
Full text loading...
Category: Microbial Genetics and Molecular Biology
Laser Scanning Microscopy, Page 1 of 2
< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555817497/9781555812232_Chap03-1.gif /docserver/preview/fulltext/10.1128/9781555817497/9781555812232_Chap03-2.gifAbstract:
This chapter provides a basis for understanding laser scanning microscopy (LSM) and the approaches that may be used to apply high-resolution digital microscopy to the study of bacteria. Additional reviews of image analysis, digital imaging, and LSM for microbiology applications are also provided in this chapter. The major focus of most courses is cell biology, although the information is of use and techniques are broadly transferable across disciplines. In practice, one of the appealing aspects of fluorescence microscopy is the lack of a requirement for fixation, dehydration, and the air drying of samples required by some other techniques. One of the goals of this approach is to examine living bacterial cells, aggregates, and biofilms. Changes in any of the microscope settings will alter the section thickness, the brightness, and the apparent size of the objects being imaged. The major advantage of LSM is the capacity to collect a series of images that allow the user to obtain 3-D spatial information. The chapter gives general protocol and considerations of lectin staining. A number of approaches have been applied to study diffusion in bacterial biofilms and serve to illustrate the application of kinetic analyses using LSM systems. The major limitation of all light microscopic systems and particularly LSM is poor axial resolution. In LSM there are both 2-D- and 3-D-related considerations regarding sampling. Application of LSM techniques results in the creation of vast image and data sets.
Full text loading...
1P and 2P LSM images of a fluidized bed reactor biofilm which degrades EDTA and is growing on pumice. The specimen was stained with SYBR green, and serial sections were taken by using both LSM systems on the same sample sequentially. Images show enhanced resolution of the 2P system relative to that of the 1P system with a 39-μm-thick biofilm specimen.
1P and 2P LSM images of a fluidized bed reactor biofilm which degrades EDTA and is growing on pumice. The specimen was stained with SYBR green, and serial sections were taken by using both LSM systems on the same sample sequentially. Images show enhanced resolution of the 2P system relative to that of the 1P system with a 39-μm-thick biofilm specimen.
Graphs showing the nature of excitation-emission spectra for an ideal series of green, red, and far-red-emitting fluors (A) and problematic fluors where excitation (Ex) and emission (Em) peaks overlap, resulting in potential cross talk or bleed through of the signal into adjacent imaging windows (B).
Graphs showing the nature of excitation-emission spectra for an ideal series of green, red, and far-red-emitting fluors (A) and problematic fluors where excitation (Ex) and emission (Em) peaks overlap, resulting in potential cross talk or bleed through of the signal into adjacent imaging windows (B).
1P LSM images showing a direct single-scan image (A) and the influence of image averaging on the quality and signal- to-noise ratio of an LSM image (B).
1P LSM images showing a direct single-scan image (A) and the influence of image averaging on the quality and signal- to-noise ratio of an LSM image (B).
1P LSM micrographs showing the nature of the 3-D stereo pair (A) and a single xy (B) and a single xz (C) scan through a microbial biofilm stained with the nucleic acid stain SYTO9 (Molecular Probes).
1P LSM micrographs showing the nature of the 3-D stereo pair (A) and a single xy (B) and a single xz (C) scan through a microbial biofilm stained with the nucleic acid stain SYTO9 (Molecular Probes).
Series of 1P LSM images showing the monitoring of pH by using a dually labeled (pH-sensitive fluorescein and pHinsensitive rhodamine) 10,000-molecular-weight dextran in a microbial biofilm. (A) pH-sensitive imaging of fluorescein. (B) pHinsensitive fluorescence of the rhodamine. (C) Grayscale representation of the standard curve for pH versus the ratio of images A and B. (D) Contour map showing the distribution of pH levels within the microbial biofilm.
Series of 1P LSM images showing the monitoring of pH by using a dually labeled (pH-sensitive fluorescein and pHinsensitive rhodamine) 10,000-molecular-weight dextran in a microbial biofilm. (A) pH-sensitive imaging of fluorescein. (B) pHinsensitive fluorescence of the rhodamine. (C) Grayscale representation of the standard curve for pH versus the ratio of images A and B. (D) Contour map showing the distribution of pH levels within the microbial biofilm.
Gallery presentation of a Z series taken through a microbial biofilm stained with SYTO9.
Gallery presentation of a Z series taken through a microbial biofilm stained with SYTO9.
3-D stereo pair presentation of the Z series shown in Fig. 6 .
3-D stereo pair presentation of the Z series shown in Fig. 6 .
Advantages and disadvantages of CLSM
Advantages and disadvantages of CLSM
Advantages and disadvantages of 2P LSM
Advantages and disadvantages of 2P LSM
Fluorescent compounds and their application in microbial studies
a Numbers in parentheses are excitation maximums in nanometers.
b FISH-MAR, FISH microautoradiography.
Fluorescent compounds and their application in microbial studies
a Numbers in parentheses are excitation maximums in nanometers.
b FISH-MAR, FISH microautoradiography.
Methods for the presentation of 3-D data sets
Methods for the presentation of 3-D data sets