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 Microscopes are used to resolve small details. Their magnification makes it easy to see these small details and to match the resolution of the microscope to the pixel size of the detector (e.g. eye, camera).
Microscopes based on wave optics (e.g. light microscope, electron microscope) are diffraction limited. The diffraction limit depends on the numerical aperture (N.A.) and the wavelength λ of the light used. Smaller details can be seen with higher N.A. and shorter wavelength (light is more blue than red). A way to overcome these limitations are scanning probe microscopes (e.g. Atomic Force Microscope (AFM) and Scanning Tunneling Microscope (STM)).
Most microscopes can detect much smaller features than they can resolve. Light microscopes can detect single dye molecules in size well below the resolution limit, but are not able to distinguish between two features which are closer to each other than the resolution limit. Some approaches to increase resolution use this fact.
In biology almost all light microscopes are either compound microscopes (high resolution) or stereo microscopes (lower resolution but direct three-dimensional viewing).
With a high end optical microscope you can expect to resolve details as small as 200nm. Typical magnifications are 100 to 1000 times (= magnification objective (10x..100x) x magnification eyepiece (10x)). The field of view at the sample is the size of the intermediate image (about 20mm, dependent on manufacturer of the microscope) divided by the magnification of the objective.
A list of microscopes at the Stower's Image center can be found here.
You can find more information about microscopy at our Microscopy and Calculate Optical Resolution pages:
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Stowers Links:
Stowers Microscopy Center: Microscopy
Image Center: Instruments
Transmitted Light Microscopy: View Powerpoint Presentation,
Download Presentation
Web Links:
Optical Microscopy Primer: Everything you want to know about microscopy.
Nobelprize.org: Microscopes
Cold Spring Harbor Protocols: Imaging/Microscopy
Literature:
M. Abramowitz: Microscope Basics and Beyond (Olympus)Download PDF
Microscopy from the very beginning (Carl Zeiss)Download PDF
The Clean Microscope (Carl Zeiss)Download PDF
M. Davidson, M. Abramowitz: Optical MicroscopyDownload PDF
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 The objective is the most important component of any light microscope. Its optical resolution is given by its numerical aperture (N.A.) and the wavelength λ of light. The N.A. = n·sinα (n = refractive index, α = half the opening angle of the objective) depends on the ratio between the diameter of the front-lens and the working distance. n is the refractive index of the medium between lens and cover slip or sample. n = 1 for air and 1.51 for oil or glass.
Using a medium with high refractive index between cover slip and objective will increase resolution. If the sample is not directly attached to the cover slip, this medium should have the same refractive index as the medium surrounding the sample to avoid artifacts. Thus live cell and animal imaging should be done with water immersion objectives. When using a mounting medium, make sure that it's refractive index is as close as possible to 1.51 to use the full potential of high resolution oil objectives.
The cover slip is part of the optical system of the microscope. Thus it should be clean and has to have the correct thickness (#1.5 = 0.170mm for almost all objectives of all brands). When using high resolution, high magnification objectives, wrong cover slips will severely impair image quality. This effect is worse with water immersion objectives than with oil immersion. Some objectives (e.g. Carl Zeiss C-Apochromat 40x and 63x) have collars to correct for differences in cover glass thickness between different batches of #1.5 cover slips.
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Stowers Links:
Image Center: Objectives
Web Links:
Optical Microscopy Primer: Objectives
Carl Zeiss: Database Filtersets and Objectives
Carl Zeiss: Objective Specifications - interactive tutorial
Leica: Microscope Objectives
Literature:
Carl Zeiss: LSM Objectives
Download PDF
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Fluorescence microscopy is the most common technique to visualize biological structures and processes. Fluorescent dyes highlight specific structures on a black background. Some dyes bind specifically to structures like membranes and mitochondria, highlight molecules like DNA or react to the environment like Ca2+ concentration. Dye labeled antibodies tag specific antigenes. In recent years fluorescent proteins expressed by cells under investigation are gaining popularity. They can be driven by unique promotors or fused to specific proteins.
Fluorescent dyes are characterized by their absorption (excitation) and emission spectra. Dyes absorb photons of higher energy (= shorter wavelength) and emit photons at lower energy (= longer wavelength). In conventional light microscopy, the spectra are defined by the difference in energy between the S0 and S1 quantum states of the molecule. The shape of the spectrum depends on vibrational states of the molecule. With standard set-ups rotational states are not resolved.
