In Darkfield microscopy oblique illumination (i.e. the zero order light is blocked out) is used to create contrast in unstained samples. It is one of the most simple contrasting techniques, with usually quite impressive results.
Here you find a detailed explanation of how darkfield illumination works.
In this half-hour video Prof. Edward Salmon describes the principles of dark field and phase contrast microscopy, two ways of generating contrast in a specimen which may be hard to see by bright field.
Phase contrast is another contrasting method for unstained samples and makes use of the fact that different structures in the sample phase shift the light passing through by different distances. These phase shifts are than made visible by being converted into different levels of brightness in the final image.
Phase contrast is especially suitable for thin samples.
This page contains a comprehensive overview of the technical and historical details of phase contrast microscopy. It also leads you to interactive java tutorials illustrating configurations and alignment.
Here you will find an illustrated step-by-step manual about how to align your micrscope for phase contrast imaging.
In this half-hour video Prof. Edward Salmon describes the principles of dark field and phase contrast microscopy, two ways of generating contrast in a specimen which may be hard to see by bright field. The lecture describes how the phase rings work to generate interference between the diffracted and undiffracted light.
The use of two crossed polarizers gives bright contrast to birefrigent objects such as anisotropic crystals and biological polymers.
On this webpage you will find a detailed explanation on how polarization of light works, how we can use the birefrigency of objects to our advantage and learn main applications of polarized light.
Here you will find an introduction to optical birefrigency, which is the prerequisite property for any specimen imaged using polarization microoscpy.
In this half-hour video Prof. Edward Salmon describes the components of a polarization microscope (e.g. polarizer, analyzer), birefringence and how it is exploited to generate images, adjusting a polarization microscope, examples of images, and new methods such the LC-Polscope.
Differential Interference Contrast (DIC) is another illumination technique to enhance contrast in unstained, transparent samples.
Orthogonally polarized light is split into two coherent beams, which pass the sample in a parallized, but sheared (spatially separated) manner. Different features in the sample (thickness, slope, refractive index) then alter the path of both beams to varying degrees, so that when combined again after the specimen, they interfere with each other. Due to the sample-altered path lengths of the light beams, the resulting image has the apparence of a 3D represention.
This page contains a comprehensive overview of the technical and historical details of DIC microscopy. It also leads you to interactive java tutorials illustrating configurations and alignment.
Here you can find step-by-step instructions on how to prepare and align your microscope for DIC microscopy.
In this half-hour video Prof. Edward Salmon discusses the mechanism of the DIC (Wollaston) prisms along with how to generate optimal contrast:
In todays life sciences, only few microscopic samples and experiments can be satisfyingly imaged using stainfree contrasting methods such as described previously. Most users that come to our facilities want to image fluorescently labeled samples.
Fluorescent substances (fluorochromes) absorb light of a specific wavelegth and (usually) emit light of a longer wavelegth (Stokes' shift). A fluorescent microscope uses different filters to distinguish between excitation and emission light. The emission light is then captured by the detector (eg. camera). Using multible fluorochromes, different structures of one microscopic specimen can be labeled, imaged and analysed simultaneously.
On these two pages you will find a very detailed introduction to the basic concepts of fluorescence and fluorescence mircroscopy: fluorescence intro 1, fluorescence intro 2
On this page fluorescence microscopy is discussed in various chapters featuring all aspects of a fluorescence microscope.
This webpage as well gives you an introduction to fluorescence microscopy, but also features on the importance of various light sources and objectives to your imaging result.
In this half an hour lecture Nico Stuurman describes the principles of fluorescence and fluorescence microscopy.
There are two common ways of getting your sample to fluoresce:
You can make a transgenic line of your model organism, thats stabely expresses a certain fluorecent protein (i.e GFP ... green fluorescent protein). If there is no established line which targets your feature of interest, than making one can cost you a lot of time and recources. Once a line is established, it is a great tool and you always have a fluorescent sample available without the need for extensive staining protocolls. However, you might also want to consider that this method makes you less flexible to react to changes on your (the facilities') fluorescent microscope (eg. changes of filters, light sources).
Here you find a comprehensive list of available fluorescent proteins combined with a great interactive graph. You may filter the plot and compare by excitation and emission wavelegth, Quantum yield, brightness, stability and and and.
If you are interested in the history of fluorescent proteins, have a look here.
And here another, well structured overview of fluorescent proteins.
Fluorescent dyes are fluorochromes which you target to your structure of interest via antibodies. For this, extensive staining protocolls are necessary, the outcome of which can vary widely regarding specificity, brightness and stability. While this might be seen as a limitation, antibody fluorescent stainings offer huge flexibility. With an elaborate amount of different first and second antibodies on offer the whole system can be easily adapted to various external factors.
In the following we provide links to comprehensive databases of both fluorescence proteins and dyes with sorted spectras and including lasers, lamps and filter sets.
In most fluorescent microscopes filters are needed to distinguish between excitation and emission light, to narrow down the wavelength band passing through or to split between several parallel excitation or emission wavelengths.
Chroma Technology Corp is one of the leading international manufacturers for optical filters.
In their "Handbook of Optical Filters for Fluorescence Microscopy" you will find an introduction to fluorescence microscopy as well as discussions about optical filters in general and fluorescence filters in specific.
Semrock is another leading international manufacturers for optical filters, giving a nice introduction to optical filters on their webpage.
Here, AHF Analysentechnik explains their Filtersystems.
Read here how best to combine fluorescence filters.
How does the overlap of fluorescence filter spectra influence your imaging? Find out in this interactive Java tutorial.