The theory of the microscope

Martin, L. C.

doi:10.1088/0959-5309/43/2/308

Cited by 42 publications

(15 citation statements)

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“…In other words, for a given vertical position of the objective lens with respect to the sample, only molecules located close to the objective focal plane are (approximately) in focus in the tube lens' image plane. The typical vertical distance from the focal plane over which a molecule can be considered approximately in focus (the depth of field) is given by [95,96]:…”

Section: (A) Wide Field Imaging Approachesmentioning

confidence: 99%

Development of new photon-counting detectors for single-molecule fluorescence microscopy

Michalet

Colyer

Scalia

et al. 2013

Phil. Trans. R. Soc. B

106

121

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Two optical configurations are commonly used in single-molecule fluorescence microscopy: point-like excitation and detection to study freely diffusing molecules, and wide field illumination and detection to study surface immobilized or slowly diffusing molecules. Both approaches have common features, but also differ in significant aspects. In particular, they use different detectors, which share some requirements but also have major technical differences. Currently, two types of detectors best fulfil the needs of each approach: single-photon-counting avalanche diodes (SPADs) for point-like detection, and electron-multiplying charge-coupled devices (EMCCDs) for wide field detection. However, there is room for improvements in both cases. The first configuration suffers from low throughput owing to the analysis of data from a single location. The second, on the other hand, is limited to relatively low frame rates and loses the benefit of single-photon-counting approaches. During the past few years, new developments in point-like and wide field detectors have started addressing some of these issues. Here, we describe our recent progresses towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. We also discuss our development of large area photon-counting cameras achieving subnanosecond resolution for fluorescence lifetime imaging applications at the single-molecule level

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Section: (A) Wide Field Imaging Approachesmentioning

confidence: 99%

Development of new photon-counting detectors for single-molecule fluorescence microscopy

Michalet

Colyer

Scalia

et al. 2013

Phil. Trans. R. Soc. B

106

121

View full text Add to dashboard Cite

show abstract

“…Such systems are deemed as ''superresolution'' since they can surpass the diffraction limit. Axial resolution (i.e., the resolution of objects along the z-axis) is much more difficult to define, and a number of formulae have been offered (Shillaber, 1944;Françon, 1961;Martin, 1966;Piller, 1977). Here, we use the formula for depth of field from Inoue and Spring (1997): Depth of field ¼ l 0 n N:A: 2 þ n M Â N:A: e where l 0 is the wavelength of light (emission wavelength in the case of fluorescence), n is the lowest refractive index, M is the lens magnification, and e is the resolution of the detector (for example, the pixel spacing of the camera).…”

Section: Optical Resolutionmentioning

confidence: 99%

A primer on the fundamental principles of light microscopy: Optimizing magnification, resolution, and contrast

Goodwin

2014

Molecular Reproduction Devel

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SUMMARYThe light microscope is an indispensable tool in the study of living organisms. Most biologists are familiar with microscopes, perhaps being first introduced to the wonders of the world of small things at a very early age. Yet, few fully comprehend the nature of microscopy and the basis of its utility. This review (re)-introduces the concepts of magnification, resolution, and contrast, and explores how they are intimately related and necessary for effective microscopy.

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“…The calculation of the total DOF has been thoroughly discussed in the last several decades. [11][12][13][14][15][16] Although a standard method of determining the total DOF has not been established to date, the following method is the most recommended in this field to compute total DOF, which is the sum of the diffractive and geometric DOF: [11][12][13][14] …”

Section: Theoretical Analysis Of Dofmentioning

confidence: 99%

Impact of the optical depth of field on cytogenetic image quality

Qiu

Chen

et al. 2012

J. Biomed. Opt

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Abstract. In digital pathology, clinical specimen slides are converted into digital images by microscopic image scanners. Since random vibration and mechanical drifting are unavoidable on even high-precision moving stages, the optical depth of field (DOF) of microscopic systems may affect image quality, in particular when using an objective lens with high magnification power. The DOF of a microscopic system was theoretically analyzed and experimentally validated using standard resolution targets under 60× dry and 100× oil objective lenses, respectively. Then cytogenetic samples were imaged at in-focused and off-focused states to analyze the impact of DOF on the acquired image qualities. For the investigated system equipped with the 60× dry and 100× oil objective lenses, the theoretical estimation of the DOF are 0.855 μm and 0.703 μm, and the measured DOF are 3.0 μm and 1.8 μm, respectively. The observation reveals that the chromosomal bands of metaphase cells are distinguishable when images are acquired up to approximately 1.5 μm or 1 μm out of focus using the 60× dry and 100× oil objective lenses, respectively. The results of this investigation provide important designing trade-off parameters to optimize the digital microscopic image scanning systems in the future.

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The theory of the microscope

Cited by 42 publications

References 0 publications

Development of new photon-counting detectors for single-molecule fluorescence microscopy

Development of new photon-counting detectors for single-molecule fluorescence microscopy

A primer on the fundamental principles of light microscopy: Optimizing magnification, resolution, and contrast

Impact of the optical depth of field on cytogenetic image quality

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