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Exploring the Depths: Matching Light Microscopy Techniques to Applications


Samuel Bonfi g* and Alec De Grand Olympus Scientifi c Solutions Americas , 48 Woerd Ave. , Waltham , MA 02453


* samuel.bonfi g@olympus.com


Abstract: Modern light microscopy techniques have specifi c advantages for the analysis of tissues and organisms that were unknown just a few decades ago. One important capability is the capture of images from greater depths within a specimen than ever before. This brief review summarizes the capabilities of fl uorescence microscopy, total internal refl ection fl uorescence (TIRF) microscopy, confocal microscopy, multiphoton microscopy, and super-resolution microscopy. In addition, clearing agents and special objective lenses are described that allow image capture from a depth of several millimeters in cleared tissue.


Introduction


Few researchers have the opportunity to build their entire laboratory from scratch; most work in laboratories where much of the equipment is already in place and only supplemental grants for select instrumentation are available. The microscopes in most life science research laboratories have become imaging workhorses, sometimes remaining in use for decades. But how does a scientist ascertain whether what is already in the laboratory is sufficient to meet the needs of the science he or she will be performing? Selecting an optimal microscopy technique depends on many things, such as the desired depth of imaging, resolution and magnifi - cation needs, ease of training, hardware expandability, and software capabilities.


All imaging techniques seek to maximize signal and decrease noise, and each experiment has its own set of challenges. For live specimens, the faster an organism moves or the deeper a region of interest is below the tissue surface, the greater the imaging challenge can be. Light scatter, absorption, background signal, and diff erences in refractive index present a variety of hurdles, but every imaging technique can be optimized with a system designed to meet particular needs. T e following is an overview of various microscope imaging techniques employed by the scientifi c community, with some of the benefi ts, drawbacks, and key applications of each technique. Of particular interest is the imaging depth associated with each method.


Fluorescence Microscopy T e go-to technique for biological imaging in many research laboratories is widefi eld epifl uorescence microscopy. Researchers use fluorescence imaging to capture the immunostaining of membranes, organelles, proteins, DNA, and many other biological structures, enabling the imaging of life processes as they occur. In fl uorescence microscopy, specimens are illuminated with specifi c wavelengths of light. T is light is absorbed by fl uorophores, also called fl uorescent probes, that have been attached to specimen features of interest; this absorption triggers the fl uorophores to emit specifi c, longer wavelengths of light. It is this emitted fl uorescent light that is detected by the microscope. Fluorescence images are routinely captured in grayscale using a photomultiplier


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tube (PMT); this light detector optimizes sensitivity for dimly fl uorescing specimens.


Widefield fluorescence microscopes offer bright, sharp images at a relatively low price point ( Figure 1 ). Fluorescence microscopy has the ability to detect low concentrations of specifi c fl uorophores over time with single-molecule resolution. A tremendous variety of fluorophores is available, allowing users to incorporate fl uorescence at diff erent wavelengths to create striking multicolor images. However, widefi eld fl uorescence has its drawbacks. Repeated exposure of the specimen to light oſt en leads to photobleaching, which reduces fl uorescence emission and degrades image contrast quickly. It also can be diffi cult to generate enough fl uorescence to gather suffi cient data. Biological specimens typically emit fl uorescence signals three to six orders of magnitude less than that of the illumination, making molecule detection challenging, particularly at greater depths [ 1 ]. Increasing the amount of light in order to get more signal out of the specimen also can be dangerous; the shorter-wavelength light used in fl uorescent imaging can be phototoxic to living cells. Finally, the inability of widefi eld epifl uorescence to reduce background noise outside the focal plane makes it generally unsuitable at depths greater than 700 nm.


TIRF Microscopy


One specialized form of fl uorescence is designed for imaging just below the surface of a cover slip where the cell membrane


Figure 1 : Mouse neurons and astrocytes captured using immunofl uorescence light microscopy. Neurons (green) were stained with anti-MAP2 and Alexa Fluor 488, astrocytes (red) were stained with anti-GFAP and Alexa Fluor 568, and nuclei (blue) were stained with DAPI. Image width = 4340 μ m. Courtesy of of James Striebel and Melissa Pathmajeyan, National Institutes of Health, Corvallis, MT. Honorable Mention, 2007 Olympus BioScapes Digital Imaging Competition.


doi: 10.1017/S1551929516000365 www.microscopy-today.com • 2016 May


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