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Upgradeable, Cerna™-Based Confocal Systems


  • Complete Upright Confocal Imaging Microscopes
  • Single-Channel Excitation and Detection
  • Large 7.74" Throat Depth Ideal for In Vivo and Intact Specimens
  • Upgradeable with More Excitation/Emission Channels and
    Widefield Imaging Capabilities

CM201

This Confocal Microscope for GFP Fluorescence Imaging includes a computer, DAQ, and ThorImage®LS Data Acquisition Software. The optical table and rack are sold separately.

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Thorlabs Imaging Systems

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400 µm Deep Z-Projections of a Dragonfly Eye and Leaf Vein Imaged using the CM100 Reflected-Light Confocal Microscope. All of the images were taken at a 1.3 µm x 1.3 µm field size.
Single-Channel Confocal Microscope Comparisona
Item # CM100 CM201
Microscope Type Reflected Light GFP Fluorescence
Laser S1FC660 660 nm SM Laser (Red) S4FC488 488 nm SM Laser (Blue)
Scan Head Galvo-Galvo
Objective RMS20X 20Xb Olympus Objective N20X-PF 20X Nikon Objective
Pinhole Ø75 µm, Optimized for Included Objective
PMT Detection One PMT1001/M Multialkali PMT
  • See the Specs tab for complete specifications.
  • The RMS20X objective provides an effective magnification of 22.2X when used with the Confocal System as it is designed for a 180 mm tube lens and Thorlabs' microscopes use a 200 mm tube lens focal length. See the Magnification & FOV tab for more information on calculating effective objective magnification.

Imaging Capabilities

  • Single-Channel Confocal Systems
    • Reflected-Light System for Examining Surface Structures
    • GFP System for Epi-Fluorescence Imaging
  • Complete with All Accessories Needed for Confocal Imaging
  • Full Frame 4096 x 4096 Pixel Images (Uni-Directional Scan)
  • 884 µm x 884 µm Field of View at 20X Magnification (FN25)
  • 2 FPS for 512 x 512 Pixel Bi-Directional Scans
  • Galvo-Galvo Scan Head with User-Selectable 1.0 to 10 µs Dwell Times

Microscope Features

  • Confocal Scan Path with Galvo-Galvo Scanners
  • One Benchtop Excitation Laser
  • One Multialkali Photomultiplier Tube (PMT)
  • Filter Set for Reflected-Light or GFP Fluorescence Imaging
  • One Objective and Matched Ø75 µm Pinhole
  • Upright Microscope Based on the Cerna™ DIY Microscopy Platform
  • Rigid Stand Slide Holder on Manual XY Stage
  • Computer with National Instruments™ (NI) PXIe-6363 X Series DAQ Card
  • ThorImage®LS Data Acquisition Software with Lifetime Support

Thorlabs' Upgradeable Single-Channel Confocal Microscopes are complete single-channel systems. 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 CM100 supports confocal reflection imaging, which can be used for viewing the surface structure of biological samples or for inspection applications. The CM201 is optimized for imaging fluorescence produced by GFP. Each system is a complete upright confocal microscope, including a laser, PMT detector, objective, and motorized Z-axis control. See the table to the right for a comparison of key features.

The top panel of each microscope has a female D1N dovetail that allows Cerna widefield viewing accessories and epi-illumination modules to be added to the system. The microscope body uses the same 95 mm dovetail as our Cerna system, making it easy to integrate trans-illumination modules, sample holders, or custom modules using body attachments built from our large catalog of optomechanics.

The CM100 reflected-light confocal microscope has a PFR14-P02 35 mm x 52 mm x 3 mm silver-coated mirror at the front of the scan path that directs light from the scanners to the objective. In order to use this microscope for widefield as well as confocal imaging, the user can replace this mirror with a beamsplitter or dichroic. The CM201 GFP confocal microscope features a movable silver-coated mirror on a manual slider at the front of the scan path that allows users to select between confocal and widefield imaging modalities without replacing the optic. The body of each confocal microscope also includes a dual-objective changer, allowing the user to easily switch between the 20X objective included for confocal imaging and a user-provided second objective for widefield imaging.

Each microscope includes a PC with a National Instruments (NI) PXIe-6363 X Series DAQ card (see the Specs tab for details) 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 Software tab for additonal information on ThorImageLS features. Upon the purchase of a confocal system, Thorlabs provides lifetime support for the ThorImageLS package.

