Transmitted Light Illumination for DIY Cerna® Systems

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
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
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

DIC Imaging
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 Contrast
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

Laser Scanning
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 some DIY Cerna microscopes.

DIY Cerna Design and Assembly

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.

• Terminology
• Microscope Body
• Illumination
• Sample Viewing/Recording
• Sample/Experiment Mounting

Terminology

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.

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Cerna Microscope Body

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Body Details

Microscope Body

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.

Microscope Bodies	Microscope Translator

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Illumination with a Cerna microscope can come from above (yellow) or below (orange). Illumination sources (green) attach to either.

Illumination

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.

Epi-Illumination Modules	Breadboards & Body Attachments	Brightfield	DIC	Dodt	Condensers	Condenser Mounting	Light Sources

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Light from the sample plane is collected through an objective (blue) and viewed using trinocs or other optical ports (pink).

Sample Viewing/Recording

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).

Objectives & Accessories	Objective Mounting

Sample Viewing	Cameras	PMTs	Breadboards & Body Attachments

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The rigid stand (purple) pictured is one of various sample mounting options available.

Sample/Experiment Mounting

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

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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.

Product Families & Web Presentations
Sample Viewing	Breadboards & Body Attachments	Cameras	PMTs

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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.

Product Families & Web Presentations
Objectives	Objective Thread Adapters	Parfocal Length Extender	Piezo Objective Scanner	Objective Mounting

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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.

Product Families & Web Presentations
Translating Platforms	Rigid Stands	Translation Stages for Rigid Stands	Motorized XY Stages	Manual XY Stage

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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.

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Trans-Illumination Kits	Solis™ High-Power LEDs	Mounted LEDs	X-Cite® Lamps	Other Light Sources

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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.

Product Families & Web Presentations
Epi-Illumination	Body Attachments	Light Sources

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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

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The microscope body provides the foundation of any Cerna microscope. The 7.74" throat depth provides a large working space for experiments. Click here for additional information about the Cerna microscope body.

Product Families & Web Presentations
Microscope Bodies	Microscope Translator