Chapter 9: Imaging

SEM vs TEM

- The magnifications that TEMs offer are also much higher compared to SEMs. TEM users can magnify their samples by more than 50 million times, while for the SEM, this is limited to 1-2 million times. - Maximum field of view (FOV) that SEMs can achieve is far larger than TEMs, meaning TEM users can only image a very small part of their sample. - Similarly, the depth of field of SEM systems is much higher than in TEM systems. - In SEMs, samples are positioned at the bottom of the electron column, and the scattered electrons (back-scattered or secondary) are captured by electron detectors. In a TEM microscope, the sample is located in the middle of the column. The transmitted electrons pass through it and through a series of lenses below the sample (intermediate and projector lenses). An image is directly shown on a fluorescent screen.

Advantages of Light Microscope

1. Cheap to purchase 2. Cheap to operate 3. Small + portable 4. Simple + easy sample preparation. Vacuum is not required. 5. Material rarely distorted by preparation 6. Natural colour of sample maintained

Disadvantages of Electron Microscope

1. Inability to analyze live specimens - As electrons are easily scattered by other molecules in the air, samples must be analyzed in a vacuum. This means that live specimens cannot be studied by this technique. 2. Black and white images - Only black and white images can be produced by an electron microscope. 3. Artefacts - These may be present in the image produced. Artefacts are left over from sample preparation and require specialized knowledge of sample preparation techniques to avoid. 4. Cost - Electron microscopes are expensive pieces of highly specialized equipment. 5. Size - Despite the advantages in technology over the years, electron microscopes are still large, bulky pieces of equipment which require plenty of space in a laboratory. 6. Training - Specialist operators are required to operate electron microscopes, and these can undergo years of training to properly use this technology.

Advantages of Electron Microscope

1. Magnification and higher resolution - as electrons rather than light waves are used, it can be used to analyze structures which cannot otherwise be seen. The resolution of electron microscopy images is in the range of up to 0.2 nm, which is 1000x more detailed than light microscopy. 2. Diverse applications 3. High-quality images

Disadvantages of Light Microscope

1. Magnifies objects up to 2000x only

Main factors that determine Optical Resolution?

1. Objective's numerical aperture (NA) 2. Wavelength of light used to examine the specimen Note: Shorter wavelengths are capable of resolving details to a greater degree than are the longer wavelengths. The greatest resolving power in optical microscopy is realized with near-ultraviolet light, the shortest effective imaging wavelength.

Main factors that determine Pixel Resolution?

3. Camera sensor used to record the output of the eyepiece.

4Pi microscopy

A 4Pi microscope is a laser scanning fluorescence microscope with an improved axial resolution. The improvement in resolution is achieved by using two opposing objective lenses, which both are focused to the same geometrical location.

Electron microscopy

An electron microscope is a microscope that uses a beam of accelerated electrons as a source of illumination. As the wavelength of an electron can be up to 100,000 times shorter than that of visible light photons, electron microscopes have a higher resolving power than light microscopes and can reveal the structure of smaller objects.

Fluorescence microscope

An optical microscope that uses fluorescence and phosphorescence instead of, or in addition to, reflection and absorption to look at organisms on a slide.

Functional Fluorescence Microscopy

As opposed to structural imaging, functional imaging centers on revealing physiological activities within a certain tissue or organ by employing medical image modalities that very often use tracers or probes to reflect spatial distribution of them within the body.

Synthetic Calcium Indicators

BAPTA

Backscattered Electrons (BSE)

Backscattered electrons (BSE) consist of high-energy electrons originating in the electron beam, that are reflected or back-scattered out of the specimen interaction volume by elastic scattering interactions with specimen atoms.

Calcium Imaging

Calcium imaging is a microscopy technique to optically measure the calcium (Ca2+) status of an isolated cell, tissue or medium. Calcium imaging takes advantage of calcium indicators, fluorescent molecules that respond to the binding of Ca2+ ions by changing their fluorescence properties.

Confocal microscopy

Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation. Resolution depends on excitation wavelength as well as image wavelength.