These systems can be installed by the user and detailed instructions are provided in each manual (included with each system and accessible by clicking on the red documents icon below). An optional installation service is available for these systems. Use our Confocal Microscopy Contact Form for more information.

Available Upgrades

Each Entry-Level Confocal Microscope is designed so that imaging capabilities can be added to accommodate new experimental needs as your research requirements grow. Some upgrades, such as adding Cerna widefield imaging accessories, can easily be performed by the user. Others, such as adding additional excitation wavelengths, require replacing part of the hardware, in which case assistance will be provided by our technical staff.

Four-Channel Upright Confocal Systems that incorporate the Confocal Capability Upgrades listed below are also available.

Thorlabs recognizes that each imaging application has unique requirements. If you have any feedback, questions, or need a quotation, use our Confocal Microscopy Contact Form or call (703) 651-1700.

Widefield Imaging Add-Ons using the Cerna Microscopy Platform
Confocal Capability Upgrades
  • Up to 4 Excitation Wavelengths
  • Up to 4 Confocal Detection Channels with Multialkali or GaAsP PMTs
  • Change the Primary Mirror to a Dichroic
  • Change Galvo-Galvo Scanner to Galvo-Resonant Scanner
  • Add a Motorized Shutter in the Scan Path
  • Motorized Wheel with 16 Sizes of Round Pinholes
  • Piezo Objective Stage or Z-Axis Piezo Sample Stage for Fast Z-Stacks
  • Fast Motorized XY Stage for Large Area Tiling

Specifications for Thorlabs' Upgradeable, Cerna™-Based Confocal Systems available from stock are provided here. If you are interested in a system with different specifications than those listed below, contact our sales team and applications engineers using our Confocal Microscopy Contact Form or at (703) 651-1700.

