Automating ZEISS Fluorescence Microscope: Axioskop 50 HBO

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Introduction

Fluorescence microscopy is an important tool in modern biology and medicine. Fluorescence is the optical phenomenon where fluorescent molecules, after beingexcited by light of a particular wavelength, emit light of a different wavelength.



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The concept of the fluorescence microscope consists of several parts: a source, 3 filters, some optics, and a detector. Light is provided by a light source, either a lamp, a laser, or lately an LED. This light passes through an excitation filter, that keeps the light wavelength we wish to use as excitation, shown in blue. A precise objective lens guides this excitation light into a sample. The fluorophores in the sample excited by the excitation light emit emission light , shown in green. This emission is collected by the objective lens and is separated from the excitation light by the dichroic filter. Finally, the emission passes through the emission filter (reject noise) and is detected in a detector (e.g. a CCD).

In fluorescence microscopy the signal that is coming from fluorescent molecules (either endogenous or exogenous like dyes, antibodies, fluorescent proteins) can be used in the study of macromolecules, cellular structures and cellular functions in fixed or living cells, in fixed tissues even in living animals.


Systems biology is the science that studies the cell functions as a system. The response of the cellular system is measured via high-throughput and multiplex covering a wide range of signals and stimuli. So far, the biomechanics and systems biology laboratory of NTUA have focused in proteomic experiments studying the expression and the state of individual proteins in a cell group. On the other hand, fluorescence microscopy allows the measurement of protein expression and structures with high spatial and temporal sampling, even to quantify the expression of each cell separately (cytometry). These data can be used to complement the proteomic data in systems biology studies. A prospective combination of proteomic experiments with fluorescent microscopy could allow the study of many samples, many proteins at high sensitivity with spatial and temporal sampling through experiments in living cells.

The main obstacle in the implementation of microscopy in systems biology is the large number of experiments necessary. This makes it difficult and tedious to perform for a single user in a non-automated microscope leading to increased error probability. There is an urgent need for automated fluorescence microscopes, which can screen hundreds of samples with minimal human presence. Commercially available automatically fluorescent microscopes have great potentials, but they are prohibitively expensive. On the other hand, the biomechanics laboratory has a simple fluorescence microscope, which does not provide automated functions.

This thesis is focused in designing a system for the automation of the laboratory's fluorescent microscope. Three key parts of the system have been automated. The shutter of the mercury lamp, the body of the optical filters and the slide base. Also the precision motion of the base is calibrated. The automation of these three main functions allows the execution of dozens of experiments low magnification imaging in fixed cells and tissue sections. Here, a simple application imaging live cell leukemia JJN3 is being presented. Finally, the next steps in order to automate the imaging in high resolution, and also of living cells, are presented.

Automation of Mercury Lamp Shutter

The automation of the Mercury Lamp Shutter is vital, in order to prevent photobleaching. Photobleaching is the photochemical alteration of a fluorophore molecule or a dye, that it is unable to fluoresce, due to light exposure. After several minutes the emition power of the fluorophore -exposed by light- fades away and imaging is no longer possible. The shutter has two discrete positions (ON/OFF) and it motorised by a servo motor. It consist of 2 components, manufactured in the LaserCutter and then glued together. The part, that is sliding in horizontal direction via the Servo, is covered with aluminium tape, in order to block the light successfully.
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Lamp Shutter Prototype



Automation of the Optical Filter's Body

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The Optical Filter's Body is the filter set. There are 3 different filter cubes, which are placed incide this filter set. In each filter cube there are enclosed three filters, which guide the light beam (excitation, dichroic, emission) from the light source to the specimen and then to the detector. Most of the experiments use more than one fluorophore for the imaging of specimens and in order for them to be imaged, the filter set is moving between 3 positions. Automating this feature will provide the user with lots of information. Each filter indicates different parts of the specimen and when we combine them we get a full structural image of the specimens (right picture).



For the development of the device that moves the filter set in the desired position, some plexiglass layers and the laser-cutting machine were used. In the picture below, the components of the device are presented in exploded view (Solidworks)

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The Filter Set is moving due to the Rack and Pinion mechanism, that was manufactured in the same way (Laser Cutter). The initialisation of this devise is made with an OPTO switch. After the combination of the components the device is shown in the picture below.
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Development of a Motorised Precision XY stage

The last part of Axioskop 50's automation is the XY stage. Changing the manual XY stage with a motorised is the ideal way for automatically positioning of a wide range of specimens and samples in many types of imaging techniques and applications. For the development of the motorised stage there are some specifications that must be satisfied:
  1. Movement in 2 directions X horizontal & Y vertical
  2. Accuracy & Repeatability of 25μm
  3. Travel Range: 130mm - 90mm
  4. Adaptor for different kind of specimens
  5. Manual Calibration of specimen's disposition

In order to satisfy these specifications, a stage from stainless steel has been developed. It consists of 4 linear bearings (2 for each direction), 2 stepper motors with high resolution, some layers of stainless steel, 4 pushbuttons for the initialization and a part that works as an adaptor for different kind os specimens.

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The adaptor, which is shown in the last sub-figure with (blue & light green colour), has been developed in a way that can adjust the height and disposition of the specimens. In addition it keeps the specimen steady and can be converted to take various specimens.

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Integration of the devices

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After the manufacture of these three devices has taken place, it was time to develop an electrical circuit, a program and a GUI in order to be easy to control. The 4 motors (1 servo, 3 steppers) are being controlled by an Arduino UNO. At first, a prototyping board was used, but due to the complexity and the large amount of jumpers, it was decided that a PCB for the Arduino UNO will replace it. The PCB was created in the program FRITZING and then it was cut in LPKF. Then the driver motors were mounted on the PCB and the software for motor control was created. The program was written in Arduino environment (Wiring). In the end, with the help of an undergraduate student (Nikolaos Koukis) the Graphic User Interface was created in MATLAB.


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Check for operating properties

After the integration of the system, some operating tests had been conducted. Three tests were made to define the accuracy and repeatability and the results were very promising. The accuracy and repeatability lays at 20-50μm, which is an excellent performance for a prototype. In addition another test was made to confirm the imaging capabilities of the microscope. For the scopes of this test a simple application imaging live cell leukemia JJN3 is being presented.




All the missing information can be found in THIS LINK(temporary link)




The following are some fundamental files that can be found here:
  • Solidworks (LampShutter - FilterSet - XY stage)
  • PCB (Fritzing)
  • Software ( Arduino - MATLAB)