The energy difference between excitation and emission (= Stokes Shift) is used to separate the excitation light from the detected fluorescence light. The goal is to choose an excitation wavelength as close to the excitation maximum as possible. This is done by selecting a band-pass filter (excitation filter) which transmits around the maximum when using a white-light-source (e.g. HBO with a standard fluorescence microscope) or a laser line with a laser based microscope (e.g. confocal laser-scanning microscope (LSM)). Exciting a dye molecule not at its optimum will decrease the amount of emitted fluorescence light, but not the characteristics of the emission spectrum. In almost all microscopes the fluorescence light is detected in the reflected light configuration. A beam splitter (dichroic) is used to separate excitation from emission light. It reflects the short wavelength excitation light and passes the long wavelength fluorescence light. To get rid of any light not originating from the dye (e.g. reflections, auto-fluorescence), an additional band pass filter is used. The emission filter passes light with wavelengths around the emission peak of the dye. When using multiple dyes, special filters and dichroics which pass and reflect multiple wavelength bands are used.
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Stowers Links:
Image Center: Constructs
Foundations of Microscopy Seminar: Illumination and Filters
Illumination and Filters: View Presentation/Download Presentation
Web Links:
Invitrogen (Molecular Probes): Handbook
Optical Microscopy Primer: Introduction to Fluorescence
Wikipedia: Energy Levels
Invitrogen (Molecular Probes): Fluorescence Spectraviewer (Java required)
Carl Zeiss: Database Filtersets and Objectives
Carl Zeiss: Matching Filter Sets with Microscope Light Sources - intereactive tutorial
Literature:
Lichtman, J. W. and J.-A. Conchello (2005). "Fluorescence microscopy." 2(12): 910-919. Download PDF
Shaner, N. C., P. A. Steinbach, et al. (2005). "A guide to choosing fluorescent proteins." Nature Methods 2(12): 905-909. Download PDF
Chroma: Handbook of Optical Filters for Fluorescence Microscopy Download PDF
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 A confocal laser scanning microscope rejects the out of focus light which blurrs images of three-dimensional samples acquired with a conventional microscope. It creates optical sections. The resolution along the optical axis is improved. The optical slices can be used to create 3D models of the sample.
The pinhole is the most important part of a confocal microscope. It is located in front of the detector, at a location confocal to the sample plane. Thus, details focused on in the sample will be in focus at the pinhole plane. In a laser scanning microscope, only one small spot at a time is illuminated by a focused laser spot. This small spot excites a dye molecule or is reflected. The light spot will be imaged as a small point to the pinhole plane and can easily pass the hole in the pinhole. Light created outside the focal plane will be out of focus at the pinhole plane. The light will be diffused over the whole area and only a negligible amount can pass through the pinhole.
In most implementations of laser scanning microscopes, a focused laser beam is moved line-by-line over the sample using scanning mirrors. The detector is a photo-multiplier tube, without any pixels (not a camera). Knowing the position of the excitation laser beam, thus knowing the origin of the detected light, one can reconstruct the image in a computer.
By changing the distance the laser beam moves one changes the magnification. How often the signal is sampled (= number of pixels) during one line and how many lines per image are acquired will define the pixel size. Thus, in contrast to a camera and the human eye, the pixel size can be changed. Thus the resolution of the objective does not have to be matched to the resolution of the detector. Therefore, when choosing an objective higher magnification will not give a benefit, but will typically decrease transmission and field of view. Important is to match the pixel size of the laser scanning microscope (given by the area scanned and the number of pixels within this area) to the optical resolution of the objective (given by its numerical aperture (N.A.) and the wavelength used).
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Stowers Links:
Image Center: Instruments
Web Links:
Optical Microscopy Primer: Laser Scanning Confocal Microscopy
Confocal Listserver: Very active discussion list
Carl Zeiss: Confocal Microscopy
Leica: Confocal Microscopy
Nikon: Confocal Microscopes
Olympus: Confocal Microscopy
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 To image thick and highly scattering samples (e.g. brain slices thicker than 100μm), the two-photon microscope is the instrument of choice.
If two photons hit a dye molecule at the same time (within several femto-seconds), their energy adds up and they can excite a dye molecule which absorbs typically at half the wavelength. In a microscope, the probability of having two photons at the same time at the same spot often enough to give a measurable signal, is only given in the focal plane of a high numerical aperture (N.A.) objective when using powerful, pulsed lasers. Thus, out-of-focus dye molecules are not excited.
The two-photon-microscope provides optical sectioning similar to a confocal laser scanning microscope, with the important exception that no out of focus light is produced. Because all the light reaching the detector originates from the focal plane, no pinhole is needed. In a confocal microscope the pinhole rejects out-of-focus light, independent of whether it was created out-of-focus (that's the purpose of the pinhole) or whether it originates from the focal plane and was scattered on its way to the detector. Thus, in a highly scattering sample, the measured signal is decreased because scattered photons are rejected. The two-photon microscope detects these photons and is therefore more sensitive when imaging these types of samples.
Two-photon microscopes use near-infrared (NIR) light to excite dyes. NIR light is reported to be less harmful to live cells and developing animals than ultra-violiet (UV) light.
The two-photon effect can be described in terms of non-linear optics (NLO). The terms NLO, two-photon microscope or multi-photon microscope (MPM) are often used interchangeably, even if their meaning is not identical.
You can find more information about Two-Photon Microscopy at our Two-Photon Microscopy, Non-Linear Optics (NLO) page:
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Stowers Links:
Stowers Microscopy Center: Two-Photon Microscopy
Image Center: LSM-510-NLO
Image Center: LSM-510-DEV
Foundations of Microscopy Seminar: Non-Linear Optics
Web Links:
User group: Multi-Photon Laser Scanning Microscopy
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 Like absorption and fluorescence spectra, fluorescence lifetime can be used to identify a specific type of dye molecules. The fluorescence lifetime is the time it takes an electron in an excited energy level of the dye molecule to return to its ground level while emitting fluorescence light. Typically, this takes several nano-seconds (ns), but depending on the energy states involved can be as long as micro-seconds (μs) or even longer. For our purposes we can neglect the time it takes to excite an electron.
The fluorescence lifetime of a molecule depends on its environment. If there are alternative routes to fluorescence to depopulate the excited energy state, the measured lifetime is reduced. One important way to depopulate an energy state is Fluorescence (Förster)-Resonance-Energy-Transfer (FRET) or quenching. Thus Fluorescence Lifetime Imaging Microscopy (FLIM) can be used to measure FRET efficiency. The advantage over intensity based methods is, that the FLIM measurement is less sensitive to changes in dye concentration and bleaching.
The system we are using to measure fluorescence lifetimes is based on Time Correlated Single Photon Counting (TCSPC). We use a pulsed laser to excite the dye molecule. The electronics measures the time between the laser pulse and the arrival of photons, the fluorescence life time.
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Stowers Links:
Image Center: LSM-510-DEV
Web Links:
Becker & Hickl: Applications
Literature:
Wolfgang Becker: TCSPC Handbooks TCSP Handbook
FLIM-LSM 510 Handbook
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 Fluorescence Correlation Spectroscopy (FCS) measures diffusion properties, local concentrations and interactions of molecules. It works with μl sample volumes (the theoretical limit is several femto-liter (fl)).The method is more sensitive at lower concentrations (the optimum is about 1 nano-molar).The measurements can be done in solution or living cells.
FCS is based on the fluctuation of light emitted by dye molecules crossing a small laser spot and detected with confocal optics. Chemical reactions or changes in the environment influence the fluctuations. One of the most common approaches is to measure the change of fluctuation, because a small molecule binds to a larger one. The small, fluorescence labeled molecule diffuses fast through the laser spot, emitting a short shower of photons. If it binds to a larger molecule it is slowed down and emits photons for a longer time.
You can find more information about FCS at our Fluorescence Correlation Spectroscopy (FCS) pages:
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Stowers Links:
Stowers Microscopy Center: FCS
Adv. Instr. & Physics: Literature FCS
Image Center: LSM-510-DEV
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To control our Carl Zeiss confocal microscopes and fluorescence correlation spectrometer and for most of our image processing tasks we are using the commercial Carl Zeiss Aim software package.s
To do symbolic mathematical computations we are using Mathematica.
The free NIH software package ImageJ adds additional image processing power.
For more advanced image processing and FCS curve fitting tasks we are using the programming environment IDL.
You can find more information about software at our Software page:
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Stowers Links:
Adv. Instr. & Physics: Software
IT Help Desk
BioInformatics
BioInformatics: Mathematica resources
BioInformatics: IDL resources
Earl Glynn's IDL resources
Web Links:
Download: Carl Zeiss LSM Image Browser
Download: AxioVision LE
Carl Zeiss: Macros
Carl Zeiss: LSM FTP Server (password required)
Wolfram Research: Mathematica
Wolfram Research: webMathematica
NIH: ImageJ download and documentation
ITT: IDL Home Page
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