Item # CM100 CM201
System Type Reflected Light Imaging GFP Fluorescence Imaging
Excitation
Item # S1FC660 Single Mode Fiber-Coupled Laser S4FC488 Single Mode Fiber-Coupled Laser
Wavelength 660 nm 488 nm
Max Output Power 15 mW (Min) 16 mW (Min)
Power Control Manual or 0 to 5 V External Signal
Scanning
Scan Head Galvo-Galvo
Mirror PFR14-P02 35 mm x 52 mm x 3 mm Mirror, Protected Silver Coated with λ/4 Surface Flatness (Peak to Valley)
Digitization / Sampling Density Up to 4096 x 4096 Pixels (Uni-Directional Acquisition)
Up to 2048 x 2048 Pixels (Bi-Directional Acquisition)
Scanning Speed  Up to 2 FPS for 512 x 512 Pixel Bi-Directional Scans with 1 µs Pixel Dwell Time
Pixel Dwell Time 1.0 - 10.0 µs, Software Selectable
Scan Zoom 1X to 32X (Continuous)
Diffraction-Limited Field of View FN25:
796 µm x 796 µm @ 22.2Xa
442 µm x 442 µm @ 40Xb
FN23:
733 µm x 733 µm @ 22.2Xa
407 µm x 407 µm FOV @ 40Xb
(Field Number is Software Selectable up to FN25)
FN25:
884 µm x 884 µm @ 20X
442 µm x 442 µm @ 40Xb
FN23:
814 µm x 814 µm @ 20X
407 µm x 407 µm FOV @ 40Xb
(Field Number is Software Selectable up to FN25)
Detection
Pinhole Ø75 µm, Optimized for Included 20X Objective
Photomultiplier Tube (PMT) PMT1001/M Multialkali PMT
Filters BSW10R 50:50 Beamsplitter
WPQ10E-670 Quarter-Wave Plate
LPVISE100-A Polarizers (2)
MD498 Dichroic: Refl. Band = 452 - 490 nm, Trans. Band = 505 - 800 nm
MF525-39 Emission Filter: 525 nm / 39 nm
Objective
Item # RMS20X Olympus Plan Achromat Objective N20X-PF Nikon Plan Fluorite Objective
Magnification 20Xa 20X
NA 0.4 0.5
Working Distance 1.2 mm 2.1 mm
Parfocal Length 45.06 mm 60 mm
Design Tube Lens Focal Length 180 mma 200 mm
Coverslip Correction 0.17 mm
Threading RMS M25 x 0.75
Fiber Patch Cables
Laser to Scan Head P1-630PM-FC-2 2 m PM Patch Cable, 620 - 850 nm, FC/PC Connectors P1-405B-FC-2 2 m SM Patch Cable, 405 - 532 nm, FC/PC Connectors
Pinhole to Detector FG910UEC MM Fiber, 1 m, Armored Stainless Steel Protective Tubing, AR-Coated End Faces, SMA Connectors
General Microscope Features
Widefield Viewing Silver-Coated Mirror can be Removed or Replaced with a Beamsplitter for Widefield Imaging Silver-Coated Mirror on a Manual Slider to Switch Between Confocal and Widefield Imaging
Female D1N Dovetail on Top of Scan Path to Mount Cerna™ Widefield Viewing Accessories
Microscope Body 95 mm Dovetail Rail to Mount Cerna Body Attachments, Transmitted Illumination Modules, and Other Accessories
7.74" Throat Depth
Nosepiece CSN1301 Dual Objective Changer
ZFM2020 Focusing Module with 1" Fine Z Translation
M32 x 0.75 Objective Threads (Two Places)
M32 x 0.75 to M25 x 0.75 (Qty. 2) and M25 x 0.75 to RMS (Qty. 2) Adapters Included
Sample Holder
Included Sample Holder MP150-RCH2 Rigid Stand Slide Holder
Sample Holder Stage Manual XY Stage, 1/2" Travel, Micrometers with 10 µm Graduations
Data Acquisition
Type National Instruments PXIe-6363 X Series DAQ Card
Analog Outputc 4 Channels
Resolution: 16 Bits
Voltage Range: ±10 V
Accuracy: 1.89 mV
Update Rate: 2.86 MS/s
Analog Inputc 1 Channel
Resolution: 16 Bits
Voltage Range: ±10 V
Accuracy: 1.66 mV
Digital I/Oc 48 Bidirectional Channels
Clock Rate 10 MHz (Max)
Frame In/Out Triggering TTL
Line Trigger Out TTL
Counter/Timersc 4
Computer and Software
Computer PC with DAQ
Software ThorImage®LS with Lifetime Support
  • The RMS20X objective provides an effective magnification of 22.2X when used with the Confocal System as it is designed for a 180 mm tube lens and Thorlabs' microscopes use a 200 mm tube lens focal length. See the Magnification & FOV tab for more information on calculating effective objective magnification.
  • For reference only. The included pinhole is not optimized for use with 40X objectives.
  • All digital channels and one counter are available for use by the user. All other channels and counters are either actively used by the confocal system or reserved to support future upgrades.

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.

CM100 Shipping List
Click to Enlarge

CM100 Components
Grid Slide, Alignment Tool, Computer Keyboard, and Mouse are Not Shown

CM100 Reflected-Light Confocal Microscope

Item # CM100 consists of:

  • Single-Channel Reflected-Light Confocal Microscope Body
  • Galvo-Galvo Scanner Control Box
  • S1FC660 660 nm Single-Channel Fiber-Coupled Laser Source
  • MP150-RCH2 Rigid Stand Slide Holder with Manual XY Translation Stage, 1/2" Travel
  • KST101 Stepper Motor Controller with USB Cable and KPS101 Power Supply
  • PMT1001/M Multialkali PMT
  • PH082E Post Holder, TR20/M Post, and CF175 Clamping Fork
  • RMS20X 20X Olympus Plan Achromat Objective
  • P1-630PM-FC-2 2 m Polarization Maintaining Patch Cable, FC/PC Connectors
  • FG910UEC Multimode Fiber in Armored Patch Cable with Stainless Steel Protective Tubing, SMA Connectors
  • 120" Long SMA to BNC Cable for Connecting PMT to NI Breakout Box
  • Alignment Tool
  • R1L3S3P Grid Slide
  • NI Breakout Box with NI SHC68-68-EPM and NI SH6868 Cables
  • Computer with 24" Monitor, Keyboard, and Mouse
  • All Hardware Required for Mounting to an Optical Table
    • 12 1/4"-20 and 12 M6 Socket Head Cap Screws
    • Six 1/4" (M6) Washers
    • 1.5 mm, 2 mm, and 2.5 mm Hex Wrenches
    • 2 mm, 5 mm, 3/32" and 3/16" Hex L-Keys

 

