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Introduction to microscopy

An instrument for viewing objects that are too small to be seen easily by the naked eye.
by

Gábor Feigl

on 1 April 2015

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Transcript of Introduction to microscopy

Recent evolution
Introduction to microscopy
Dyes
Immunostaining / antibodies
Illumination
Lasers
Electronics
Cameras
Computers
Algorythms & software
Live cell environmental control
Gábor Feigl
Definition
An instrument for viewing objects that are too small to be seen easily by the naked eye.
The history of microscopes
The first vision aid was invented (inventor unknown) called a reading stone
Circa 1000 AD
Circa 1284
Italian, Salvino D'Armate is credited with inventing the first wearable eye glasses
1590
Two Dutch eye glass makers, Zaccharias Janssen and son Hans Janssen experimented with multiple lenses placed in a tube
1665
English physicist, Robert Hooke (aka the father of microscopy) looked at a sliver of cork through a microscope lens and noticed some "pores" or "cells" in it
1674
Anton van Leeuwenhoek built a simple microscope with only one lens to examine blood, yeast, insects and many other tiny objects
18th century
Technical innovations improved microscopes, leading to microscopy becoming popular among scientists
1830
Joseph Jackson Lister reduces spherical aberration or the "chromatic effect" by showing that several weak lenses used together at certain distances gave good magnification without blurring the image. This was the prototype for the compound microscope
1872
Ernst Abbe, then research director of the Zeiss Optical Works, wrote a mathematical formula called the "Abbe Sine Condition".
1903
Richard Zsigmondy developed the ultramicroscope that could study objects below the wavelength of light
1932
Frits Zernike invented the phase-contrast microscope that allowed for the study of colorless and transparent biological materials for which he won the Nobel Prize in Physics in 1953
1931
Ernst Ruska co-invented the electron microscope for which he won the Nobel Prize in Physics in 1986
1981
Gerd Binnig and Heinrich Rohrer invented the scanning tunneling microscope that gives three-dimensional images of objects down to the atomic level
Huygens
Dutch mathematician, astronomer any physicist, developed an improved two lens eye piece.
1893
In 1893 Augus Köhler invented a method of providing optimum illumination of a microscope specimen. Improved resolution and evenness of light illumination made photomicrography possible.
Nomarski
Georges Nomarski developed the differential interference contrast (DIC) microscopy technique
Marvin Minsky
Patented the confocal scanning microscope in 1957. Practical confocal microscope systems became available in the lat 1980's. Yields improved contrast, resolution and optical sectioning.
Better resolution
More sensitivity
Lower noise
Faster detection
Greater specificity
Easier analysis
New capabilities
Increased complexity
Increased cost
More raw data
Optical/light microscope
The optical microscope, often referred to as the "light microscope", is a type of microscope which uses visible light and a system of lenses to magnify images of small samples
Oldest design of microscope
Basic optical microscopes can be very simple
There are many complex designs which aim to improve resolution and sample contrast
There are two basic configurations of the conventional optical microscope:
Simple microscope
A simple microscope is a microscope that uses a lens or set of lenses to enlarge an object through angular magnification alone, giving the viewer an erect enlarged virtual image.
Not capable of high magnification
Compound microscope
A compound microscope is a microscope which uses a lens close to the object being viewed to collect light (called the objective lens) which focuses a real image of the object inside the microscope. (image 1)

