Microscopy Lecture outline • Compound microscope and its variants – Physical principles of microscopy – Phase contrast – Fluorescence microscope • Optical scanning microscopes – Confocal scanning microscope – Near field optical scanning microscope • Electron microscopy – Transmission electron microscopy – Scanning electron microscopy – Scanning tunnelling microscope • Microscopes based on other physical principles – Atomic force microscopy – Acoustic microscopy Prerequisites • What should you know? ØFundamentals of geometric and wave optics. Compound microscope and its variants Physical principles of microscopy • The spatial resolution (SR) of the unaided eye at a distance of 25 cm is about 14 lines per mm. • The magnifying glass can increase this substantially (for high SR we require large diameter of lens and smaller focal length) However, it does not have a high enough SR to allow us to study the microstructure of living matter. • The first microscope was manufactured in The Netherlands in the end of 16^th century. Anthony van Leuwenhoek (1632-1723) improved its construction after 1650 (could see say 2000 lines per mm). • The construction of the electron microscope (in 30^th of 20^th century) was the next milestone of microscopy. The SR of microscopes improved about 1000-times more than the optical microscope, so it was possible to see big molecules. Today we can resolve even individual atoms. • In principle, we can use any wave motion to depict microscopic objects. The only condition is that the wavelength must be shorter than the dimensions of the observed object. The first usable microscope (17^th century) Scheme of the microscope and properties of its optical system • Basic parts: two systems of lenses - objective and eyepiece. (Both approx. converging lenses). • Considering the quality of the image, the most important part is the objective which forms a real, magnified and inverted image. The observed object must be placed between F (position of the focus) and 2F. The objective can be considered as a convex lens of very short focal length for high SR. • The mechanical piece connecting the objective with the eyepiece is called the drawtube. The image formed by the objective (positioned just behind the front focus of the eyepiece) is observed by the eyepiece in the manner of a simple magnifying glass. A magnified, inverted and virtual image results. • The condenser optical system focuses light onto the observed object, and ensures its perfect illumination. Optical scheme and magnification of the microscope Microscope Objectives Objective Specifications • Numerical Aperture (NA, max. value approx. 1.5) - This is the most important specification: it determines the light acceptance angle (which determines the brightness of the image, the higher the NA the higher the acceptance angle), the limiting SR (prop. to NA), and the depth of field (inversely prop. to NA^2). • Linear Magnification • Optical Corrections - Achro and Achromat (achromatic), Fl, Fluar, Fluor, Neofluar, Fluotar (fluorite, better spherical and chromatic corrections), Apo (apochromatic, highest correction for spherical and chromatic aberrations) • Cover Glass Thickness (standard thickness 0.17 mm). Some objectives have a correction collar to compensate for any variation from this standard. • Working Distance - Distance between objective front lens and top of cover glass when the specimen is in focus. Decreases as magnification increases. Newer objectives have the working distance in mm inscribed on the barrel. • Immersion Medium - Most objectives are designed to image specimens with air as the medium between the objective and the cover glass. To attain higher NA, many objectives are designed to image the specimen through another medium that reduces refractive index differences between glass and the imaging medium. High-resolution planapochromatic objectives can achieve numerical apertures up to 1.40 when the immersion medium is special oil with a refractive index of 1.51. Other media are water and glycerin. • Color Codes - Microscope manufacturers label their objectives with color codes to help in rapid identification of the magnification and immersion media requirements. Use of Immersion Media SR limit of the microscope • The SR limit is proportional to the NA and inversely proportional to the wavelength l of light used (German physicist Abbe,1840-1905). • In some textbooks of microscopy, the SR is calculated by the formula: d = l/NA where d is distance of two still distinguishable points (NA = n.sina, n is refraction index of medium between the