Visual Fluo Bacteria: a fluorescence image analisys software

Promoter activity can be revealed using fluorescent proteins as reporters. An image of fluorescence bacteria can be obtain thanks to a microscopy system and a video camera ( Figure 1 shows an acquired bacterial image).

Figure 1:Fluorescence field

In this image we can achieve bacteria's morphology informations, size and the number of bacteria in a single view. Additionally we can assess the promoter activation dynamic in terms of fluorescence mean value per bacterium and standard deviation.
The software we created is easy and intuitive and in the Figure 2 it is possible contemplate the main software frame.

Figure 2:Main frame

The algorithm reads fluorescence images and converts it into a "black and white" one that is then filtered by Top Hat filter to correct uneven illumination when the background is dark. The following step is needed to compute the global threshold in order to convert an intensity image to a binary image using Otsu’s method. The image is then ready to be scanned, pixel by pixel, to detect bacteria (cluster of pixels) and obtain final informations about their area, fluorescence mean (in RGB channel: R for RFP, G for GFP, B for CFP) and standard deviation. All the data are processed with area and focus efficiency parameters to estimate the population fluorescence mean, standard deviation, median, minimal and maximal fluorescence levels.

Our software consists in a filtering of bacteria that are on the acquired image and it based on an algorithm that avail different parameters. In particular the user can set two indicators:

• Area dimension range ( definite by a superior and an inferior limit): This range defers to selected the bacteria that have same size.
• Fluorescence intensity ( definite by the std\mean ratio referred to each bacteria): this values consent to throw away the bacteria that lie on another focal layer or that aren’t focused correctly.

At this point, the filter based on these values discard the bacteria that have a std\mean higher than the threeshold and the bacteria that have a dimension out of the range.

Image before filtering

Image after filtering

To download the program and relative user manual, you can click on the following icons.

Visual Fluo Bacteria 1.0
Note: after decompacting file, execute and run classificazione.m
User Manual

Microscopy system for fluorescence image analysis

Acquisition system

The illumination system is composed of a 75 Watt Xenon arc lamp connected to a Photon Technology Instruments DeltaRAM X monochromator, which breaks up a single polychromatic light beam into several monochromatic light beams (with only one wavelength each). Only the selected wavelength can pass through the output port and reach the microscope.

The system’s core is a Nikon Eclipse TE2000-U inverted fluorescence microscope. For GFP image acquisition we used a B-2A filter by Nikon with an excitation band between 450 and 490 nm and the optimal emission placed at 520 nm.

The camera used to acquire images and film segments is a Nikon DS-5m with a DS-U1 controller. This one receives the acquired signal form the camera through a serial connection and sends it to the PC through an USB slot. Nikon also supplied an interface software for image acquisition and elaboration.

The control software is implemented in a Labview environment and permits the regulation of the excitation wavelength and the calibration of the system. It also pictures the output signal from the photomultiplier, which can be memorized and elaborated.

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A.R.Q.: an Automatic Registry Query generator software

The idea from which was born this application is to realize a database registry like, in order to share with all the faculty of the bologna university parts and interesting project. The devolpment starts from the registry, which is well structured and an important source of information, we want so to create a tool that permits to query automatically the web page of the registry to achieve the data of a part and compare with ours. We start to develop a tools that scans the registry page to find three parameters:the registry code, the partial description of the parts and the sequence.
There are three research modality included in this tools:

• INPUT :code OUTPUT:sequence and partial description;
• INPUT:parts or subparts of the name OUTPUT:all the parts which share that name;
• INPUT:sequence OUTPUT:all tha parts that match that sequence;

It's possible to imit the search to a selected categories of the registry or to extends to all with a drop down menù.
The software was developed in java language to give the possibility to run it on all the type of machine provided thath you have a java virtual machine.
The user's interface is friendly an very simple:

the REGISTRY CODE PARTS requires the standard registy code BBa_XXXXX, the PARTIAL DESCRIPTION is a free text field, where can be writted any keywords of the research the TYPE PARTS is a drop down menù with twelve option:

• measurement
• generator
• composite
• rna
• dna
• conjugation
• reporters
• signalling
• rbs
• regulatory
• terminator
• all
  

to limit only in a restricted arts of the registry the search.
the SEQUENCE in a text area in which insert the sequence of the parts without space.
When we use the parameters partial description and sequence the search will not be unique so the resutl will show in a new window teh result page:

this page show a list of name of the registry parts and when we query the tools with a sequence is possible through a tooltip to view in order :

• the name of the parts
• the first and the last index of the alignment between the query and the answer sequence
• the length of the answer sequence
• the group of the registry parts(regulatory,conjugation,signalling....)

When a part is selected, by a double mouse double click, the program automatically update the windows loading the empty fields with the part's data. The sequence field show the match between the parts changing to upper case the shared base.