CM201 Shipping List
Click to Enlarge

CM201 Components
Fluorescent Bead Slide, Fluorescence Slide, Alignment Tool, Computer Keyboard, and Mouse are Not Shown

CM201 GFP Confocal Microscope

Item # CM201 consists of:

  • Single-Channel Fluorescence Confocal Microscope Body
  • Galvo-Galvo Scanner Control Box
  • S4FC488 488 nm Single-Channel Fiber-Coupled Laser Source
  • MP150-RCH2 Rigid Stand Slide Holder with Manual XY Translation Stage, 1/2" Travel
  • KST101 Stepper Motor Controller with USB Cable and KPS101 Power Supply
  • PMT1001/M Multialkali PMT
  • PH082E Post Holder, TR20/M Post, and CF175 Clamping Fork
  • N20X-PF 20X Nikon Plan Fluorite Objective
  • P1-405B-FC-2 2 m Single Mode Patch Cable, FC/PC Connectors
  • FG910UEC Multimode Fiber in Armored Patch Cable with Stainless Steel Protective Tubing, SMA Connectors
  • 120" Long SMA to BNC Cable for Connecting PMT to NI Breakout Box
  • Alignment Tool
  • Fluorescent Bead Slide with 0.1, 0.2, 0.5, 1.0, and 4.0 µm Bead Sizes
  • Fluorescence Slide
  • NI Breakout Box with NI SHC68-68-EPM and NI SH6868 Cables
  • Computer with 24" Monitor, Keyboard, and Mouse
  • All Hardware Required for Mounting to an Optical Table
    • 12 1/4"-20 and 12 M6 Socket Head Cap Screws
    • Six 1/4" (M6) Washers
    • 1.5 mm, 2 mm, and 2.5 mm Hex Wrenches
    • 2 mm, 5 mm, 3/32" and 3/16" Hex L-Keys

We are pleased to announce that ThorImageLS' full source code is available. E-mail us for your copy.

ThorImageLS Brochure

ThorImage®LS: 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
Download

Version 3.0 - November 2, 2016

Please contact ImagingTechSupport@thorlabs.com to obtain the latest ThorImageLS version compatible with your microscope. Because ThorImageLS 3.0 adds significant new features over 2.x and 1.x versions, it may not be compatible with older microscopes. We continue to support older software versions for customers with older hardware.

New Features

  • Added support for DDR05(/M) fast power control device, which allows for faster power ramping acquisitions
  • Added support for the entry level Galvo-Galvo Confocal system (no separate digital acquisition board necessary)
  • Added fine two-way calibration, which provides fewer two-way adjustments when changing field sizes
  • Added ability to save Galvo-Galvo offset and scale values to ThorConfocalGalvoSettings.xml file
  • Added option to save .tiff files compressed or uncompressed
  • Added option to save only enabled channels as raw image files
  • Added ability to save snapshot image either as a single image or as an experiment
  • Added ability for multi-location imaging on platforms with supporting stages (Bergamo, MCM3000 and High-Speed Motorized XY Scanning Stages)
  • Includes new UI for defining and navigating multi-location regions
  • Added ability to invert scanners (flip horizontal and vertical) for Galvo-Resonant and Galvo-Galvo systems
  • Added ability to invert stage directions for Bergamo, MCM3000 and High-Speed Motorized XY Scanning Stages
  • Added ability to use an ROI mask (ROIMask.raw) as a Pockels mask. This includes updating the mask in the UI
  • Added ability to use Pockels power ramping during fast Z acquisitions
  • Added high temporal resolution image capture spacing. Allows the user to set a delay between frame acquisitions
  • Added ability to sequentially capture images using different hardware settings, such as Channel, PMT, Laser and Power
  • Added ability for High-Speed Motorized XY Scanning Stages laser calibration to linearize power output
  • Added support for a secondary Z panel
  • Added offset control support for PMT1000 and PMT2100 devices
  • Added ability to image and bleach simultaneously
  • Added option to save bleach images in Raw image format
  • Added ability to turn off the computer monitor when starting experiment capture
  • Added ability to synchronize the Start and Stop of ThorSync with ThorImage Capture start and stop
  • Added orthogonal view functionality for Z stack acquisitions in Image Review
  • Added new script commands to move X, Y and Z motors between script acquisitions
  • Added support for Tiberius two-photon laser

