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Image acquisition

Fluorescence

Images acquisition



In any microscopy technique - brightfield, DIC, fluorescence ..., optical media recording to be used must be carefully selected to offer the best possible picture quality for the application. This selection is often not trivial, since the researcher must consider a myriad of variables, most of which are mutually exclusive. Often, the use of a variable is positive for one reason and negative for another. For example, in most situations involving live cell microscopy, the primary task becomes the balance between the health of cells and image quality, taking into account factors such as cell viability, signal to noise ratio and resolution request or the speed of acquisition. The aims and objectives of the type of investigation should determine the order of priority of these factors.

The options for recording media include color CCD cameras to capture images in brightfield or for samples containing multiple fluorophores, infrared cameras often used in imaging mode DIC, cooled CCD cameras with a sensor to capture low level signals as fluorescent sensors for high resolution black and white for TIRF and other techniques and many others.

Shift and multiband filter set


When you make a photograph with multiple exposures or more digital images of samples labeled with different fluorochromes, using different filters in a standard microscope-cube, there are inevitable changes in registration between exposures.

These changes contribute to the following factors:

-) variability of the peaks in the emission filter and dichroic filter;
-) variations in thickness and alignment of the dichroic filter;
-) mechanical vibrations that occur when the filters are changed on cube.


Although for some applications these effects can be reduced to acceptable levels, many others require a design-cube filters more sophisticated and more complex optical devices. Table 1 lists a few methods available for applications involving the use of multiple investigations. These include multi-band filter set and various configurations using both multi-band filters that single-band filters


Methods for applications that use multiple investigations


All these methods are designed to eliminate registration errors mentioned above. Method 2 offers the ability to visually analyze up to three colors simultaneously. (Methods 3 to 6 also have this possibility if they are added to the filter set of multiband filters.)

METHODS

COMPONENTS

ADVANTAGES

DISADVANTAGES

1. Single-band filter set

Standard microscope.

No extra equipment required. Brighter images.

The simultaneous processing of images is not possible. is not perfect combination of images.

2. Multi-band filter set

Standard microscope.

Simultaneous observation of error-free recording.

No extra equipment required (special filter set excluded).

Lower brightness. reccomended for use with XENON arc light.color balance.


3. Single-band excitation filter, multi-band dichroic and emission filter.

Microscope with a filter or rotating filter-slide in the path of the light.

Camera or digital camera with image processing.

Observation sequential error-free recording. Precise record of combined images, color balance adjustment. The optimization of excitation filter helps to provide a brighter image than that obtained with method 2.

Required extra equipment (rotary filter or filter-slide).

4. Multi-band dichroic filter, single-band emission and excitation filters.
Single camera.

Microscope with a filter or rotating filter-slide in the path of the light and that of the image processing.

Camera or digital camera with image processing.

The excitation and emission filters can be designed to have a brightness similar to that obtained in step 1. Reduction of errors in registration (by eliminating the movement of dichroic filter).

Required extra equipment (rotary filter or filter-slide).

There may still be registration errors between the emission filters.

5. Multi-band dichroic filter, single-band emission and excitation filters.
More cameras.

Microscope with a filter or rotating filter-slide in the path of the light.

Dichroic filter assembly for the separation of channels, each channels has a separate emission filter.

In addition to the advantage of the method 4 registry errors from emission filters can be eliminated. Further application are supported (ratiometry).

More complex devices require additional dichroic filters and more cameras.

6. As the point 4 and 5 by replacing the multi-band dichroic filter with a neutral dichroic filter.

 

Any valid configuration can be used for emission and excitation filters.

The brightness can be reduced by as much as 80%, are recommended special light sources (ie. laser).

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