All Research Areas

Autoconfocal microscopy

ACM is a nonlinear-optical microscopy that uses a femtosecond-pulsed infrared laser beam to trans-illuminate a tissue sample. As opposed to other nonlinear-optical techniques such as two-photon excited fluorescence (TPEF) microscopy, the laser beam does not generate signal within the sample. Instead, the laser beam traverses the sample and is then re-focused onto a nonlinear crystal. The detected signal is the SHG produced by the crystal. Because this signal is nonlinear, the crystal acts as a “virtual pinhole” and an ACM exhibits 3D resolution and background rejection similar to a transmission-confocal microscope. An ACM requires no sample labeling, is technically simple, and can be readily combined with existing nonlinear imaging modalities such as TPEF microscopy.

We have shown that the equivalent effect of a “virtual pinhole” can be achieved by thermionic emission in a standard PMT photocathode. Because of the nonlinear dependence of thermionic emission on absorbed laser power, a PMT is found to produce a virtual pinhole effect that rejects unfocused light at least as strongly as a physical pinhole. This virtual pinhole effect can be exploited in a scanning transmission confocal microscope equipped with a cw laser source. Because the area of the PMT photocathode is large, signal de-scanning is not required and thermionic detection acts as a self-aligned pinhole.


Publications Related to this Research Area

Autoconfocal microscopy with a cw laser and thermionic detection

Daryl Lim, Kengyeh K. Chu, and Jerome Mertz,

Optics Letters

We introduce an application of thermionic emission in a PMT photocathode. Because of the nonlinear dependence of thermionic emission on absorbed laser power, a conventional PMT is found to produce a virtual pinhole effect that rejects unfocused light at least as strongly as a physical pinhole. This virtual pinhole effect is exploited in a scanning transmission confocal microscope equipped with a cw laser source. Because the area of the PMT photocathode is large, signal descanning is not required and thermionic detection acts as a self-aligned pinhole. Our technique of thermionic-detection autoconfocal microscopy is further implemented with graded-field contrast to obtain enhanced phase-gradient sensitivity in unlabeled samples, such as rat hippocampal brain slices.

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Graded-field autoconfocal microscopy

Kengyeh K. Chu, Ran Yi, and Jerome Mertz,

Optics Express

Autoconfocal microscopy (ACM) is a simple implementation of a transmitted-light confocal microscopy where a nonlinear detector plays the role of a virtual self-aligned pinhole. We report here a significant improvement of ACM based on the use of graded-field (GF) imaging. The technique of GF imaging involves introducing partial beam blocks in the illumination and detection apertures of an imaging system. These partial beam blocks confer phase-gradient sensitivity to the imaging system and allow control over its background level. We present the theory of the GF contrast in the context of ACM, comparing it to GF contrast in a non-scanning widefield microscope, and discuss various performance characteristics of GF-ACM in terms of resolution, sectioning strength, and an “under-detection” light collection geometry. An advantage of ACM is that it can be readily combined with two-photon excited fluorescence (TPEF) microscopy. We present images of rat brain hippocampus using simultaneous GF-ACM and TPEF microscopy. These images are inherently co-registered.

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Autoconfocal microscopy with nonlinear transmitted light detection

T. Pons and J. Mertz,

Journal of the Optical Society of America B - Optical Physics

We describe a simple and robust technique for transmission confocal laser scanning microscopy wherein the detection pinhole is replaced by a thin second-harmonic generation crystal. The advantage of this technique is that self-aligned confocality is achieved without the need for signal descanning. We derive the point-spread function of our instrument and quantify both signal degradation and background rejection when imaging deep within a turbid slab. As an example, we consider a slab whose index of refraction fluctuations exhibit Gaussian statistics. Our model is corroborated by experiment.

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Autoconfocal Setup

Combined ACM and TPEF movie

Simultaneous graded-field ACM (gray) and TPEF (green) images of a rat hippocampus slice (400 μm thickness) stained with sulforhodamine 101, which preferentially labels astrocytes.