objective and the cover glass and a is the above mentioned acceptance angle). • SR increases with magnification. By combining strong converging lenses, we could construct a microscope with almost arbitrary magnification, however we find that beyond a certain limit (limit of ‘useful magnification’) there is no further increase in the limiting SR (just ‘empty’ magnification’). • SR decreases if the condenser aperture is reduced however the contrast resolution (CR) increases! Hence for a given specimen one must choose a condenser aperture to provide a balance between SR and CR. If one just needs to reduce brightness it is best to turn down the voltage to the lamp then decrease the condenser aperture so that one does not reduce the SR. Depth of Field Z ^• This is the thickness of objects along the z-axis which is simultaneously in focus. Important for thicker specimens.^ Special optical microscopes • Observation in bright or dark field • Stereomicroscope (two microscopes with individual objectives and eyepieces with optical axes at an angle of about 15°) - stereoscopic vision. In medicine: microsurgery. The image must not be inverted. The surgery field is illuminated by optical fibres. The possibility of changes in the focal length of the objective produces zooming – a variable spatial resolution. • Modern research microscopes ere equipped with digital cameras for microphotography or microcinematography (video recording). • Image processing software: performs changes of contrast, brightness, sharpness etc. Advanced software enables quantitative analysis of images, searching for typical patterns etc. • Most kinds of microscopes can be set up by changing the objectives, eyepieces, condensers, or by addition of some special optical elements. Many accessories are available, e.g. micromanipulators used to place microelectrodes into cells, separate organelles etc. Stereomicroscope Phase Contrast Microscopes A technique that produces contrast images of biological specimens, which structures have similar light attenuation (all equally transparent and therefore produce little contrast in normal transmission microscopy) but have slight differences in refractive index (and hence produce differential phase). The phase contrast technique changes the phase differences into amplitude differences. Living cells can be examined without being fixed, and stained. Principle: The annular diaphragm is added in condenser frontal focus plane - light passes through a narrow, ring-shaped slot. As the light passes through the object, the rays are deflected from the original direction. In the objective back focal plane is a phase plate, shaped like the annulus again, which shifts the phase by +p/2 or -p/2), i.e. by 1/4 of the wavelength. This plate transmits rays which did not change their direction on phase objects. Other rays miss the plate, their phase is not shifted. The picture is formed by interference of the phase-shifted and non-shifted rays. The phase objects seem dark or bright in comparison with their surrounding (positive or negative contrast). Phase contrast microscope Fluorescence microscope •Fluorescence microscopy is based on the ability of some substances to emit visible light after irradiation by light of shorter wavelength (UV radiation or violet light). •The optics of the condenser must be adapted to UV light, which can be also supplied through the objective (upper illumination). The remaining part of the microscope is identical with the same part of a common microscope. Eye protecting UV filters are needed. •The fluorescence is exhibited e.g. by tryptophan or other compounds with an aromatic ring or heterocycle. In most cases, fluorescent dyes specifically interacting with various cell structures are added to the observed biological objects. Sometimes the dye (fluorochrome, fluorescence probe) is bound to an antibody specific for some protein. This immunofluorescence method can selectively visualise e.g. the cytoskeleton, chromatin, membrane proteins. FM Fluorescence microscope Confocal laser scanning microscope •L - laser, C – diaphragms with small circular opening, PPZ – semitransparent mirror, DET – light detector (photomultiplier), SM – scanning mechanism, Č - objective (projective), O – point object, PREP – microscopic section. •Only rays reflected from point structures in the focus can pass through the diaphragm C in front of the detector. Other rays (scattered) are stopped by the diaphragm. These rays would lower the image quality in a common microscope since they lower the contrast. Using this microscope, we can study relatively