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Syllabus for

Academic year
TIF045 - Technical bioimaging
 
Owner: TTFYA
5,0 Credits (ECTS 7,5)
Grading: TH - Five, Four, Three, Not passed
Level: D
Department: 16 - PHYSICS


Teaching language: English

Course module   Credit distribution   Examination dates
Sp1 Sp2 Sp3 Sp4 No Sp
0105 Project 5,0 c Grading: TH   5,0 c    

In programs

TTFYA ENGINEERING PHYSICS, Year 4 (elective)

Examiner:

Bitr professor  Annika Enejder



Eligibility:

For single subject courses within Chalmers programmes the same eligibility requirements apply, as to the programme(s) that the course is part of.

Aim

To provide knowledge of modern techniques for optical imaging and of spectroscopical studies on a molecular level, with applications toward molecular biology.

Goal

To bring up concepts from a wide range of fundamental physics courses and use them to understand how a 3-dimensionell picture is created in a modern light microscope, how cell structures and biomolecules selectively can be imaged and manipulated in fluorescence and spectroscopically based microscopes, and how important image analysis and sample preparation is for the final results.

Content

The light microscope has traditionally been our most important instrument to catch a glimpse of the fascinating micro-cosmos of biological cells. The fundamental optics of the microscope and its function - to image with an ever increasing magnification and resolution - has been well-known for several hundred years. However, the information provided by the conventional -snapshots- of transmitted light is no longer enough within the modern biosciences in order to gain full understanding of the complex processes life comprises. The requirements on resolution and contrast in order to visualize the -invisible- have the past decade drastically been changed; we now want to be able to monitor biochemical processes in living cells on molecular level and with high temporal resolution rather than merely visualizing cellular structures. This has motivated physicists in close collaboration with biologists to develop a range of sophisticated microscopy methods, where the conventional light source is replaced by technically advanced laser systems. Micro-spectroscopic analysis and imaging of biomolecules and molecular markers in the sample is done by inducing fluorescence and vibrational processes. Living cells are captured in the probe volume and manipulated with laser tweezers. The light emitted from the sample, sometimes only single photons, is wavelength-selectively filtered and projected onto a sensitive detector. An image is formed and finally analyzed by means of powerful image analysis software. These advanced laser-based imaging systems set high and new requirements on the user of modern microscopes, one of which is good knowledge in physics. Following issues will be treated:

- The light microscope
How is the modern light microscope constructed and how is a three-dimensional image of the object formed? How can we optically improve the quality of a picture and which limitations must then be considered? The aim is to get familiar with the different optical parts of the modern microscope (widefield and confocal) such as the objective, condenser, light source etc. and how they together can form a picture of sub-cellular structures with a size close to the wavelength. In addition, we will bring up how the characteristic properties of light can be utilized to improve the image contrast in for instance polarization and differential interference contrast microscopy.

- Fluorescence microscopy
By labeling specific structures and molecules in biological cells with fluorescing markers, they can selectively be visualized in a fluorescence microscope. Different types of lasers are here employed as the light source and the emitted fluorescence light is collected and projected onto a suitable detector. Important concepts are e.g. excitation/emission wavelength, optical filters and photo-bleaching. Beside conventional fluorescence microscopy will more advanced techniques be discussed, such as fluorescence life-time imaging, TIRF, and FRET.

- Spectroscopic and non-linear microscopy methods
To get a -true- picture of biochemical processes in living cells, it is crucial to be able to monitor biomolecules and cellular structures without the need for artificial marker molecules. This is possible by inducing a vibration to the biomolecule with laser light through various interaction processes (Raman scattering, CARS, Second-harmonic generation etc.). The scattered light is then wavelength shifted, characteristic for the atoms and chemical bonds of the target molecule. This wavelength-shifted signal is only detected at locations of the biomolecule. Thus, a selective image of the distribution of lipids/proteins/carbohydrates in a biological cell can be obtained. The aim is to get a deep understanding for advanced interaction processes light-biomolecules and how these can be used for chemically selective imaging in a microscope.

- Imaging analysis
As the novel microscopy methods provide enormous amounts of data, it is essential to be able to make the most of all the information. Thus, imaging analysis has become a vital part of microscopy. We will demonstrate various analysis techniques and how they improve the image quality. Tools for generating three-dimensional reconstructions of the object, identifying cells and analyze time-series of images will be discussed. Other central concepts are noise reduction and co-localization.

- Sample preparation
Microscopy does not only involve imaging; the actual sample preparation plays an important role. We will demonstrate different labeling techniques and give further advice how to establish optimal conditions for biological microscopy.

- Optical manipulation
The microscopist has traditionally always been as a passive observer. Optical manipulation has changed this drastically. The target object can today be fully controlled by means of tightly focused laser beams, i.e. optical tweezers. Cells can be captured and kept at a specific position in the probe volume of the microscope. Laser scalpels can be used to modify cell membranes and chromosomes. Power, wavelength, temperature and photo-induced damages are topics which will be brought up.

A common theme for all sections is light interaction with media on microscopical and molecular level. Rayleigh scattering, light absorption, fluorescence emission, Raman scattering and various non-linear optical processes are some of the most important phenomena behind modern microscopy techniques and thus important to understand. These processes will be discussed in general and primarily illustrated by examples from the fascinating world of biological cells and molecules.

Organisation

The course consists of full-time studies in the daytime corresponding to five weeks (5 p). The teaching will be given as lectures combined with laboratory lessons. The latter are compulsory, as well as the oral presentations of the written reports.

Literature

Handbook of Biological Confocal Microscopy (Kluwer Academic Publishers), James B. Pawley (Editor), 2005

Introduction to Optical Microscopy, Digital Imaging, and Photomicrography:
see http://micro.magnet.fsu.edu/primer/index.html

Examination

A written exam will be given in the end of the course. Passed exam and laboratory lessons/reports will each be graded with 3, 4 or 5. A total laboratory grade will be formed from the individual report grades by averaging. A final grade requires completed laboratory lessons/reports and passed exam and is given as a rounded average of the two separate grades. An additional exam will be given for students not passing the regular exam.


Page manager Published: Mon 28 Nov 2016.