Fixed Bugs

  • Fixed bleaching capture fields from being editable during capture
  • Fixed raw image review not supporting 3D display
  • Fixed PMT3 and PMT4 voltage range setting saved in the template not being used when running a Script
  • Fixed galvo-galvo line scan setting the Y scale incorrectly when changing the X scale
  • Fixed error when galvo-galvo snapshot appears to lock up with large pixel density setting and dwell time, and added abort button
  • Fixed standard sensitivity PMTs not being set to 0 when the display indicates 0 after a hardware reset
  • A 4 channel snap-shot image is now saved as a multi-page image instead of RGB
  • Added an adjustable phase shift parameter to correct the pockels waveform sometimes being out of phase across the image 
  • Removed un-necessary files to correct for the application appearing locked up when selecting certain .xml files in the settings editor takes a long time

User Interface (UI) Improvements

  • Changed the layout for the hardware setup window
  • Added mouse scrolling functionality in settings editor
  • Removed un-necessary .xml files in settings editor view
  • Moved center scanners and resonance scanner from always visible to the area control advanced panel in Capture Setup
  • Added second column option for capture setup display
  • Moved field size entry to under scan area cartoon
  • Added dropdown list for most popular pixel density settings.
  • Added +/- buttons for Galvo-Galvo angle control
  • Changed the Z slider bar to objective graphical
  • Replaced coarse/fine buttons with buttons labeled increase/decrease
  • Changed Z units from mm to μm
  • Added visibility option for "Set Zero" feature in XY and Z panels
  • Added visibility for invert option in Z control panel
  • Added visibility option individual light path controls
  • Renamed the coherent control panel to multiphoton laser control
  • Display summary and status for collapsed panels in Capture Setup
  • Added features to histogram control:
    • Black and white point fields
    • Connector between black and white point fields to help locate mid-point
    • Ability to enlarge single or all histograms
    • Log scale display option
  • Stats chart and stats window changes:
    • Change how they are displayed. Selecting to close the window now de-selects visibility
    • Added ability to save a chart as a .jpg
    • Added ability to save table data as .csv, .txt or .raw
    • Changed the chart Y scale to scientific notation
    • Set the chart X axis limit to the range of data
  • Added display option for line profile window
  • Color settings changes:
    • Added more look up table (LUT) colors: BlueStat, CyanHot, GrayStat, GreenStat and RedStat
    • Allow the same LUT for multiple colors
    • Enhanced display of min and max when viewing the gray scale of single image features
  • Eliminated zoom-level edit dialog and replaced with user-entered zoom field
  • Changed experiment naming by adding a separate iteration field
  • Added dialog to suppress the "File Name Exists" prompt
  • Changed browsers to a more useful interface
  • Removed the intermediate menu when selecting image review
  • Added the ability to save more experiment information, such as Galvo-Galvo angle value and pinhole size
  • Z stack experiments open Z slider to mid-range
  • Added Z and T unit display for Z and time index
  • Image review play is now in a continuous loop until manually stopped
  • Changed Galvo-Galvo pixel dwell time scale bar to single bar

 

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. Upon request, an SDK can be 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
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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.

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
Click to Enlarge

Figure 2 Optical Sections (Visualization of Thin Planes within a Bulk Sample)

Optical Sectioning in Confocal Microscopy

Optical Sectioning in Confocal Microscopy
Click to Enlarge


Optical Sectioning in Multiphoton Microscopy

Optical Sectioning in Multiphoton Microscopy
Click to Enlarge

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 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.

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

Many components and modules in Thorlabs' Cerna Microscopy Platform use dovetails for mechanical mating and optical port alignment. These components are connected by inserting one dovetail into another, then tightening one or more locking setscrews.

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.

Dovetail Reference
Type Shape Outer Dimension Angle
95 mm Linear 95 mm 45°
D1N Circular Ø2.018" 60°
D2Na Circular Ø1.50" 90°
D2NBa Circular Ø1.50" 90°
D3N Circular Ø45 mm 70°
D3T Circular Ø1.65" 90°
D5N Circular Ø1.58" 90°
D1Y Circular Ø107 mm 60°
  • 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).

To help ensure that only compatible components can be mated, different dovetail types are used on different sections of the microscope. 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. Each 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 left gives the outer diameter and angle (as defined by the drawings below) of each dovetail type used by the Cerna platform. 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

These cross sections show two examples of how circular male dovetails can be manufactured.
Female Microscope Dovetails
Click to Enlarge

These cross sections show two examples of how circular female dovetails can be manufactured.

 

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 D3T D5N D1Y C-Mount
(1.00"-32)
SM1c
(1.035"-40)
SM30
(M30.5x0.5)
SM2d
(2.035"-40)
30 mmc 60 mmd
2SCM1-DC Internal & External Internal Yes Yes
BSA2000 Female
CEA1350 Male Female Yes
CEA1400 Male Female Yes
CEA1500 Male Female Yes
CEA1600 Male Female Yes
CFB1500 Male
CSA1000 Female
CSA1001 Female Internal Yes
CSA1002 Female Internal Yes
CSA1003 Female Yes
CSA1051 Male
CSA1100e Yes
CSA1500f
CSA2000 Female Internal Yes
CSA2001 Female External
CSA2100 Internal Yes
CSA3000(/M) Male
CSA3010(/M) Male Yes Yes
CSC1001 Male
CSC1002 Male
CSC1003 Male
CSD1001 Male & Female Female
CSD1002 Male & Female External
CSE1000g Male & Female
CSE2000 Male & Female Yes
CSE2100 Male & Female Female Internal Yes Yes
CSN1301e Yes
CSS2001 Female
LCPN1 Male Internal Yes Yes
LCPN2 Male Internal Yes Yes
OPX2400(/M) Male & Female Internal Yes
SM1A58 Male Male Internal External Yes
Item # 95 mm D1N D2N D2NB D3N D3T D5N D1Y C-Mount SM1 SM30 SM2 30 mm 60 mm
WFA0150 Female
WFA1000 Yes
WFA1010 Internal Yes
WFA1020 Internal Yes
WFA1051 Internal Yes
WFA1100 Yes
WFA2001 Male & Female Internal & External
WFA2002 Male & Female Internal Yes
WFA4000 Male Female
WFA4001 Male Female
WFA4002 Male Female
WFA4003 Male Female
WFA4100 Male External Internal
WFA4101 Male External Internal
WFA4102 Male External Internal
WFA4105 Male External
WFA4106 Male External
WFA4107 Male External
WFA4108 Male External
WFA4109 Male External
WFA4110 Male External
WFA4111 Male External
WFA4112 Male External
XT95P11(/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.
  • This nosepiece directly accepts M32 x 0.75 objective threads and ships with ring-type thread adapters for M25 x 0.75 and RMS (0.800"-36) objective threads.
  • This blank arm is designed for custom DIY machining.
  • This epi-illuminator module has a female Nikon bayonet mount, which can be connected to our SM1 lens tubes, SM2 lens tubes, and 30 mm cage system using our lamphouse port adapters.
Widefield Viewing Optical Path
When viewing an image with a camera, the system magnification is the product of the objective and camera tube magnifications. When viewing an image with trinoculars, the system magnification is the product of the objective and eyepiece magnifications.
Magnification & FOV Calculator
Manufacturer Tube Lens
Focal Length
Leica f = 200 mm
Mitutoyo f = 200 mm
Nikon f = 200 mm
Olympus f = 180 mm
Thorlabs f = 200 mm
Zeiss f = 165 mm

The rows highlighted in green denote manufacturers that do not use f = 200 mm tube lenses.

Magnification and Sample Area Calculations

Magnification

The magnification of a system is the multiplicative product of the magnification of each optical element in the system. Optical elements that produce magnification include objectives, camera tubes, and trinocular eyepieces, as shown in the drawing to the right. It is important to note that the magnification quoted in these products' specifications is usually only valid when all optical elements are made by the same manufacturer. If this is not the case, then the magnification of the system can still be calculated, but an effective objective magnification should be calculated first, as described below.

To adapt the examples shown here to your own microscope, please use our Magnification and FOV Calculator, available for download by clicking on the red button above.

Example 1: Camera Magnification
When imaging a sample with a camera, the image is magnified by the objective and the camera tube. If using a 20X Nikon objective and a 0.75X Nikon camera tube, then the image at the camera has 20X × 0.75X = 15X magnification.

Example 2: Trinocular Magnification
When imaging a sample through trinoculars, the image is magnified by the objective and the eyepieces in the trinoculars. If using a 20X Nikon objective and Nikon trinoculars with 10X eyepieces, then the image at the eyepieces has 20X × 10X = 200X magnification. Note that the image at the eyepieces does not pass through the camera tube, as shown by the drawing to the right.

