Photonic Science EMCCD cameras

EMCCD cameras and scientific Detector Systems

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Western Blots / Northern Blot Gel Imaging

The Western Blot (alternately, immunoblot) is used for detecting specific proteins.

This uses gel electrophoresis order to separate proteins according to their molecular weight and conformal state. The proteins are then transferred to a membrane where they are detected using antibodies to the target protein.

Good dynamic range is required for sensing both bright and low intensities in the same image; typically a 16 bit image digitisation allows good contrast resolution.
Reasonable spatial resolution, typically 1.4 megapixel is necessary in order to allow image segmentation for quantitative analysis.
Alternatively, a variant technique with a chemiluminescent agent can be used for producing luminescence in proportion to the amount of protein.

A high sensitivity camera is then used to record an image of the antibodies bound to the blot. Usually longer exposures up to minutes are necessary hence a very low dark current / read out noise must be achieved on the camera.

Western blots / northern blot Gel imaging

Western Blots / Northern Blot Gel Imaging

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Confocal Fluorescence Imaging

In a confocal microscope, the detector aperture obstructs the light that is not coming from the focal point. The out-of-focus light is blocked by the pinhole and in order to produce crisp image with haze contribution introduced by the depth of field from the objective. The smaller the pinhole, the sharpest the image will be as it will block effectively the fluorescence from the nearest neighbouring planes, but also the less light will be caught by the detector.
Therefore there is a requirement for a very sensitive camera in order to capture the fluorescence arising from the confocal plane.

Typical integration time must be kept as short as possible in order to avoid cell damages under extended periods of digital recording. Typically 100ms to 1 second is used per image, depending on sensitivity settings chosen on the camera.

Three dimensional re construction is achieved by acquiring multiple images at different Z positions using a piezo / stepper motor driven z axis on a motorized microscope. Z stacks acquired over time allows to build multidimensional data sets taking into account multiples variables such as wavelengths, x,y,z positions and time. Multiple cameras can be synchronized as one single detector in order to produce parallel acquisition.

Confocal fluorescence imaging

Confocal Fluorescence Imaging

TIRF

A Total Internal Reflection Fluorescence Microscope uses evanescent waves to selectively illuminate and excite fluorophores in a restricted region of the sample. Evanescent waves are generated only when the incident light is totally reflected. The evanescent electromagnetic field decays exponentially and thus penetrates to a depth of only approximately 100 nm into the sample.

Thus the TIRFM enables a selective visualization of surface regions such as plasma membrane. TIRF can also be used to observe the fluorescence of a single molecule.
Large magnification objectives are used routinely: typically 63 up to 100x objectives are combined with x2 projective optics in order to reach less than 100nm resolution.

As the volume probed is very small, the amount of fluorescence recorded is small and hence requires very high sensitivity cameras. Exposure time from 10ms to less than 1sec are also necessary in order to record live data from biological samples.

 

TIRF

TIRF

Luminescence / Small Animal Imaging

Luminescence emissions from reporter genes provide a quantitative model for the study of development of human diseases.

As the amount of light collected using this method is very faint, an ultra sensitive camera is required to record live luminescence emissions propagating through soft tissues.

Cameras with near single photon counting sensitivity are used for this type of experiment. They provide a live image of very low photonic emissions, typically down to few hundred photons per second per steradian and cm2.

These measurements are usually combined with fluorescence as well as X-ray CT scans in order to provide an accurate 3D model of tumour / location propagation.

Quantification of luminescence emission to derive gene reporter activity is also used with transgenic plants. Monitoring live luminescence on Arabidopsis samples according to varying physiological conditions is possible using a real time recording intensified digital camera system. `

 

Luminescence / small animal imaging

Luminescence / Small Animal Imaging

High Throughput Cell Screening

Green Fluorescent Protein (GFP) has many applications as a marker in living cells, and has become widely used as a reporter gene in microbial, plant and animal cells.

Screening microbial colonies for GFP expression enables various types of assays (e.g. for mutations).

Fast automated imaging data collection routines enable discrimination between colonies, based on the level of fluorescence activity and the picking function automatically transfers cells to microplate wells.

Measuring fluorescent activity allows quantification of fluorescent tag concentration/expression. Fast acquisition requires a high sensitivity camera to collect images at high speed with as little as 1ms integration time.

Gated cameras can be used with pulsed fluorescence sources in order to reduce noise and synchronise accurately exposure to excitation sequences.

 

High thoughput cell screening

High Thoughput Cell Screening

Ophtalmology Fluorescence Imaging

Detecting fluorescence from retinal lipofuscin chromophores allows to indirectly quantify and spatially image the distribution of Macular Pigment (MP). The lipofuscin fluorescence intensity is reduced at all retinal locations containing Macular Pigment (MP), since MP has a competing absorption in the blue-green wavelength region.

By projecting a large diameter, 488 nm excitation spot onto the retina centred on the fovea, extending into the macular periphery, and comparing lipofuscin fluorescence intensities outside and inside the foveal area, it is possible to spatially map out the distribution of MP.
A very high resolution camera is required in order to capture the fluorescence signal with the best possible spatial resolution.

The camera can be used with pulsed sources by gating the camera keeping temporal resolution down to few microseconds with good repetition rate so as to keep the signal to noise high and integrated power low. Two versions with 11 and 16 million pixels versions are available.

Ophtalmology fluorescence imaging

Ophtalmology Fluorescence Imaging

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Photonic Science Limited - EMCCD detector cameras and Scientific detector systems