In this iBiology video Dr. Ron Vale (UCSF. HHMI) provides a survey of various techniques that are used in Light Microscopy. The goal is to find the technique that is most relevant for a particular set of experimental goals. He begins with describing the pros and cons of Transmitted Light and Fluorescence Microscopy methods. Within each of these two broad categories of Light Microscopy, he drills down further to examine the strengths and weaknesses of specific imaging modalities, showing images and live sequences that show use-cases of the techniques. Many of these techniques are described in more detail in other videos in this series, but this video provides an excellent overview of techniques in microscopy.
The benefit of using transmitted light microscopy techniques, e.g. Dark field microscopy, phase contrast microscopy is that they are simple and relatively inexpensive. No genetic encoding of fluorescence is needed. Also, these techniques can be less damaging to the sample than fluorescence excitation. This makes these techniques ideal for the long-term observation of live cells, while causing minimal damage..
Fluorescence Microscopy is a very popular technique, and there are many different versions. The selection of the most suitable method often depends on whether Live or Fixed samples are involved: this impacts the rate of acquisition and level of cellular damage that the experiment can tolerate. The thickness of specimens is also an important factor in this selection since different techniques are ideally suited for different imaging depths.
Epifluorescence (wide-field) imaging is ubiquitous in research laboratories today because it is easy to use and does not involve the cost or complexity of laser-based illumination. The disadvantage of this modality is that there can be a high background caused by out-of-focus fluorescence, effectively lowering the signal-to-background ratio which can degrade the contrast and clarity of an image. UV excitation can also be damaging to live cells.
Deconvolution Microscopy is a way to implement “sectioning” without using Laser illumination. It involves the addition of a motorized x-axis stage to a conventional epi-fluorescence microscope – but it also requires computational software to process a z-stack of images.
There are several imaging modalities within the broad category of Fluorescence Microscopy that can be sub-categorized as Laser Based Sectioning techniques
- Total Internal Reflection Fluorescence (TIRF): a particularly useful technique for imaging details of the sample that are near the coverslip surface (within ~200nm of the coverslip surface). It is interesting to note that a version of this technique is used in some Next Generation Sequencing systems from Illumina.
- Pinhole microscopy: these can be sub-categorized into (a) point or line scanning methods (well suited for fixed samples that are <100um thick, in which photodamage is less of a concern) or (b) spinning disk confocal methods (well suited for fast acquisition with live cell imaging, in samples that are <30um thick).
- Two photon imaging: well suited for samples that are > 100um thick (e.g. tissue, brain section)
- Light Sheet Microscopy: applicable to cleared specimens. Very low photodamage, allows fast acquisition of live, cleared specimens, e.g. zebrafish embryos.
Super Resolution Microscopy: these are techniques that seek to achieve better optical resolution than conventional techniques which are limited to 200~250nm (at best).
- Structured Illumination: optical resolution of ~100nm, well suited for Live Imaging
- STORM/PALM and STED: optical resolution of ~40nm, well suited for fixed samples
Dr. Nico Stuurman (UCSF) is a co-developer of Micromanager, the popular image acquisition plugin for ImageJ.
In this iBiology lecture, Dr. Stuurman provides an overview of the importance of the various components that are used in quantitative image analysis. Building on this framework, he shows that since the goal of quantitative image analysis is the acquisition of data, one must always be mindful of the interactions between the components of a system. In addition, he focuses on the computational resources that drive the components of the system and also analyze the resulting data.
Dr. Nico Stuurman next iBiology video provides an overview of Micromanager, a software package that is freely available. This video showcases Micromanager, but it illustrates basic principles that are used in many software packages that are used with research microscopes. Most laboratory setups involved in research microscopy typically include sCMOS and/or CMOS cameras. More complex setups also include software controlled light sources and motorized components to control microscope objectives, filters, shutters and stages.
For cameras that are compatible with Micromanager software: