Dodt Gradient Contrast for DIY Cerna® Systems
- Generate High-Contrast Images of Thick, Unlabeled Specimens
- Illumination Module Conditions Sample Illumination
- Compatible with Collimated Light Sources in 30 mm Cage Systems
Dodt Contrast Module
Dodt Contrast Image of a Mouse Retina
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The WFA1100 Illumination Module is positioned in the optical path by the WFA0150 Dovetail Clamp.
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Drawing of Trans-Illumination Module
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Simplified Dodt Contrast Module Schematic
- Module Accepts Collimated Illumination Through Ø1" Optical Input Port
- Optical Input and Output Ports are 30 mm Cage Compatible
- Pre-Aligned Quarter Annulus and Diffusers Generate Dodt Illumination Gradient
- Includes Five Quarter Annuli to Match Specific Objective NAs
- 95 mm Dovetail Clamp Attaches Illumination Module to Microscope Body
- Designed for 7.74" Throat Depth of DIY Cerna® Systems
Thorlabs' WFA1100 Trans-Illumination Module is designed to enable Dodt gradient contrast for DIY Cerna systems. Dodt gradient contrast, also known more simply as Dodt contrast, can be understood as an improvement upon oblique illumination. Both methods use a mask to generate an illumination gradient, but in Dodt contrast, the mask occurs much earlier in the optical path. This configuration improves the image contrast to a point where it is comparable to that obtained using differential interference contrast (DIC).
Designed for use in the 400 - 1000 nm wavelength range, the WFA1100 module accepts collimated illumination through a Ø1" input port on the side of the module. It generates a Dodt illumination gradient by sending the collimated input through a pre-aligned ED1-C50 Engineered Diffuser™, a quarter annulus, and a DG10-220 Ground Glass Diffuser, as shown in the drawing to the right.
We include five quarter annuli with this module, which are pictured below. Each annulus is matched to a specific objective NA (0.3, 0.5, 0.65, 0.8, or 1.0), and the 1.0 NA quarter annulus is pre-installed. The annulus can be exchanged by following the procedure on page 3 of the mechanical drawing (PDF link). When replacing the annulus, it is important to use the hard stops inside the module in order to maintain the optical alignment. The module can be ordered with a different pre-installed annulus by contacting Tech Support prior to purchase.
To mount an illumination source to the input port, the module accepts cage rods via four Ø6 mm bores spaced for our 30 mm cage system. Locking setscrews for these cage rods can be accessed by removing a dust cover that is held in place with a 1.5 mm hex button head screw, as shown in the drawing to the right. Our illumination kits are specifically designed for use with this input port, and four ER025 cage rods for these kits are included with the module. If using another light source, note that for best performance, the input port should be filled.
The rotating knob on the front controls an integrated field stop diaphragm, which can be used to adjust the illumination intensity and match the back aperture of a condenser. Because Dodt contrast relies on oblique light rays to provide contrast, the condenser NA should be at least as large as the objective NA. 30 mm cage compatibility for the optical output port is provided by four 4-40 taps.
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Quarter annuli for objective NAs of 0.3, 0.5, 0.65, 0.8, and 1.0 are included with the WFA1100 Trans-Illumination Module.
The WFA0150 Dovetail Clamp, sold separately, is used to connect the trans-illumination module to the microscope body. This 95 mm dovetail clamp is attached to the trans-illumination module by the included adapter plate, as shown in the image above. The clamp and adapter plate together position the optical output port at the 7.74" throat depth used in DIY Cerna systems.
To complement our trans-illumination modules, Thorlabs offers several condensers that collect the output light to illuminate a specimen.
Other Transmitted Light Imaging Modalities
We also support brightfield and oblique illumination and DIC for DIY Cerna systems. As a rule of thumb, Dodt contrast generates images with slightly less maximum contrast than DIC, but it also maintains its performance over a greater range of sample thicknesses. In addition, it is easier to add Dodt contrast to DIY Cerna systems than to add DIC, because it requires fewer specific microscope modules. Brightfield illumination can be performed on DIY Cerna systems that have Dodt contrast, but the aligned quarter annulus and engineered diffuser will have to be removed first. See the Imaging Modalities tab above for details on these techniques.
Cerna® microscopes support several imaging modalities, including epi-fluorescence, brightfield illumination, differential interference contrast (DIC) imaging, and Dodt gradient contrast imaging. Each of these methods requires different accessories and confers different advantages to the microscopist, as described below.
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Epi-Fluorescence Image of Mouse Kidney with Multiple Labels
Epi-fluorescence makes use of fluorescent labels and intrinsic fluorescence in a specimen to identify sample features. To create an epi-fluorescence image, light that has been passed through an excitation filter is directed through an objective and absorbed by a sample. This excitation causes fluorophores within the sample to emit light of a longer wavelength (i.e., lower energy) than the excitation light. Some of this emitted light is collected by the objective, which helps direct the emission onto a camera for observation. Additional details on this imaging modality are available here.
For performing epi-fluorescence measurements in DIY Cerna systems, we offer a range of widefield viewing and epi-illumination accessories, as well as fluorescence filter sets targeted at common fluorophores.
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Brightfield Image of Onion Mitosis
Brightfield illumination is the simplest method of trans-illumination. In this modality, light from an illumination source is collected by a condenser and passed through a sample, which is observed by its effect on the intensity of the transmitted light. Brightfield illumination only requires an illumination source (i.e., an illumination kit) and a condenser to be attached to a DIY Cerna system.
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DIC Image of a Buttercup Root
In differential interference contrast (DIC) imaging, light transmitted through the sample is manipulated by a number of polarization optics. Light from the illumination source is polarized and then split into two orthogonally polarized beams before it reaches the sample. Small differences in the optical path length experienced by the two beams cause interference when the beams are recombined, providing enhanced contrast for sample features that would be transparent under brightfield illumination. In addition to an illumination source and a condenser, DIC imaging requires several additional optical elements: a DIC polarizer, a condenser prism, an objective prism, and an analyzer.
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Dodt Contrast Image of a Mouse Retina
Dodt gradient contrast, also known more simply as Dodt contrast, can be understood as an improvement upon oblique illumination. Both methods use a mask to generate an illumination gradient, but in Dodt contrast, the mask occurs much earlier in the optical path. This configuration improves the image contrast to a point where it is comparable to that obtained using DIC.
The Dodt illumination gradient is generated using a specially shaped quarter annulus and diffusers, and reveals thickness changes in a sample over the field of view. Compared to brightfield illumination, Dodt contrast offers improved resolution of sample features, and compared to DIC, it allows thicker samples to be studied. Thorlabs manufactures a pre-configured, pre-aligned illumination module for Dodt contrast that generates the desired gradient; it requires an illumination source and a condenser for operation.
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Laser Scanned Image of a Flower Bud
Like epi-fluorescence, laser scanning makes use of fluorescent labels and intrinsic fluorescence in a specimen to identify sample features. Unlike epi-fluorescence, laser scanning is able to resolve thin individual planes relatively deep into a thick sample, enabling 3D volumetric images and opening the door to in vivo studies.
Laser scanning techniques (e.g., multiphoton and confocal microscopy) rely upon the coherence of laser beams to provide significantly improved axial resolution. In confocal microscopy, a pinhole eliminates the out-of-focus light that would reduce the axial resolution (as it does in epi-fluorescence), while in multiphoton microscopy, the necessity of two- or three-photon absorption by the fluorophore, a low-probability event, effectively creates optical sections.
Additional details are available at our laser scanning microscopy tutorial.
Building a Cerna® Microscope
The Cerna microscopy platform's large working volume and system of dovetails make it straightforward to connect and position the components of the microscope. This flexibility enables simple and stable set up of a preconfigured microscope, and provides easy paths for later upgrades and modification. See below for a couple examples of the assembly of preconfigured and DIY Cerna microscopes.
Preconfigured Microscope Kit Design and Assembly
Walkthrough of Cerna® Microscope Kit 4
This Cerna microscope configuration is equipped with both epi- and trans-illumination modules. All Cerna preconfigured microscope kits enable individual components to be removed or substituted for complete customization.
The D1N and D2N circular dovetails align the sample viewing and epi-illumination apparatus along the optical path. The microscope body's 95 mm linear dovetail is used to secure the objective mounts and condenser mounts, as well as the transmitted light illumination module. The dovetail allows components to slide along the vertical rail prior to lockdown.
DIY Cerna Design and Assembly
Walkthrough of a DIY Microscope Configuration
This DIY microscope uses a CSA3000(/M) Breadboard Top, a CSA2001 Dovetail Adapter, our CSA1001 and CSA1002 Fixed Arms, and other body attachments and extensions. These components provide interfaces to our lens tube and cage construction systems, allowing the rig to incorporate two independent trans-illumination modules, a home-built epi-illumination path, and a custom sample viewing optical path.
DIY Microscope Configuration Assembly
The simplicity of Thorlabs optomechanical interfaces allows a custom DIY microscope to be quickly assembled and reconfigured for custom imaging applications.
Click on the different parts of the microscope to explore their functions.
Elements of a Microscope
This overview was developed to provide a general understanding of a Cerna® microscope. Click on the different portions of the microscope graphic to the right or use the links below to learn how a Cerna microscope visualizes a sample.
Arm: Holds components in the optical path of the microscope.
Bayonet Mount: A form of mechanical attachment with tabs on the male end that fit into L-shaped slots on the female end.
Bellows: A tube with accordion-shaped rubber sides for a flexible, light-tight extension between the microscope body and the objective.
Breadboard: A flat structure with regularly spaced tapped holes for DIY construction.
Dovetail: A form of mechanical attachment for many microscopy components. A linear dovetail allows flexible positioning along one dimension before being locked down, while a circular dovetail secures the component in one position. See the Microscope Dovetails tab or here for details.
Epi-Illumination: Illumination on the same side of the sample as the viewing apparatus. Epi-fluorescence, reflected light, and confocal microscopy are some examples of imaging modalities that utilize epi-illumination.
Filter Cube: A cube that holds filters and other optical elements at the correct orientations for microscopy. For example, filter cubes are essential for fluorescence microscopy and reflected light microscopy.
Köhler Illumination: A method of illumination that utilizes various optical elements to defocus and flatten the intensity of light across the field of view in the sample plane. A condenser and light collimator are necessary for this technique.
Nosepiece: A type of arm used to hold the microscope objective in the optical path of the microscope.
Optical Path: The path light follows through the microscope.
Rail Height: The height of the support rail of the microscope body.
Throat Depth: The distance from the vertical portion of the optical path to the edge of the support rail of the microscope body. The size of the throat depth, along with the working height, determine the working space available for microscopy.
Trans-Illumination: Illumination on the opposite side of the sample as the viewing apparatus. Brightfield, differential interference contrast (DIC), Dodt gradient contrast, and darkfield microscopy are some examples of imaging modalities that utilize trans-illumination.
Working Height: The height of the support rail of the microscope body plus the height of the base. The size of the working height, along with the throat depth, determine the working space available for microscopy.
Cerna Microscope Body
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The microscope body provides the foundation of any Cerna microscope. The support rail utilizes 95 mm rails machined to a high angular tolerance to ensure an aligned optical path and perpendicularity with the optical table. The support rail height chosen (350 - 600 mm) determines the vertical range available for experiments and microscopy components. The 7.74" throat depth, or distance from the optical path to the support rail, provides a large working space for experiments. Components attach to the body by way of either a linear dovetail on the support rail, or a circular dovetail on the epi-illumination arm (on certain models). Please see the Microscope Dovetails tab or here for further details.
Illumination with a Cerna microscope can come from above (yellow) or below (orange). Illumination sources (green) attach to either.
Using the Cerna microscope body, a sample can be illuminated in two directions: from above (epi-illumination, see yellow components to the right) or from below (trans-illumination, see orange components to the right).
Epi-illumination illuminates on the same side of the sample as the viewing apparatus; therefore, the light from the illumination source (green) and the light from the sample plane share a portion of the optical path. It is used in fluorescence, confocal, and reflected light microscopy. Epi-illumination modules, which direct and condition light along the optical path, are attached to the epi-illumination arm of the microscope body via a circular D1N dovetail (see the Microscope Dovetails tab or here for details). Multiple epi-illumination modules are available, as well as breadboard tops, which have regularly spaced tapped holes for custom designs.