thick native sections. The scanning mechanism is a system of rotating mirrors which can move the focus along dense parallel lines. Confocal scanning microscope Confocal microscope • Live Cell Imaging- we can follow the growth of cell cultures in a flow-through cell in real time • We can follow direct effects of chemical of physical factors on cell cultures. • Development: + simultaneous use of a built-in spectrophotometer, common laser replaced by a „white laser“, i.e. laser tunable continuously in wavelength range of 470-670 nm (Leica). + Raman spectrometer- contactless, non-destructive chemical analysis of materials; (see the lecture on devices used in molecular biophysics) Optical scanning microscopes Near field optical scanning microscope NFOSM = NSOM = SNOM Electron microscopy • „Classical“ electron microscopes (EM) use beams of accelerated electrons for imaging. The electrons have wavelength of the so called de Broglie matter waves. Let us remind following formulas: Electron microscopy TEM – transmission electron microscope according to: http://www.vetref.net/emscope/theorysch.html Transmission electron microscopy Brookhaven TEM Magnification 50 000 000x, resolution 0,1 nm, X-ray spectrophotometry for chemical analysis is simultaneously possible. TEM – preparation and staining of sections • The need of very thin sections (max. hundreds of nm) and positioning of the sections in vacuum requires special methods of preparation. Native (wet) sections can be observed only by modern environmental EM in which the sections are located in a relatively high pressure medium. • The biological materials must be prepared by means of a special fixation – impregnated by different substances (epoxy resins) before cutting. • The biological specimens are often metal-coated in vacuum from the side so that a „shadow“ appears behind the elevated parts of the specimen. • To increase the scattering of electrons in the specimen, salts or oxides of heavy metals (osmium, tungsten, uranium) are used. TEM – preparation and staining of sections • Kryofixation = an attempt to replace slow chemical fixation by faster fixation by freezing • Rapid freezing of a native sample under high pressure (MPa) and temperature of liquid nitrogen (-190 °C) • Disadvantage: the samples have to be manipulated using cooled instruments (from –190°C to – 4°C), TEM is cooled by liquid nitrogen. • Freeze-etching method: metallic replicas of surfaces of cellular membrane structures are prepared • The sample is fractured in high vacuum (10^-5 Pa) at temperature of –100°C. The exposed structures are coated by a thin layer of a heavy metal (Pt, Ta) under the angle of (45°), so that a „shadow“ appears behind the elevated parts of the specimen. Then a layer of carbon is added under an angle of 90°, which is necessary for fixation of the metallic structures • Total layer thickness is about 25 nm • Biological material is then removed chemically TEM- section vs. replica TEM working at low accelarating voltage LV-STEM vs. LV-TEM • Relatively thick ultrathin sections: 30-40 nm for biological material • No need to use heavy metal contrast • Spatial resolution in LV-STEM 1-2 nm TEM -http://www.ualberta.ca/~mingchen/tem.htm Electron microscopy Scanning electron microscopy SEM According: http://www.rpi.edu/dept/materials/COURSES/NANO/shaw/BigSEM.gif SEM – preparation of specimens • Biological specimens have to be dried at low temperature in CO[2] atmosphere • Non-conducting materials have to be coated by thin metal layer (Au, C, Ag – vaporised metals are applied) • The microscopes with low accelerating voltage - advantages: less artefacts, smaller damage of specimens, smaller or no metal layer (MIRA produced by TESCAN) • Possibility to study chemical composition of the specimen by X-ray spectrometry (structures bigger than 200 nm) • Development of microscopes enabling simultaneous observation of the surface and cutting SEM – MIRA, crystals prepared for X-ray analysis • Ant leg detail in SEM - http://www.wtn.org/ss/story.phtml?storyId=33&type=EdOutreach Electron microscopy Scanning tunnelling microscope (STM) Microscopes based on other physical principles Atomic force microscopy AFM DNA image from AFM - http://spm.phy.bris.ac.uk/research/DNA/images/dna2.jpg Microscopes based on other physical principles Acoustic microscopy • Neurons growing on a plastic plate http://transducers.stanford.edu/stl/Projects/ControlledPatt.htm Authors: Vojtěch Mornstein, Naděžda Vaškovicová Content collaboration and language revision: Carmel J. Caruana Presentation design: - Last revision: September 2009