That image is then magnified by a second lens or group of lenses (called the eyepiece) that gives the viewer an enlarged inverted virtual image of the object. (image 2)
Much higher magnification
Reduced chromatic aberration
Exchangeable objective lenses to adjust the magnification
A compound microscope also makes more advanced illumination setups possible.
Lighting techniques
Köhler illumination
Phase contrast
Fluorescence microscopy
Modern biological microscopy depends heavily on the development of fluorescent probes for specific structures within a cell. In contrast to normal transilluminated light microscopy, in fluorescence microscopy the sample is illuminated through the objective lens with a narrow set of wavelengths of light. This light interacts with fluorophores in the sample which then emit light of a longer wavelength. It is this emitted light which makes up the image.
Key parts of a typical microscope
Eyepiece - ocular lens
The eyepiece, or ocular lens, is a cylinder containing two or more lenses; its function is to bring the image into focus for the eye.
An eyepiece consists of several "lens elements" in a housing, with a "barrel" on one end.
The barrel is shaped to fit in a special opening of the instrument to which it is attached.
The image can be focused by moving the eyepiece nearer and further from the objective.
Modern research-grade microscopes do not use eyepieces. Instead, they have high-quality CCD sensors mounted at the focal point, and the images are viewed on a computer screen.
Nose piece - objective turret
Objective turret, revolver, or revolving nose piece is the part that holds the set of objective lenses.
It allows the user to switch between objective lenses
Objective
At the lower end of a typical compound optical microscope, there are one or more objective lenses that collect light from the sample
The objective is usually in a cylinder housing containing a glass single or multi-element compound lens
Microscope objectives are characterized by two parameters, namely,
magnification
and
numerical aperture
Magnification
Numerical aperture
Normally ranges from 2,5X - 100X
More magnification gives less light intensity at the detector
Objective lenses with higher magnifications normally have a higher numerical aperture and a shorter depth of field in the resulting image
Optical magnification is the ratio between the apparent size of an object (or its size in an image) and its true size, and thus it is a dimensionless number.
In microscopy, NA is important because it indicates the resolving power of a lens. The size of the finest detail that can be resolved is proportional to lambda/2NA, where lambda is the wavelength of the light.
A lens with a larger numerical aperture will be able to visualize finer details than a lens with a smaller numerical aperture. Assuming quality (diffraction limited) optics, lenses with larger numerical apertures collect more light and will generally provide a brighter image, but will provide shallower depth of field.
Capacity of an instrument to resolve two points which are close together.
Optical resolution describes the ability of an imaging system to resolve detail in the object that is being imaged.
In optics, particularly as it relates to film and photography, depth of field (DOF) is the distance between the nearest and farthest objects in a scene that appear acceptably sharp in an image.
Oil immersion objective
Some microscopes make use of oil-immersion objectives or water-immersion objectives for greater resolution at high magnification.
These are used with index-matching material such as immersion oil or water and a matched cover slip between the objective lens and the sample.
The refractive index of the index-matching material is higher than air allowing the objective lens to have a larger numerical aperture (greater than 1) so that the light is transmitted from the specimen to the outer face of the objective lens with minimal refraction
The larger numerical aperture allows collection of more light making detailed observation of smaller details possible.
An oil immersion lens usually has a magnification of 40 to 100×
Focus knob
Adjustment knobs move the stage up and down with separate adjustment for coarse and fine focusing.
The same controls enable the microscope to adjust to specimens of different thickness.
Stage
The stage is a platform below the objective which supports the specimen being viewed. In the center of the stage is a hole through which light passes to illuminate the specimen.
The stage usually has arms to hold slides
Light source
Many sources of light can be used. At its simplest, daylight is directed via a mirror. Most microscopes, however, have their own adjustable and controllable light source – often a halogen lamp, although illumination using LEDs and lasers are becoming a more common provision.
Condenser
The condenser is a lens designed to focus light from the illumination source onto the sample.

The condenser may also include other features, such as a diaphragm and/or filters, to manage the quality and intensity of the illumination.
Operation
The optical components of a modern microscope are very complex and for a microscope to work well, the whole optical path has to be very accurately set up and controlled.

Despite this, the basic operating principles of a microscope are quite simple.
The
objective
lens is a lens with a very short focal length.
This is brought very close to the specimen being examined so that the light from the specimen comes to a focus about 160 mm inside the microscope tube. This creates an
enlarged
image of the subject.
This image is
inverted
and can be seen by removing the eyepiece and placing a piece of tracing paper over the end of the tube.
By carefully
focusing
a brightly lit specimen, a
highly enlarged
image can be seen.
It is this
real image
that is viewed by the
eyepiece lens
that provides
further enlargement
.
Illumination techniques
Many techniques are available which modify the light path to generate an improved contrast image from a sample. Major techniques for generating increased contrast from the sample include:
The "standard" bright field illumination
cross-polarized light
dark field
phase contrast
differential interference contrast illumination.
A recent technique (Sarfus) combines cross-polarized light and specific contrast-enhanced slides for the visualization of nanometric samples.
Bright field
Cross polarised
Dark field
Phase contrast
Differential interference contrast illumination
Other illumination techniques
Microspectroscopy (where a UV-visible spectrophotometer is integrated with an optical microscope)
Ultraviolet microscopy
Near-Infrared microscopy
Multiple transmission microscopy, for contrast enhancement and aberration reduction.
Automation (for automatic scanning of a large sample or image capture)
Optical microscope variants
There are many variants of the basic compound optical microscope design for specialized purposes:
Stereo microscope, a low powered microscope which provides a stereoscopic view of the sample, commonly used for dissection.
Comparison microscope, which has two separate light paths allowing direct comparison of two samples via one image in each eye.
Inverted microscope, for studying samples from below; useful for cell cultures in liquid, or for metallography.
Student microscope, designed for low cost, durability, and ease of use.
Fiber optic connector inspection microscope, designed for connector end-face inspection
Other microscope variants are designed for different illumination techniques:
Petrographic microscope, whose design usually includes a polarizing filter, rotating stage and gypsum plate to facilitate the study of minerals or other crystalline materials whose optical properties can vary with orientation.
Polarizing microscope, similar to the petrographic microscope.
Phase contrast microscope, which applies the phase contrast illumination method.
Epifluorescence microscope, designed for analysis of samples which include fluorophores.
Confocal microscope, a widely used variant of epifluorescent illumination which uses a scanning laser to illuminate a sample for fluorescence.
Alternatives
In order to overcome the limitations set by the diffraction limit of visible light other microscopes have been designed which use other waves
Atomic force microscope (AFM)
Scanning electron microscope (SEM)
Scanning ion-conductance microscopy (SICM)
Scanning tunneling microscope (STM)
Transmission electron microscopy (TEM)
Ultraviolet microscope
X-ray microscope
Scanning thermal microscopy
Atomic force microscopy
Atomic force microscopy (AFM) or scanning force microscopy (SFM) is a very high-resolution type of scanning probe microscopy, with demonstrated resolution on the order of fractions of a nanometer, more than 1000 times better than the optical diffraction limit.
When the tip is brought into proximity of a sample surface, forces between the tip and the sample lead to a deflection of the cantilever
Typically, the deflection is measured using a laser spot reflected from the top surface of the cantilever into an array of photodiodes.
Scanning electron microscope
A scanning electron microscope (SEM) is a type of electron microscope that produces images of a sample by scanning it with a focused beam of electrons. The electrons interact with atoms in the sample, producing various signals that can be detected and that contain information about the sample's surface topography and composition.
SEM can achieve resolution better than 1 nanometer.
Scanning ion-conductance microscopy
The scanning ion-conductance microscope (SICM) consists of an electrically charged glass micro- or nanopipette probe filled with electrolyte lowered toward the surface of the sample (which is non-conducting for ions) in an oppositely charged bath of electrolyte.
As the tip of the micropipette approaches the sample, the ion conductance and therefore current decreases since the gap through which ions can flow, is reduced in size.
Variations in the ion current are measured by an amplifier, and are used as a feedback signal by a scanner control unit to keep the distance between pipette tip and sample constant by applying corresponding voltages to the Z-piezo drive during the scanning procedure.
Therefore, the path of the tip follows the contours of the surface.
Scanning tunneling microscope (STM)
A scanning tunneling microscope (STM) is an instrument for imaging surfaces at the atomic level.
The STM is based on the concept of quantum tunneling.
When a conducting tip is brought very near to the surface to be examined, a bias (voltage difference) applied between the two can allow electrons to tunnel through the vacuum between them.
The resulting tunneling current is a function of tip position, applied voltage, and the local density of states (LDOS) of the sample.
Information is acquired by monitoring the current as the tip's position scans across the surface, and is usually displayed in image form.
Transmission electron microscopy
Transmission electron microscopy (TEM) is a microscopy technique in which a beam of electrons is transmitted through an ultra-thin specimen, interacting with the specimen as it passes through. An image is formed from the interaction of the electrons transmitted through the specimen; the image is magnified and focused onto an imaging device.
Thank you for your attention!
The numerical aperture of a microscope objective is a measure of its ability to gather light and resolve fine specimen detail at a fixed object distance.
In August 1893 August Köhler developed Köhler illumination. This method of sample illumination gives rise to extremely even lighting and overcomes many limitations of older techniques of sample illumination.
Before development of Köhler illumination the image of the light source, for example a lightbulb filament, was always visible in the image of the sample.
Lighting techniques
The Nobel Prize in physics was awarded to Dutch physicist Fritz Zernike in 1953 for his development of phase contrast illumination which allows imaging of transparent samples. By using interference rather than absorption of light, extremely transparent samples, such as live mammalian cells, can be imaged without having to use staining techniques.
Just two years later, in 1955, Georges Nomarski published the theory for differential interference contrast microscopy, another interference-based imaging technique.
Resolving power
(http://virtual.itg.uiuc.edu/training/AFM_tutorial/)
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