Trans-illumination illuminates from the opposite side of the sample as the viewing apparatus. Example imaging modalities include brightfield, differential interference contrast (DIC), Dodt gradient contrast, oblique, and darkfield microscopy. Trans-illumination modules, which condition light (on certain models) and direct it along the optical path, are attached to the support rail of the microscope body via a linear dovetail (see Microscope Dovetails tab or here). Please note that certain imaging modalities will require additional optics to alter the properties of the beam; these optics may be easily incorporated in the optical path via lens tubes and cage systems. In addition, Thorlabs offers condensers, which reshape input collimated light to help create optimal Köhler illumination. These attach to a mounting arm, which holds the condenser at the throat depth, or the distance from the optical path to the support rail. The arm attaches to a focusing module, used for aligning the condenser with respect to the sample and trans-illumination module.
& Body Attachments
|Brightfield||DIC||Dodt||Condensers||Condenser Mounting||Light Sources|
Light from the sample plane is collected through an objective (blue) and viewed using trinocs or other optical ports (pink).
Once illuminated, examining a sample with a microscope requires both focusing on the sample plane (see blue components to the right) and visualizing the resulting image (see pink components).
A microscope objective collects and magnifies light from the sample plane for imaging. On the Cerna microscope, the objective is threaded onto a nosepiece, which holds the objective at the throat depth, or the distance from the optical path to the support rail of the microscope body. This nosepiece is secured to a motorized focusing module, used for focusing the objective as well as for moving it out of the way for sample handling. To ensure a light-tight path from the objective, the microscope body comes with a bellows (not pictured).
Various modules are available for sample viewing and data collection. Trinoculars have three points of vision to view the sample directly as well as with a camera. Double camera ports redirect or split the optical path among two viewing channels. Camera tubes increase or decrease the image magnification. For data collection, Thorlabs offers both cameras and photomultiplier tubes (PMTs), the latter being necessary to detect fluorescence signals for confocal microscopy. Breadboard tops provide functionality for custom-designed data collection setups. Modules are attached to the microscope body via a circular dovetail (see the Microscope Dovetails tab or here for details).
The rigid stand (purple) pictured is one of various sample mounting options available.
Various sample and equipment mounting options are available to take advantage of the large working space of this microscope system. Large samples and ancillary equipment can be mounted via mounting platforms, which fit around the microscope body and utilize a breadboard design with regularly spaced tapped through holes. Small samples can be mounted on rigid stands (for example, see the purple component to the right), which have holders for different methods of sample preparation and data collection, such as slides, well plates, and petri dishes. For more traditional sample mounting, slides can also be mounted directly onto the microscope body via a manual XY stage. The rigid stands can translate by way of motorized stages (sold separately), while the mounting platforms contain built-in mechanics for motorized or manual translation. Rigid stands can also be mounted on top of the mounting platforms for independent and synchronized movement of multiple instruments, if you are interested in performing experiments simultaneously during microscopy.
|Translating Platforms||Rigid Stands||Translation Stages for Rigid Stands||Motorized XY Stages||Manual XY Stage|
For sample viewing, Thorlabs offers trinoculars, double camera ports, and camera tubes. Light from the sample plane can be collected via cameras, photomultiplier tubes (PMTs), or custom setups using breadboard tops. Click here for additional information about viewing samples with a Cerna microscope.
Microscope objectives are held in the optical path of the microscope via a nosepiece. Click here for additional information about viewing a sample with a Cerna microscope.
Large and small experiment mounting options are available to take advantage of the large working space of this microscope. Click here for additional information about mounting a sample for microscopy.
Thorlabs offers various light sources for epi- and trans-illumination. Please see the full web presentation of each to determine its functionality within the Cerna microscopy platform.
Epi-illumination illuminates the sample on the same side as the viewing apparatus. Example imaging modalities include fluorescence, confocal, and reflected light microscopy. Click here for additional information on epi-illumination with Cerna.
Trans-illumination illuminates from the opposite side of the sample as the viewing apparatus. Example imaging modalities include brightfield, differential interference contrast (DIC), Dodt gradient contrast, oblique, and darkfield microscopy. Click here for additional information on trans-illumination with Cerna.
|Product Families & Web Presentations|
|Brightfield||DIC||Dodt||Condensers||Condenser Mounting||Illumination Kits||Other Light Sources|