Epi-FluorescenceEpi-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.

Objective prisms, also known as sliders, are designed for use with particular objectives. The table to the right summarizes the objective prism compatibility. All of our objective prisms are Nomarski modified.

Differential interference contrastmicroscopy principle

The WFA3110 DIC Analyzer is designed to fit into a specially designed slot in the CSE2000, CSE2100, and CSE2200 Epi-Illuminator Modules, which have detents that stop the WFA3110 at each of its three slots. It is important to insert this analyzer right-side up (i.e., with the labels facing upward), or it can become stuck in the epi-illuminator module. If using another epi-illuminator module, a custom mounting solution will be required.

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.

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.

DIC ImagingIn 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.

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.

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.

Turret Mounting AdapterThe WFA3000 Turret Mounting Adapter secures the WFA3100 polarizer turret onto the WFA1000 trans-illumination module and aligns the module's optical output port with one of the positions on the turret. It contains four M3 counterbores that are spaced to mate with four M3 tapped holes on top of the module, as well as one M5 tapped hole that mates with the polarizer turret. Four M3 button head screws are included with the adapter.

After the two spatially separated, orthogonally polarized beams generated by the condenser prism travel through the sample, they are recombined by the objective prism. If the sample has changes in refractive index and/or thickness across the field of view, as described in the Overview tab, the two beams will have experienced different optical path lengths.

The WFA3130 Condenser Prism is used with N1 objectives, while the WFA3131 Condenser Prism is used with N2 objectives. The housings of DIC-compatible objectives are typically engraved with either "N1" or "N2". You should verify that this engraving matches the condenser prism to ensure compatibility. Both of our prisms are Nomarski modified.

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.

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.

The relative thickness of the polarizer turret and turret mounting adapter is a key reason why microscope bodies with rail heights less than 400 mm do not support DIC imaging.

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.

Differential interference contrastvs phasecontrast

Dodt ContrastDodt 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.

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Laser ScanningLike 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.

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.

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).

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.

Brightfield IlluminationBrightfield 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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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.

Bellows: A tube with accordion-shaped rubber sides for a flexible, light-tight extension between the microscope body and the objective.

The final DIC element in the optical path is the DIC analyzer. It effectively converts the differences in optical path length across the field of view into differences in intensity that can be resolved by the widefield viewing apparatus (e.g., a camera or trinoculars). One slot on the analyzer is intentionally left empty for situations that only require brightfield illumination. The other two slots are filled by a visible analyzer (i.e., a non-rotating polarizer) and an IR analyzer.

The objective also has a hemispherical front lens and a meniscus second lens, which work synchronously to assist in capturing light rays at high numerical ...

Differential interference contrastmicroscope images

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).

Condenser prisms accept the polarized light from the DIC polarizers described above. When the optical axis of the prism is aligned at 45° with respect to the input polarization, the prism will split the input beam into two spatially separated, orthogonally polarized beams of equal intensity. These spatially separated, orthogonally polarized beams make the DIC method sensitive to refractive index and thickness changes across the field of view, as described in the Overview tab.

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.

The WFA1000 Trans-Illumination Module steers the light from a user-provided illumination source into the transmitted light optical path. It is designed for the 400 - 1000 nm wavelength range.

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.

Differential interference contrastmicroscope

DIC PolarizersThe WFA3120 and WFA3121 consist of a polarizer and a quarter-wave plate (i.e., a de Sénarmont compensator) that have been combined into a single housing. Each housing has gear teeth that enable smooth rotation of the polarizer through 360° without changing the orientation of the quarter-wave plate. Each polarizer therefore controls whether linearly polarized, circularly polarized, or elliptically polarized light is incident on the specimen, letting the experimenter fine tune the system for the greatest possible sensitivity. To allow the polarizers to rotate, the WFA3100 polarizer turret includes two knurled rings. The knurled ring that is located clockwise from the polarizer rotates it.

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.

