the 561 nm laser.

 

Scan Head Dimensions

Confocal Scanning System Dimensions


System Schematic

System Schematic

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.

ThorImageLS: Intuitive Workflow-Oriented Software Suite

ThorImageLS™ 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, the laser power at the sample, and even the wavelength of a tunable Coherent Chameleon™ Ti:Sapphire laser. 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.

Capture Setup Tab
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Layout of Capture Setup Tab

Selected Capture Setup Tab Panels and Features

ThorImageLS Galvo-Galvo Control
Click to Enlarge

Galvo-Galvo Scanner Control
ThorImageLS Laser Power Control
Click to Enlarge

Laser Power Control

Galvo-Resonant or Galvo-Galvo Scanner Control
(Shown at Left)

  • Choose Small Scan Areas for High Frame Rates or Large Scan Areas for High Resolution
  • Line, Square, or Rectangular Scans
  • Assign a Color to Each Detection Channel (Up to Four)
  • Calculator Instantly Determines Pixel and Optical Resolution
  • Change Pixel Dwell Time (of Galvo Axes) and Perform Frame Averaging

Laser Power Control
(Shown at Right)

  • Exponential Power Ramping for Increasing Laser Power with Sample Depth
  • Independently Control Power of All Input Lasers (Up to Four in Confocal Systems)
  • Edge Blanking and Masking (Available with Pockels Cells)

Light Path Control
(Shown at Right)

  • Insert and Remove Dichroic Mirrors for Different Scan Types
    • Epi-Fluorescence
    • Photoactivation/Uncaging
    • Widefield Illumination
    • Streaming Exposure
  • Intuitive Layout Shows the Physical Arrangement of the Mirrors

Pinhole Control (Confocal Systems Only)
(Shown at Right)

  • Select 1 of 16 Pinhole Diameters
  • Align Pinhole to Boost Image's Signal-to-Noise
ThorImageLS Light Path Control
Click to Enlarge

Light Path Control
ThorImageLS Light Path Control
Click to Enlarge

Pinhole Control (Confocal Systems Only)

 

Capture Tab

Streaming Mode with Triggered Exposure
Click to Enlarge

Streaming Mode with Triggered Exposure
Bleaching Mode
Click to Enlarge

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. An SDK is provided that permits custom acquisition sequences to be programmed by the user.

Capture Tab
Click to Enlarge

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.

Review Tab
Click to Enlarge

Layout of Review Tab
(Image Courtesy of Dr. Hajime Hirase and Katsuya Ozawa, RIKEN Brain Science Institute, Wako, Japan)
Channel Color Selection and Image Sliders
Click to Enlarge

Channel Color Selection and Image Sliders
Intensity Readout
Click to Enlarge

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.

ThorImageLS Interface
Click to Enlarge

ThorImageLS Capture Setup Tab

ThorImageLS

Please contact ImagingTechSupport@thorlabs.com to obtain the latest ThorImageLS version compatible with your microscope.

ThorImageLS™ is powerful data acquisition and analysis software for Thorlabs' multiphoton and confocal microscopes and microscope kits. Its workflow-oriented interface minimizes visual clutter by only displaying the parameters you need for the scan series you wish to perform. Please see the ThorImageLS tab for more details on the user interface.

Data generated by ThorImageLS can be exported for analysis in programs such as ImageJ. Application programming interfaces (APIs) and a software development kit (SDK) are also included for the development of custom applications by the end user. For example, the open-source ScanImage package is implemented using our APIs.

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 Wide Field 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

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 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 called an Airy disk. The spot size is the distance between the first 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 determined by the distance required to observe two points as two distinct entities. According to the Rayleigh criterion, these two points are said to be resolvable when the maximum of one point falls no closer than the first zero of the Airy pattern of the adjacent point—that is, when the center of one bullseye falls no closer than the middle of the first ring around the center of the second bullseye. Lateral resolution (Rlateral) is therefore:

Lateral Resolution, Confocal LSM

Equation 2 Lateral Resolution, Confocal LSM

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. The axial resolution (Raxial) of a confocal microscope is given as:

Axial Resolution, Confocal LSM

Equation 3 Axial Resolution, Confocal LSM

where n is the refractive index of the immersion medium.

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.

The longer wavelength used for excitation would lead one to believe (from Equation 2 above) that the resolution in nonlinear microscopy would be reduced 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. The lateral resolution of a nonlinear microscope is:

Lateral Resolution, Multiphoton LSM

Equation 5 Lateral Resolution, Multiphoton LSM

and the axial resolution is:

Axial Resolution, Multiphoton LSM

Equation 6 Axial Resolution, Multiphoton LSM

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 the Rayleigh criterion (Equation 2) 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 297 nm (Equation 2) and the pixel size in the displayed image should be ~129 nm. Therefore, for a 1024 x 1024 pixel capture resolution, the scan field on the specimen would be ~132 μm x 132 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.

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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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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
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