Using an Objective with a Microscope from a Different Manufacturer

Magnification is not a fundamental value: it is a derived value, calculated by assuming a specific tube lens focal length. Each microscope manufacturer has adopted a different focal length for their tube lens, as shown by the table to the right. Hence, when combining optical elements from different manufacturers, it is necessary to calculate an effective magnification for the objective, which is then used to calculate the magnification of the system.

The effective magnification of an objective is given by Equation 1:

Equation 1 (Eq. 1)

Here, the Design Magnification is the magnification printed on the objective, fTube Lens in Microscope is the focal length of the tube lens in the microscope you are using, and fDesign Tube Lens of Objective is the tube lens focal length that the objective manufacturer used to calculate the Design Magnification. These focal lengths are given by the table to the right.

Note that Leica, Mitutoyo, Nikon, and Thorlabs use the same tube lens focal length; if combining elements from any of these manufacturers, no conversion is needed. Once the effective objective magnification is calculated, the magnification of the system can be calculated as before.

Example 3: Trinocular Magnification (Different Manufacturers)
When imaging a sample through trinoculars, the image is magnified by the objective and the eyepieces in the trinoculars. This example will use a 20X Olympus objective and Nikon trinoculars with 10X eyepieces.

Following Equation 1 and the table to the right, we calculate the effective magnification of an Olympus objective in a Nikon microscope:

Equation 2

The effective magnification of the Olympus objective is 22.2X and the trinoculars have 10X eyepieces, so the image at the eyepieces has 22.2X × 10X = 222X magnification.


Image Area on Camera

Sample Area When Imaged on a Camera

When imaging a sample with a camera, the dimensions of the sample area are determined by the dimensions of the camera sensor and the system magnification, as shown by Equation 2.

Equation 5 (Eq. 2)

The camera sensor dimensions can be obtained from the manufacturer, while the system magnification is the multiplicative product of the objective magnification and the camera tube magnification (see Example 1). If needed, the objective magnification can be adjusted as shown in Example 3.

As the magnification increases, the resolution improves, but the field of view also decreases. The dependence of the field of view on magnification is shown in the schematic to the right.

Example 4: Sample Area
The dimensions of the camera sensor in Thorlabs' 1501M-USB Scientific Camera are 8.98 mm × 6.71 mm. If this camera is used with the Nikon objective and trinoculars from Example 1, which have a system magnification of 15X, then the image area is:

Equation 6

Sample Area Examples

The images of a mouse kidney below were all acquired using the same objective and the same camera. However, the camera tubes used were different. Read from left to right, they demonstrate that decreasing the camera tube magnification enlarges the field of view at the expense of the size of the details in the image.

Image with 1X Camera Tube
Click to Enlarge

Acquired with 1X Camera Tube (Item # WFA4100)
Image with 1X Camera Tube
Click to Enlarge

Acquired with 0.75X Camera Tube (Item # WFA4101)
Image with 1X Camera Tube
Click to Enlarge

Acquired with 0.5X Camera Tube (Item # WFA4102)

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.

Showroom Icon

Showroom and Customer Support Sites

 

Sterling, Virginia, USA
Thorlabs Imaging Systems HQ

Thorlabs Imaging Systems
108 Powers Court
Sterling, VA 20166

Appointment Scheduling and Customer Support

 

China

Thorlabs China
Room A101, No. 100, Lane 2891, South Qilianshan Road
Shanghai 200331

Appointment Scheduling and Customer Support

 

Japan

Thorlabs Japan, Inc.
Higashi-Ikebukuro Q Building, 2-23-2
Higashi-Ikebukuro, Toshima-ku, Tokyo 170-0013

Appointment Scheduling and Customer Support

Customer Support Sites

 

Newton, New Jersey, USA
Thorlabs HQ

Thorlabs, Inc.
56 Sparta Avenue
Newton, NJ 07860

Customer Support

 

United Kingdom

Thorlabs Ltd.
1 Saint Thomas Place, Ely
Ely CB7 4EX

Customer Support

 

Germany

Thorlabs GmbH
Hans-Boeckler-Str. 6
Dachau/Munich 85221

Customer Support

 

France

Thorlabs SAS
109, rue des Cotes
Maisons-Laffitte 78600

Customer Support

 

Brazil

Thorlabs Vendas de Fotônicos Ltda.
Rua Riachuelo, 171
São Carlos, SP 13560-110

Customer Support

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).