The WFA3100 Polarizer Turret and WFA3000 Turret Mounting Adapter are used to mount DIC polarizers onto the WFA1000 Trans-Illumination Module shown above.

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 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.

Our CSC1001, CSC1002, and CSC2001 condensers contain internal turrets or slots for trays that accept these condenser prisms.

Differential interference contrastprinciple

These objective prisms are designed to fit into specially designed slots in the CSN1201 and CSN1202 Objective Nosepieces. If using another nosepiece, a custom mounting solution will be required.

Please note that the CSD1001 Double Camera Port includes a built-in IR analyzer for the rear camera port. If your Cerna system has this double camera port and visible DIC imaging is not needed, then the WFA3110 analyzer is not required.

Differential interference contrastmicroscopy PDF

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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.

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).

Within the sample, each beam experiences a different optical path length. This path length difference can be caused by changes in the refractive index, such as those between cell interfaces or those due to birefringence, and it can also be caused by variations in the specimen's thickness over the field of view. The two beams are spatially recombined at the Nomarski-modified objective prism, allowing optical interference between them to occur. This (sample-induced) interference pattern is resolved by the analyzer, which creates differences in intensity that can be viewed on a camera.

Differential interference contrastmicroscopy application

When mounted to a Cerna microscope body using the WFA0150 Dovetail Clamp, our WFA1000 Trans-Illumination Module will direct illumination along an optical axis 7.74" away from the edge of the vertical support rail. The module is compatible with any collimated illumination source coupled into a 30 mm cage system, such as an illumination kit. To complement the trans-illumination module, we offer DIC polarizers, condenser prisms, objective prisms (sliders), and an analyzer that manipulate the polarized light which goes through the sample. Our condenser and objective prisms are Nomarski modified.

As shown in the drawings to the right, the collimated illumination is accepted through a Ø1" input port on the side of the module. To mount an illumination source to this 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.

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.

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.

Bayonet Mount: A form of mechanical attachment with tabs on the male end that fit into L-shaped slots on the female end.

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Other Transmitted Light Imaging ModalitiesWe also support Dodt contrast and brightfield and oblique illumination for DIY Cerna systems. These imaging modalities are easier to add to Cerna systems because they do not require a particular minimum rail height, objective nosepiece, or epi-illuminator module. 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. For more details, see the Imaging Modalities tab.

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 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.

Differential interference contrastmicroscopy advantages and disadvantages

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.

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This webpage contains Thorlabs' selection of components for performing differential interference contrast (DIC) imaging in DIY Cerna systems. DIC imaging is an extremely popular method for obtaining contrast from thin unlabeled samples in vitro.

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.

System RequirementsPlease note that when configuring a Cerna system for DIC, the microscope body needs to be tall enough to make room for all of the needed optical elements. In addition, the condenser, objective mounting apparatus, and epi-illuminator module need to have slots for the condenser prism, objective prism, and analyzer, respectively. Our recommendations are shown in the "System Requirements for DIC" table above.

Polarizer TurretThe WFA3100 Polarizer Turret's bottom wheel has four positions (empty, ND 2, ND 4, and ND 16), while the top wheel ships with three empty slots and a color-temperature-balancing filter. Two of the empty slots on the top wheel are sized to fit the WFA3120 Visible Polarizer and the WFA3121 IR Polarizer, which are sold separately, while the third empty slot is intentionally left empty for situations that only require brightfield illumination. An M5 thumbscrew connects the turret to the WFA3000 mounting adapter.

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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.

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. A diffuser comes pre-installed inside the module in order to homogenize the output light. If desired, the user can access and easily remove this diffuser by unscrewing four #1 Phillips-head screws on the side of the module. 30 mm cage compatibility for the output port is provided by four 4-40 taps.

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.

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.

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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DIC imaging generates contrast using several polarization-manipulating optics. The illumination source is first polarized, then split into two spatially separated, orthogonally polarized beams of equal intensity by the Nomarski-modified condenser prism. These two beams are directed onto the sample.