Review of advanced imaging techniques

Abstract

Pathology informatics encompasses digital imaging and related applications. Several specialized microscopy techniques have emerged which permit the acquisition of digital images ("optical biopsies") at high resolution. Coupled with fiber-optic and micro-optic components, some of these imaging techniques (e.g., optical coherence tomography) are now integrated with a wide range of imaging devices such as endoscopes, laparoscopes, catheters, and needles that enable imaging inside the body. These advanced imaging modalities have exciting diagnostic potential and introduce new opportunities in pathology. Therefore, it is important that pathology informaticists understand these advanced imaging techniques and the impact they have on pathology. This paper reviews several recently developed microscopic techniques, including diffraction-limited methods (e.g., confocal microscopy, 2-photon microscopy, 4Pi microscopy, and spatially modulated illumination microscopy) and subdiffraction techniques (e.g., photoactivated localization microscopy, stochastic optical reconstruction microscopy, and stimulated emission depletion microscopy). This article serves as a primer for pathology informaticists, highlighting the fundamentals and applications of advanced optical imaging techniques.

Keywords: 2-photon microscopy; 4Pi microscopy; advanced imaging; confocal microscopy; digital; microscopy; optical coherence tomography; optics; photoactivated localization microscopy; spatially modulated illumination microscopy; stimulated emission depletion microscopy.

Figure 1

Jablonski diagram for single photon (a) and two-photon (b) excitation

Figure 2

High-speed volumetric imaging of chromosomes in mitosis using scanned Bessel beams in conjunction with two-photon excitation. The images shown demonstrate 3D tracing of two chromatids (green and purple) in a living cell over a series of eight image planes. (Reproduced with permission from reference 35.)

Figure 3

Schematic diagram of an OCT system using a Michelson interferometer

Figure 4

Line-scanning OCM images (a,c,e) and corresponding histology (b,d,f) at different depths: 40 mm (a,b); 100 mm (c,d); 150 mm (e,f). Bar = 100 m. (g) 3D isosurface view of two central crypts, including their lumens (l) and the adjacent goblet cells (g). 3D object size is 360 mm ´ 170 mm × 145 mm (depth). (Reproduced with permission from reference 65)

Figure 5

(a) A 21-gauge needle OCT probe overlying the human basal ganglia (PUT = putamen, GPe= globus pallidus externa, GPi = globus pallidus interna). Above it is the full-track reconstructed expanded OCT image that was created ex vivo. (b-d) Simultaneous OCT and Doppler OCT (DOCT) imaging of an anesthetized sheep brain in vivo showing real-time monitoring of vessel compression by the OCT probe. (Reproduced with permission from reference 79)

Figure 6

Live 3D SIM and conventional wide-field microscopy images showing the mitochondria dynamics in living HeLa cells. (a) Maximum-intensity projection along z dimension through the cell. (b) One x–z cross-section of the same volume sliced through the dashed line shown in a. (c–f) Single-plane x–y slices corresponding to the boxed regions in a. (g) Eight time frames of the region boxed with a dashed line in (a). Each frame is a maximum-intensity projection along z over a 1.3 m thickness that contains the featured ‘Y’-shaped mitochondrion. Scale bars are 2 μm (a,b) and 1 μm (c–g). (Reproduced with permission from reference 100.)

Figure 7

(a) The process of stimulated emission. A ground state (S0) fluorophore can absorb a photon from excitation light and jump to the excited state (S1). Spontaneous fluorescence emission brings the fluorophore back to the ground state. Stimulated emission happens when the excited-state fluorophore encounters another photon with a wavelength comparable to the energy difference between the ground and excited state. (b) Schematic drawing of a STED microscope. The excitation laser and STED laser are combined and focused into the sample through the objective. A phase mask is placed in the light path of the STED laser to create a specific pattern at the objective focal point. (c) In the xy mode, a donut-shaped STED laser is applied with the zero point overlapped with the maximum of the excitation laser focus. With saturated depletion, fluorescence from regions near the zero point is suppressed, leading to a decreased size of the effective PSF. (Reproduced with permission from reference 4.)

Figure 8

An example of two-color 3D STORM images of transferrin and clathrin in live cells. (a) Conventional image of clathrin coated pits (CCPs) and transferrin in a live cell. (b) A 3D STORM image x-y projection of the same area taken in 30 seconds. (c,d) STORM images of CCPs indicated in b: x-y cross-section near the plasma membrane (left), x-z cross-section cutting through the middle of the invaginating CCP (middle) and corresponding x-z cross-section of the clathrin channel only (right). Scale bars are 500 nm (a,b) and 100 nm (c,d). (Reproduced with permission from reference 114.)

Figure 9

The SL-QPM system. (Xe: Xenon-arc lamp; L: lens; A: aperture; BS: beam splitter; OB: objective; ST: sample stage; M: mirror; RM: removal mirror; TL; tube lens; CAM: camera; SP: spectrograph; SS: scanning stage; CCD: charged coupled device (CCD) camera.)

Figure 10

(a) Representative histology images of breast tissue and (b) the corresponding optical path length (OPL) maps of cell nuclei are shown from normal, uninvolved (i.e., histologically normal cells adjacent to tumor) and malignant cells. (c) By analyzing approximately 30-40 cell nuclei per group, the average optical path length of normal, uninvolved and malignant cell nuclei are significantly increased, when compared to the normal cell nuclei (t-test, two-sided P-value < 0.001)