eTraining Introduction

Specimen Preparation

Leica Ultracut UCT

Histology

JEOL JSM-6400 SEM

Hitachi S-4700 FE-SEM

Hitachi FB-2000A FIB

Microanalysis

Veeco Dim 3000 AFM

Fluorescence Microscopes

Support

Tips and Troubleshooting

Phase Plates

Differential Interference Contrast (DIC)

DIC results in the construction of a “3D” image. This technique is commonly used when imaging live specimens. The objectives available for DIC imaging are 10X and 20X.

Phase Contrast

Phase Contrast is based on the change in phase and diffraction by the specimen. There are several plate options for phase contrast imaging:

Ph1 and Ph2

Ph1 and Ph2 both add three dimensional characteristics to the edges of samples but to different degrees. The user will have to determine which plate provides the best image. Ph1 is typically used with the 10X objective and Ph2 is used with the 20X objective.

Ph3—Darkfield

In darkfield imaging, light waves that are not diffracted by the sample are removed from the image. The resulting image represents the sample against a very dark background. This technique is useful for very small samples that are efficient at diffracting light such as, diatoms, cilia, filaments, microtubules, and so on. This phase plate is very sensitive. It is commonly used for adding contrast when viewing tissue sections.

Brightfield Imaging

Brightfield imaging is used when the sample has enough intrinsic contrast to not necessitate adding contrast filters. Such samples include stained tissue, colored specimens, and microorganisms.

 

DIC Phase Contrast Brightfield Darkfield
transparent samples
(bacteria, protozoa, mites, etc.)
diatoms
fibers
hairs
powders
liquids
transparent samples
(bacteria, protozoa, mites, etc.)
diatoms
fibers
hairs
powders
liquids
tissues
organelles
stained tissue
insects
algae
tissue sections
diatoms
fibers
hairs
flagella
filaments
microtubules

What is exposure?

Exposure determines the amount of time the camera is open for the collection of photons. When an image is overexposed, too many photons are collected. This results in brightness where there should be none, or a “false positive”. Underexposure is the opposite.

Choosing the correct stage plate

There are two stage plates available: one is designed for slides and dishes, and the other is used for plates.

Choosing the Fluorescence Filter

Filter Set Emission Range (nm)
01
404-582
10
502-579
18
455-549
20
567-637

 

Use the Filter Set information binder in the lab room to select the filter corresponding to the dye used in your sample. In the guide, the excitation and emission wavelengths are listed for each fluorescent dye. The excitation wavelength indicates the wavelength of light which will result in fluorescence from the dye. The emission wavelength is the wavelength at which fluorescent light leaves the sample, indicating the color of visible light observed.

Objective Lenses

2.5X
5X
10X
20X DIC
40X DIC

Oil Lenses

Oil immersion lenses provide superior detail and magnification. As the name suggests, these lenses utilize a barrier of oil between the surface of the sample and the objective lens. This barrier increases the refractive index of the barrier between the sample and the lens. By increasing the refractive index, the numerical aperture of the objective lens increases, allowing for the collection of a greater number of refracted light rays. More light rays means more detail and resolution. It is important to use only the oil which corresponds to the lens to maximize the capabilities of the lens and avoid negative effects of a mismatched oil-lens combination. The oil lenses available are used only with fluorescent light; not transmitted light imaging.

63X Oil
100X Oil

Troubleshooting

The technical support number is 1-800-233-2343.

Difficulty finding a sample that is stained with fluorescent dye.

It is often difficult to find a sample in white light because of a lack of contrast. You may want to begin with fluorescence microscopy in order to find the sample.

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