Signal strength and noise in confocal microscopy: Factors influencing selection of an optimum detector aperture

Abstract

In confocal microscopy, several factors influence the selection of an optimum size and geometry of detector aperture. These include (1) strength of signal from the specimen, (2) noise level in the system, (3) optical configuration of the microscope (e.g., reflection or fluorescence), (4) time available for signal accumulation, ( 5 ) specimen thickness, and (6) amount of reduction in axial and transverse resolution (from the theoretical maximum) that can be tolerated. It is shown both theoretically and experimentally that the size of the detector aperture critically influences the type and amount of system noise that is detected along with the specimen signal. It is also demonstrated that increasing the size of the detector pinhole does not appreciably increase the signal strength from any single thin plane, but only increases the sampling depth, enhancing brightness at the cost of a reduction in axial resolution. As a result, it is shown that there is no advantage, from the standpoint of signal strength, to using a slit aperture rather than a circular detector pinhole. Finally, it is concluded that all confocal microscopes should be designed to allow the user the capability of selecting an optimum compromise detector aperture setting based on the particular specimen properties, type of microscopy (e.g., fluorescence or reflection) and resolution required.

How has confocal microscopy helped us in refractive surgery?

“…Since the outgoing light path and incoming light path use the same set of conjugate pinholes in the Nipkow disk, any misalignment of the Nipkow disk results in severe image degradation [28]. Thus, alignment of the disk requires a stable, reliable, high-speed Nipkow disk system from the manufacturer or a significant amount of the operator's time is lost in adjusting alignment.…”

“…These studies and many others demonstrate the unique optical-sectioning capabilities of the confocal microscope and provide the required initial analysis and comparison with prior standard histological examinations of the normal anatomy [15,23,28,34,35].…”

Fluorescent Labels for Confocal Microscopy

“…In principle, a fraction of the emitted light (roughly 25-50% or more) is lost to a confocal detection aperture, but in fact this loss is desirable to achieve enhanced signal-to-background ratios Webb, 1991, 1994;Sheppard et al, 1991;Sheppard, 1991;Webb et al, 1990). In principle, a fraction of the emitted light (roughly 25-50% or more) is lost to a confocal detection aperture, but in fact this loss is desirable to achieve enhanced signal-to-background ratios Webb, 1991, 1994;Sheppard et al, 1991;Sheppard, 1991;Webb et al, 1990).…”

Evaluating the performance of fluorescence microscopes

“…(Overall throughput = 0.86 × 0.26 × 0.70 × 0.50 = 0.078.) The pinhole itself will pass ≈ 75% of the in‐focus signal when opened to a diameter corresponding to one Airy disc ( Shuman, 1988; Sheppard et al ., 1991 ; Sandison et al ., 1995). Of the 5.8 × 10 4 photons per pixel passing through the pinhole, ≈ 10% will actually be detected (photomultiplier with S20 photocathode, 530 nm light), giving a maximum signal of 5800 ‘counts’ per pixel.…”

“…The confocal pinhole is opened to a size corresponding to 1.2 λ/NA in the specimen plane [1 Airy disc diameter; a larger pinhole would be more appropriate for comparisons between wide‐field and confocal systems, ( Sheppard et al ., 1991 ; Sandison et al ., 1995 )]. The magnification is chosen to give a pixel size in the final image corresponding to 0.05 μm in the specimen plane.…”