Connectomics

Connectomics is the study of the brain's structural and functional connections between cells, which is visualized as a connectome. The connectome is a map of all neural connections in a brain and connectomics is the mapping of these connections.

Limits of Resolution: Rayleigh Criterion

For a circular aperture, lens, or mirror, the Rayleigh criterion states that two images are just resolvable when the center of the diffraction pattern of one is directly over the first minimum of the diffraction pattern of the other.

Genetic Calcium Indicator

GCamp6

Light sheet fluorescence microscopy (LSFM)

In contrast to epifluorescence microscopy only a thin slice (usually a few hundred nanometers to a few micrometers) of the sample is illuminated perpendicularly to the direction of observation. For illumination, a laser light-sheet is used, i.e. a laser beam which is focused only in one direction (e.g. using a cylindrical lens).

Ojective Lens

In microscopy, the objective lenses are the optical elements closest to the specimen. The objective lens gathers light from the specimen, which is focused to produce the real image that is seen on the ocular lens.

Numerical Aperture (NA)

In optics, the numerical aperture of an optical system is a dimensionless number that characterizes the range of angles over which the system can accept or emit light.

Two-Photon Excitation microscopy

In two-photon microscopy, two photons of light with double the wavelength are used to excite the same or similar fluorescent dyes. 2P can track activity of cells in real time. The principal advantages of 2P are reduced phototoxicity, increased imaging depth (of up ~1.5mm), and the ability to initiate highly localized photochemistry in thick samples. Resolution depends only on excitation wavelength.

Microendoscopy: Imaging of Large Neuronal Populations

Microendoscopy is a type of endoscopy that uses optical fibres or fibre bundles in conjunction with microscopy techniques to obtain microscopic images of tissue immediately adjacent to the imaging end of the endoscope inserted into the body.

Optogenetics

Optogenetics most refers to a biological technique that involves the use of light to control neurons that have been genetically modified to express light-sensitive ion channels.

Scanning Electron microscope (SEM)

SEM creates an image by detecting reflected or knocked-off electrons. Useful magnification is up to 100,000x

Stochastic Optical Reconstruction microscopy (STORM)

STORM is based on the principle of single molecule localization. STORM uses stochastic activation of relatively small numbers of fluorophores using very low-intensity light. This random stochastic "activation" of fluorophores allows temporal separation of individual molecules, enabling high precision Gaussian fitting of each fluorophore image in XY. Disadvantages: - Very slow - Needs very high leaser intensities for excitation/depletion (PALM) - Not well suited for in-vivo applications. - STORM resolution is not the 'real' resolution. (Interpolation between dye molecules)

Wide-field microscopy

Since widefield microscopy permanently illuminates the whole sample, it can be distinguished from confocal microscopy where only one single focal spot is illuminated and recorded at a time. Typically, widefield microscopy utilizes light sources such as halogen, metal halide lamps or LED for sample illumination.

Super-resolution microscopy

Super-resolution microscopy is a series of techniques in optical microscopy that allow such images to have resolutions higher than those imposed by the diffraction limit, which is due to the diffraction of light.

Transmission Electron microscope (TEM)

TEM uses transmitted electrons (electrons that are passing through the sample) to create an image. Useful magnification is up to 250,000x

Light microscopy

The optical microscope, also referred to as a light microscope, is a type of microscope that commonly uses visible light and a system of lenses to generate magnified images of small objects.

Magnification

The ratio of an object's image size to its real size

Ocular Lens or Eyepiece

This eyepiece lens magnifies the image formed by the large objective lens and directs the light to your eye. Basically, the eyepiece works a lot like a magnifying glass; it enables your eye to focus much more closely than you normally can.

Voltage Imaging

This movement of ions across the membrane can be measured, and results in voltage changes. These changes are marked by voltage indicators, and the imaging of these dyes is known as voltage imaging. Voltage-sensitive dyes, genetically-encoded indicators and light-sensitive opsins can be used to image the flow of voltage across neurons.

One-Photon Excitation microscopy

Traditional fluorescence microscopy uses a single photon to excite fluorescent dyes using mainly visible excitation wavelengths (390-700 nm).