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Reflected-Light Imaging Confocal Microscope

The front panel of the microscope can be removed to access the mirror at the front of the scan path. The user may replace this mirror with a beamsplitter or dichroic, enabling widefield imaging with the addition of DIY Cerna components.
  • Ideal for Imaging Surface Structures of Biological Samples and for Inspection Applications
  • Red 660 nm Single Mode Laser
  • Silver-Coated Mirror at Front of Scan Path can be Removed or Replaced with a Dichroic to Enable Widefield Imaging
  • Compatible with DIY Cerna Platform for Later Upgrades

Thorlabs' CM100 Reflected-Light Confocal Microscope includes a 660 nm single mode laser, galvo-galvo scanner, a removable silver-coated mirror at the front of the scan path, polarizers and a quarter-wave plate to minimize unwanted reflections for improved image quality, and a single multialkali PMT. A manual dual objective changer with motorized focus control accepts the included 20X objective, while the sample stage provides 1/2" of manual XY translation to adjust the sample position. Each system includes a computer with DAQ and the ThorImage®LS data acquisition software.

This microscope includes everything needed for reflected-light confocal imaging. Additionally, the 95 mm dovetail on the microscope body and female D1N dovetail on top of the scan path allow the user to upgrade the microscope with widefield imaging capabilities using our wide selection of DIY Cerna components. The included dual objective changer allows the user to easily switch between the included 20X objective and a second user-provided objective with a different magnification when moving between widefield and confocal imaging.

The microscope body features the same 7.74" throat depth as our widefield Cerna systems. To achieve this large free space beneath and around the objective, the microscope is not designed to be free-standing and must be bolted to an optical table. For this purpose, 1/4"-20 and M6 cap screws are included to provide compatibility with both metric and imperial optical tables.

Based on your currency / country selection, your order will ship from Newton, New Jersey  
+1 Qty Docs Part Number - Universal Price Available / Ships
CM100 Support Documentation
CM100NEW!Single-Channel Confocal Microscope for Reflected-Light Imaging
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GFP Fluorescence Confocal Microscope


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The D1N dovetail on the top of the housing allows Cerna accessories to be added, such as the WFA2001 epi-illumination module, WFA4100 camera tube, and a scientific camera shown here. A silver-coated mirror mounted on a slider in the confocal scan head allows the user to switch between imaging modalities.
  • Ideal for Green Fluorescent Protein (GFP) Microscopy
  • Blue 488 nm Single Mode Laser
  • Silver-Coated Mirror on Manual Slider at Front of Scan Path for Switching Between Imaging Modalities
  • Compatible with DIY Cerna Platform for Later Upgrades

Thorlabs' CM201 GFP Fluorescence Confocal Microscope includes a 488 nm single mode laser, galvo-galvo scanner, a silver-coated mirror on a two position slider at the front of the scan path, a dichroic and emission filter for GFP confocal fluorescence imaging, and a single multialkali PMT. A manual dual objective changer with motorized focus control accepts the included 20X objective, while the sample stage provides 1/2" of manual XY translation to adjust the sample position. Each system includes a computer with DAQ and the ThorImage®LS data acquisition software.

This microscope includes everything needed for GFP fluorescence confocal imaging. Additionally, the 95 mm dovetail on the microscope body and female D1N dovetail on top of the scan path allow the user to upgrade the microscope with widefield imaging capabilities using our wide selection of DIY Cerna components. The included dual objective changer allows the user to easily switch between the included 20X objective and a second user-provided objective with a different magnification when moving between widefield and confocal imaging.

The microscope body features the same 7.74" throat depth as our widefield Cerna systems. To achieve this large free space beneath and around the objective, the microscope is not designed to be free-standing and must be bolted to an optical table. For this purpose, 1/4"-20 and M6 cap screws are included to provide compatibility with both metric and imperial optical tables.

Based on your currency / country selection, your order will ship from Newton, New Jersey  
+1 Qty Docs Part Number - Universal Price Available / Ships
CM201 Support Documentation
CM201NEW!Single-Channel Confocal Microscope for GFP Fluorescence Imaging
$42,200.00
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